Renato V. Iozzo

This review focuses on the extracellular proteoglycans. Special emphasis is placed on the structural features of their protein cores, their gene organization, and their transcriptional control. A simplified nomenclature comprising two broad groups of extracellular proteoglycans is offered: the small leucine-rich proteoglycans or SLRPs, pronounced “slurps,” and the modular proteoglycans. The first group encompasses at least five distinct members of a gene family characterized by a central domain composed of leucine-rich repeats flanked by two cysteine-rich regions. The second group consists of those proteoglycans whose unifying feature is the assembly of various protein modules in a relatively elongated and often highly glycosylated structure. This group is quite heterogeneous and includes a distinct family of proteoglycans, the “hyalectans,” that bind hyaluronan and contain a C-type lectin motif that is likely to bind carbohydrates, and a less distinct group that contains structural homologies but lacks hyaluronan-binding properties or lectin-like domains.—Iozzo, R. V., Murdoch, A. D. Proteoglycans of the extracellular environment: clues from the gene and protein side offer novel perspectives in molecular diversity and function. FASEB J. 10, 598-614 (1996)

The focus of this review is on conceptual and functional advances in our understanding of the small leucine-rich proteoglycans. These molecules belong to an expanding gene class whose distinctive feature is a structural motif, called the leucine-rich repeat, found in an increasing number of intracellular and extracellular proteins with diverse biological attributes. Three-dimensional modeling of their prototype protein core proposes a flexible, arch-shaped binding surface suitable for strong and distinctive interactions with ligand proteins. Changes in the properties of individual proteoglycans derive from amino acid substitutions in the less conserved surface residues, changes in the number and length of the leucine-rich repeats, and/or variation in glycosylation. These proteoglycans are tissue organizers, orienting and ordering collagen fibrils during ontogeny and in pathological processes such as wound healing, tissue repair, and tumor stroma formation. These properties are rooted in their bifunctional character: the protein moiety binding collagen fibrils at strategic loci, the microscopic gaps between staggered fibrils, and the highly charged glycosaminoglycans extending out to regulate interfibrillar distances and thereby establishing the exact topology of fibrillar collagens in tissues. These proteoglycans also interact with soluble growth factors, modulate their functional activity, and bind to cell surface receptors. The latter interaction affects cell cycle progression in a variety of cellular systems and could explain the purported changes in the expression of these gene products around the invasive neoplastic cells and in regenerating tissues.

Basement membranes are delicate, nanoscale and pliable sheets of extracellular matrices that often act as linings or partitions in organisms. Previously considered as passive scaffolds segregating polarized cells, such as epithelial or endothelial cells, from the underlying mesenchyme, basement membranes have now reached the center stage of biology. They play a multitude of roles from blood filtration to muscle homeostasis, from storing growth factors and cytokines to controlling angiogenesis and tumor growth, from maintaining skin integrity and neuromuscular structure to affecting adipogenesis and fibrosis. Here, we will address developmental, structural and biochemical aspects of basement membranes and discuss some of the pathogenetic mechanisms causing diseases linked to abnormal basement membranes.

Abstract Decorin (dcn) and biglycan (bgn), two members of the family of small leucine-rich proteoglycans (SLRPs), are the predominant proteoglycans expressed in skin and bone, respectively. Targeted disruption of the dcn gene results in skin laxity and fragility, whereas disruption of the bgn gene results in reduced skeletal growth and bone mass leading to generalized osteopenia, particularly in older animals. Here, we report that bgn deficiency leads to structural abnormality in collagen fibrils in bone, dermis, and tendon, and to a “subclinical” cutaneous phenotype with thinning of the dermis but without overt skin fragility. A comparative ultrastructural study of different tissues from bgn- and dcn-deficient mice revealed that bgn and dcn deficiency have similar effects on collagen fibril structure in the dermis but not in bone. Ultrastructural and phenotypic analysis of newly generated bgn/dcn double-knockout (KO) mice revealed that the effects of dcn and bgn deficiency are additive in the dermis and synergistic in bone. Severe skin fragility and marked osteopenia characterize the phenotype of double-KO animals in which progeroid changes are observed also in the skin. Ultrastructural analysis of bone collagen fibrils in bone of double-KO mice reveals a complete loss of the basic fibril geometry with the emergence of marked “serrated fibril” morphology. The phenotype of the double-KO animal mimics directly the rare progeroid variant of human Ehlers-Danlos syndrome (EDS), in which skin fragility, progeroid changes in the skin (reduced hypodermis), and osteopenia concur as a result of impaired glycosaminoglycan (GAG) linking to bgn and dcn core proteins. Our data show that changes in collagen fibril morphology reminiscent of those occurring in the varied spectrum of human EDS are induced by both bgn deficiency and dcn deficiency in mice. The effects of an individual SLRP deficiency are tissue specific, and the expression of a gross phenotype depends on multiple variables including level of expression of individual SLRPs in different tissues and synergisms between different SLRPs (and likely other macromolecules) in determining matrix structure and functional properties.

Research Article| September 15 1994 The biology of perlecan: the multifaceted heparan sulphate proteoglycan of basement membranes and pericellular matrices R V Iozzo; R V Iozzo 1Department of Pathology and Cell Biology and the Jefferson Cancer Institute, Thomas Jefferson University, Room 249, Jefferson Alumni Hall, 1020 Locust Street, Philadelphia, PA 19107, U.S.A. Search for other works by this author on: This Site PubMed Google Scholar I R Cohen; I R Cohen 1Department of Pathology and Cell Biology and the Jefferson Cancer Institute, Thomas Jefferson University, Room 249, Jefferson Alumni Hall, 1020 Locust Street, Philadelphia, PA 19107, U.S.A. Search for other works by this author on: This Site PubMed Google Scholar S Grässel; S Grässel 1Department of Pathology and Cell Biology and the Jefferson Cancer Institute, Thomas Jefferson University, Room 249, Jefferson Alumni Hall, 1020 Locust Street, Philadelphia, PA 19107, U.S.A. Search for other works by this author on: This Site PubMed Google Scholar A D Murdoch A D Murdoch 1Department of Pathology and Cell Biology and the Jefferson Cancer Institute, Thomas Jefferson University, Room 249, Jefferson Alumni Hall, 1020 Locust Street, Philadelphia, PA 19107, U.S.A. Search for other works by this author on: This Site PubMed Google Scholar Biochem J (1994) 302 (3): 625–639. https://doi.org/10.1042/bj3020625 Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Facebook Twitter LinkedIn MailTo Cite Icon Cite Get Permissions Citation R V Iozzo, I R Cohen, S Grässel, A D Murdoch; The biology of perlecan: the multifaceted heparan sulphate proteoglycan of basement membranes and pericellular matrices. Biochem J 15 September 1994; 302 (3): 625–639. doi: https://doi.org/10.1042/bj3020625 Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu toolbar search search input Search input auto suggest filter your search All ContentAll JournalsBiochemical Journal Search Advanced Search This content is only available as a PDF. © 1994 The Biochemical Society, London1994 Article PDF first page preview Close Modal You do not currently have access to this content.

The extracellular matrix (ECM) constitutes a highly dynamic three-dimensional structural network comprised of macromolecules, such as proteoglycans/glycosaminoglycans (PGs/GAGs), collagens, laminins, fibronectin, elastin, other glycoproteins and proteinases. In recent years, the field of PGs has expanded rapidly. Due to their high structural complexity and heterogeneity, PGs mediate several homeostatic and pathological processes. PGs consist of a protein core and one or more covalently attached GAG chains, which provide the protein cores with the ability to interact with several proteins. The GAG building blocks of PGs significantly influence the chemical and functional properties of PGs. The primary goal of this comprehensive review is to summarize major achievements and paradigm-shifting discoveries made on the PG/GAG chemistry-biology axis, focusing on structural variability, structure–function relationships, metabolic, molecular, and epigenetic mechanisms underlying their synthesis. Recent insights related to exosome biogenesis, degradation, and cell signaling, their status as diagnostic tools and potential pharmacological targets in diseases as well as current applications in nanotechnology and biotechnology are addressed. Moreover, issues related to docking studies, molecular modeling, GAG/PG interaction networks, and their integration are discussed.

The overall structure and architecture of the extracellular matrix undergo dramatic alterations in composition, form, and functionality over time. The stochasticity begins during development, essential for maintaining organismal homeostasis and is heavily implicated in many pathobiological states including fibrosis and cancer. Modeling and remodeling of the matrix is driven by the local cellular milieu and secreted and cell-associated components in a framework of dynamic reciprocity. This collection of expertly-written reviews aims to relay state-of-the-art information concerning the mechanisms of matrix modeling and remodeling in physiological development and disease.

Like the major theme of a Mozart concerto, the immense and pervasive extracellular matrix drives each movement and ultimately closes the symphony, embracing a unique role as the fundamental mediator for most, if not all, ensuing intracellular events. As such, it comes as no surprise that the mechanism of just about every known disease can be traced back to some part of the matrix, typically in the form of an abnormal amount or activity level of a particular matrix component. These defects considerably affect downstream signaling axes leading to overt cellular dysfunction, organ failure, and death. From skin to bone, from vessels to brain, from eyes to all the internal organs, the matrix plays an incredible role as both a cause and potential means to reverse diseases. Human malaises including connective tissue disorders, muscular dystrophy, fibrosis, and cancer are all extracellular matrix-driven diseases. The ability to understand and modulate these matrix-related mechanisms may lead to the future discovery of novel therapeutic options for these patients.

The small leucine-rich proteoglycans (SLRPs), decorin and biglycan, are key regulators of collagen fibril and matrix assembly. The goal of this work was to elucidate the roles of decorin and biglycan in tendon homeostasis. Our central hypothesis is that decorin and biglycan expression in the mature tendon would be critical for the maintenance of the structural and mechanical properties of healthy tendons. Defining the function(s) of these SLRPs in tendon homeostasis requires that effects in the mature tendon be isolated from their influence on development. Thus, we generated an inducible knockout mouse model that permits genetic ablation of decorin and biglycan expression in the mature tendon, while maintaining normal expression during development. Decorin and biglycan expression were knocked out in the mature patellar tendon with the subsequent turnover of endogenous SLRPs deposited prior to induction. The acute absence of SLRP expression was associated with changes in fibril structure with a general shift to larger diameter fibrils in the compound knockout tendons, together with fibril diameter heterogeneity. In addition, tendon mechanical properties were altered. Compared to wild-type controls, acute ablation of both genes resulted in failure of the tendon at lower loads, decreased stiffness, a trend towards decreased dynamic modulus, as well as a significant increase in percent relaxation and tissue viscosity. Collagen fiber realignment was also increased with a delayed and slower in response to load in the absence of expression. These structural and functional changes in response to an acute loss of decorin and biglycan expression in the mature tendon demonstrate a significant role for these SLRPs in adult tendon homeostasis.

We have determined the complete nucleotide and deduced amino acid sequence of the major protein core of the human heparan sulfate proteoglycan HSPG2/perlecan of basement membranes. Eighteen overlapping cDNA clones comprise 14.35 kilobase pairs (kb) of contiguous sequence with an open reading frame of 13.2 kb. The mature protein core, without the signal peptide of 21 amino acids, has a M(r) of 466,564. This large protein is composed of multiple modules homologous to the receptor of low density lipoprotein, laminin, neural cell adhesion molecules, and epidermal growth factor. Domain I, near the amino terminus, appears unique for the proteoglycan since it shares no significant homology with any other proteins. It contains three Ser-Gly-Asp sequences that could act as attachment sites for heparan sulfate glycosaminoglycans. Domain II is highly homologous to the LDL receptor and contains four repeats with perfect conservation of all 6 consecutive cysteines. Next is domain III which shares homology to the short arm of laminin A chain and contains four cysteine-rich regions intercalated among three globular domains. Domain IV, the largest module with greater than 2000 residues, contains 21 repeats of the immunoglobulin type as found in neural cell adhesion molecule. Near the beginning of this domain, there is a stretch of 29 hydrophobic amino acids which could allow the molecule to interact with the plasma membrane. Domain V, similar to the carboxyl-terminal globular G-domain of laminin A and to the related protein merosin, contains three globular regions and four EGF-like repeats. In situ hybridization and immunoenzymatic studies show a close association of this gene product with a variety of cells involved in the assembly of basement membranes, in addition to being localized within the stromal elements of various connective tissues. Our studies show that this proteoglycan is present in all vascularized tissues and suggest that this unique molecule has evolved from the utilization of modular structures with adhesive and growth regulatory properties.

Cell-surface heparan sulfate proteoglycans have been shown to participate in lipoprotein catabolism, but the roles of specific proteoglycan classes have not been examined previously. Here, we studied the involvement of the syndecan proteoglycan family. First, transfection of CHO cells with expression vectors for several syndecan core proteins produced parallel increases in the cell association and degradation of lipoproteins enriched in lipoprotein lipase, a heparan-binding protein. Second, a chimeric construct, FcR-Synd1, that consists of the ectodomain of the IgG Fc receptor Ia linked to the highly conserved transmembrane and cytoplasmic domains of syndecan-1 directly mediated efficient internalization, in a process triggered by ligand clustering. Third, internalization of lipase-enriched lipoproteins via syndecan-1 and of clustered IgGs via the chimera showed identical kinetics (t1/2 = 1 h) and identical dose-response sensitivities to cytochalasin B, which disrupts microfilaments, and to genistein, which inhibits tyrosine kinases. In contrast, internalization of the receptor-associated protein, which proceeds via coated pits, showed a t1/2 < 15 min, limited sensitivity to cytochalasin B, and complete insensitivity to genistein. Thus, syndecan proteoglycans can directly mediate ligand catabolism through a pathway with characteristics distinct from coated pits, and might act as receptors for atherogenic lipoproteins and other ligands in vivo.

The small leucine-rich proteoglycan decorin interacts with the epidermal growth factor receptor (EGFR) and triggers a signaling cascade that leads to elevation of endogenous p21 and growth suppression. We demonstrate that decorin causes a sustained down-regulation of the EGFR. Upon stable expression of decorin, the EGFR number is reduced by ∼40%, without changes in EGFR expression. However, EGFR phosphorylation is nearly completely abolished. Concurrently, decorin attenuates the EGFR-mediated mobilization of intracellular calcium and blocks the growth of tumor xenografts by down-regulating the EGFR kinase in vivo. Thus, decorin acts as an autocrine and paracrine regulator of tumor growth and could be utilized as an effective anti-cancer agent. The small leucine-rich proteoglycan decorin interacts with the epidermal growth factor receptor (EGFR) and triggers a signaling cascade that leads to elevation of endogenous p21 and growth suppression. We demonstrate that decorin causes a sustained down-regulation of the EGFR. Upon stable expression of decorin, the EGFR number is reduced by ∼40%, without changes in EGFR expression. However, EGFR phosphorylation is nearly completely abolished. Concurrently, decorin attenuates the EGFR-mediated mobilization of intracellular calcium and blocks the growth of tumor xenografts by down-regulating the EGFR kinase in vivo. Thus, decorin acts as an autocrine and paracrine regulator of tumor growth and could be utilized as an effective anti-cancer agent. epidermal growth factor receptor epidermal growth factor the cyclin-dependent kinase inhibitor p21WAF1/CIP1 cytosolic [Ca2+] The factors that control tumor progression in vivoinvolve, among others, the interplay between the invading neoplastic cells and the tumor stroma, a newly formed connective tissue highly enriched in proteoglycans, growth factors, and cytokines. Proteoglycans not only constitute a physical barrier to the invading tumor cells but also influence their behavior by binding and storing growth factors or by activating cell surface receptors (1Conrad H.E. Heparin-binding Proteins. Academic Press, Inc., San Diego1998Google Scholar, 2Bernfield M. Götte M. Park P.W. Reizes O. Fitzgerald M.L. Lincecum J. Zako M. Annu. Rev. Biochem. 1999; 68: 729-777Crossref PubMed Scopus (2332) Google Scholar). Decorin, a prototype member of an expanding family of small leucine-rich proteoglycans, plays key roles in regulating matrix assembly and cell proliferation (3Iozzo R.V. Annu. Rev. Biochem. 1998; 67: 609-652Crossref PubMed Scopus (1349) Google Scholar, 4Iozzo R.V. J. Biol. Chem. 1999; 274: 18843-18846Abstract Full Text Full Text PDF PubMed Scopus (577) Google Scholar). Most of decorin's biological interactions occur via the central leucine-rich repeat region, an arch-shaped structure whose concave surface is well suited to bind both globular and nonglobular proteins (5Weber I.T. Harrison R.W. Iozzo R.V. J. Biol. Chem. 1996; 271: 31767-31770Abstract Full Text Full Text PDF PubMed Scopus (307) Google Scholar). Our working hypothesis is that the increased expression of decorin around invasive carcinomas represents a mechanism designed to counteract the invading neoplastic cells (6Iozzo R.V. Crit. Rev. Biochem. Mol. Biol. 1997; 32: 141-174Crossref PubMed Scopus (454) Google Scholar). This hypothesis is based on several observations. For instance, decorin levels are markedly elevated during growth arrest and quiescence, its expression is abrogated by viral transformation, and its transcription is suppressed in most tumorigenic cell lines (4Iozzo R.V. J. Biol. Chem. 1999; 274: 18843-18846Abstract Full Text Full Text PDF PubMed Scopus (577) Google Scholar). Upon transgenic expression of decorin, tumor cells with diverse histogenetic backgrounds revert to their normal phenotype; they loose anchorage-independent growth, fail to generate tumors in immunocompromised animals, and become arrested in G1 (7Santra M. Skorski T. Calabretta B. Lattime E.C. Iozzo R.V. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7016-7020Crossref PubMed Scopus (206) Google Scholar,8Santra M. Mann D.M. Mercer E.W. Skorski T. Calabretta B. Iozzo R.V. J. Clin. Invest. 1997; 100: 149-157Crossref PubMed Scopus (184) Google Scholar). Lack of decorin expression is permissive for tumor development insofar as bitransgenic mice lacking both decorin and the tumor suppressor p53 develop an accelerated lymphoma tumorigenesis (9Iozzo R.V. Chakrani F. Perrotti D. McQuillan D.J. Skorski T. Calabretta B. Eichstetter I. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3092-3097Crossref PubMed Scopus (122) Google Scholar). We discovered that decorin causes dimerization and autophosphorylation of the epidermal growth factor receptor (EGFR),1 and that it binds both the soluble EGFR ectodomain and the immunopurified EGFR (10Moscatello D.K. Santra M. Mann D.M. McQuillan D.J. Wong A.J. Iozzo R.V. J. Clin. Invest. 1998; 101: 406-412Crossref PubMed Scopus (245) Google Scholar, 11Iozzo R.V. Moscatello D. McQuillan D.J. Eichstetter I. J. Biol. Chem. 1999; 274: 4489-4492Abstract Full Text Full Text PDF PubMed Scopus (322) Google Scholar). This interaction triggers a signal cascade leading to activation of mitogen-activated protein kinases (10Moscatello D.K. Santra M. Mann D.M. McQuillan D.J. Wong A.J. Iozzo R.V. J. Clin. Invest. 1998; 101: 406-412Crossref PubMed Scopus (245) Google Scholar), mobilization of intracellular calcium (12Patel S. Santra M. McQuillan D.J. Iozzo R.V. Thomas A.P. J. Biol. Chem. 1998; 273: 3121-3124Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar), up-regulation of p21WAF1/CIP1 (p21), a potent inhibitor of cyclin-dependent kinases (13El-Deiry W.S. Tokino T. Velculescu V.E. Levy D.B. Parsons R. Trent J.M. Lin D. Mercer W.E. Kinzler K.W. Vogelstein B. Cell. 1993; 75: 817-825Abstract Full Text PDF PubMed Scopus (7957) Google Scholar), and ultimately to growth suppression (7Santra M. Skorski T. Calabretta B. Lattime E.C. Iozzo R.V. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7016-7020Crossref PubMed Scopus (206) Google Scholar, 8Santra M. Mann D.M. Mercer E.W. Skorski T. Calabretta B. Iozzo R.V. J. Clin. Invest. 1997; 100: 149-157Crossref PubMed Scopus (184) Google Scholar, 14Nash M.A. Loercher A.E. Freedman R.S. Cancer Res. 1999; 59: 6192-6196PubMed Google Scholar). Despite the above findings, the mechanism by which decorin exerts its cytostatic effects is not understood. It is also unclear how activation of the EGFR by decorin would result in protracted growth suppression. In this investigation, we discovered that decorin leads to a profound and sustained down-regulation of the EGFR. Concurrently, decorin attenuates the EGFR-mediated mobilization of intracellular calcium and blocks the growth of tumor xenografts by acting as an in vivo paracrine growth inhibitor. These results provide an explanation for the long term effects of decorin on tumor suppression and suggest novel therapeutic approaches against cancer. Media and fetal bovine serum were purchased from Mediatech (Herndon, VA). Hybond ECL membranes were purchased from Amersham Pharmacia Biotech, and AG1478 was purchased from Calbiochem. Antibodies include polyclonal rabbit antibodies against the N-terminal region of decorin (15Fisher L.W. Stubbs III, J.T. Young M.F. Acta Orthop. Scand. 1995; 66: 61-65Crossref Scopus (0) Google Scholar) and monoclonal antibodies against human p21 (6B6; Pharmingen), against the human EGFR (Ab-12; NeoMarkers, Inc.), and against phosphotyrosine (PY20, Transduction Laboratories). Recombinant human decorin proteoglycan and decorin protein core were purified as before (16Ramamurthy P. Hocking A.M. McQuillan D.J. J. Biol. Chem. 1996; 271: 19578-19584Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). 125I-EGF was purchased from Amersham Pharmacia Biotech, and EGF was from R & D Systems. A431 squamous carcinoma cells were obtained from ATCC. Generation of A431 clones stably expressing decorin in pcDNA3.1 (Invitrogen) and screening procedures by Northern blotting were described before (8Santra M. Mann D.M. Mercer E.W. Skorski T. Calabretta B. Iozzo R.V. J. Clin. Invest. 1997; 100: 149-157Crossref PubMed Scopus (184) Google Scholar). Cells, rendered quiescent by serum deprivation for 24–36 h, were incubated with various ligands for 5–10 min, washed extensively, and processed for Western immunoblotting as described before (7Santra M. Skorski T. Calabretta B. Lattime E.C. Iozzo R.V. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7016-7020Crossref PubMed Scopus (206) Google Scholar, 8Santra M. Mann D.M. Mercer E.W. Skorski T. Calabretta B. Iozzo R.V. J. Clin. Invest. 1997; 100: 149-157Crossref PubMed Scopus (184) Google Scholar). The growth of various clones was monitored by CellTiter 96TM, a nonradioactive cell proliferation assay (Promega). Fluorescence-activated cell sorting analysis was performed as described before (8Santra M. Mann D.M. Mercer E.W. Skorski T. Calabretta B. Iozzo R.V. J. Clin. Invest. 1997; 100: 149-157Crossref PubMed Scopus (184) Google Scholar). A431, AD13, AD14, and AD15 cells (5 × 104 cells/2 cm2) were plated in complete medium (Dulbecco's modified Eagle's medium, 10% fetal bovine serum) into 24-well plates (Costar; Corning, NY). EGF binding was conducted with confluent cell cultures essentially as described previously (17Nugent M.A. Lane E.A. Keski-Oja J. Moses H.L. Newman M.J. Cancer Res. 1989; 49: 3884-3890PubMed Google Scholar). Briefly, cells were washed with ice-cold binding buffer (Dulbecco's modified Eagle's medium, 1 mg/ml bovine serum albumin, 25 mm Hepes, pH 7.2), and incubated for 10 min at 4 °C. 125I-EGF was added directly to the binding buffer along with the appropriate amount of unlabeled EGF (0.01–50 ng/ml). A large excess (2 μg/ml) of unlabeled EGF was added to replicate wells in the presence of each concentration of125I-EGF to empirically determine nonspecific binding levels. Cells were incubated for 3 h at 4 °C and washed three times with cold binding buffer, and bound 125I-EGF was extracted with 10 mm Tris, pH 7.4, 1 mm EDTA, 0.5% SDS for 15 min at room temperature. 125I-EGF was quantified in a γ-counter (Packard Auto γ 5650). Nonspecific binding, generally <2% of the input radioactivity, was subtracted from each experimental value. Specific binding was determined by the method of Scatchard using a single site model to determine theK d and number of EGF receptors per cell. While our data showed a somewhat curvilinear profile indicative of two classes of binding sites, the higher affinity class represented a very small percentage (<3%) of the total and could not be accurately resolved by our analysis. Thus, we applied a single-site linear regression analysis to the data, which yielded better fits with less statistical error. A431 cells were cultured in 12-well culture plates (∼105cells/well), while the decorin-expressing clones were cultured in microporous inserts (3 μm) and placed on the top of the A431 cells. After several days in co-culture, the target cells were tested for growth and EGFR phosphorylation as described above. For fluorescence imaging, the A431 cells and two of their decorin-expressing subclones, AD13 and AD14, were plated onto poly-d-lysine-coated coverglasses and grown to confluence in Dulbecco's modified Eagle's medium supplemented with 5% fetal calf serum and serum-starved for 24–36 h. The cells were loaded with the Ca2+-insensitive hydrophobic acetoxymethylester form of fura2 for 25–30 min at room temperature in an extracellular medium containing 121 mmNaCl, 5 mm NaHCO3, 10 mm Na-HEPES, 4.7 mm KCl, 1.2 mmKH2PO4, 1.2 mm MgSO4, 2 mm CaCl2, 10 mm glucose, and 2% bovine serum albumin, pH 7.4, supplemented with 0.003% pluronic acid and sulfinpyrazone (200 μm). After loading with fura2/AM, cells were washed into an extracellular medium with 0.25% bovine serum albumin supplemented with sulfinpyrazone. Subsequently, the coverslips were mounted on the thermostated stage (35 °C) of an Olympus IX70 inverted epifluorescence microscope coupled to a high quantum efficiency cooled CCD camera driven by a customized computer program that also controlled a scanning monochromator (DeltaRAM, PTI) to select multiple excitation wavelengths (18Csordás G. Thomas A.P. Hajnóczky G. EMBO J. 1999; 18: 96-108Crossref PubMed Scopus (454) Google Scholar). Fura2 fluorescence was measured at 340- and 380-nm excitation using a 400-nm long pass dichroic and a 510/80-nm band pass emission filter. At the end of each measurement, cells were permeabilized with an intracellular medium composed of 120 mm KCl, 10 mm NaCl, 1 mmKH2PO4, 20 mm Tris-Hepes, 2 mm MgATP, and 1 μg/ml each of antipain, leupeptin, and pepstatin, pH 7.2, supplemented with 40 μg/ml digitonin, and the fluorescence remaining in the cells was accounted as background fluorescence. Images of the background fluorescence were subtracted from the images collected from intact cells prior to calculation of the fluorescence ratios. To calculate [Ca2+]c in nm, [Ca2+]fura2 was calibrated in intracellular medium. To test the ability of decorin to function as a tumor repressor in vivo, decorin-producing cells were co-injected with wild-type A431 human squamous carcinoma cells into 4–6-week-old male nu/nu mice (Taconic) and treated in accordance with institutional guidelines. AD13 cells (A431 cells stably transfected with the human decorin transgene) were mixed with A431 cells in 1:2, 1:4, or 1:8 ratios and injected either alone or in the various combinations described in the text (see Fig. 7legend). Mice were carefully examined every two or three days for up to 35 days postinjection, and any tumor growth was measured with a microcaliper using the formula V =a(b 2/2), wherea is the width at the widest point and b is the width perpendicular to a. Tumors were frozen in liquid nitrogen, pulverized in a mortar, and boiled in SDS-polyacrylamide gel electrophoresis buffer containing 100 mmβ-mercaptoethanol before processing for SDS-PAGE and Western immunoblotting. Parallel samples were analyzed by conventional light microscopy (9Iozzo R.V. Chakrani F. Perrotti D. McQuillan D.J. Skorski T. Calabretta B. Eichstetter I. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3092-3097Crossref PubMed Scopus (122) Google Scholar).Figure 7Decorin acts as an autocrine and paracrine inhibitor of tumor growth in vivo. A, growth of tumor xenografts in nude mice, following injection of A431 or AD13 cells alone or in combination as indicated. In these experiments, the number of the cells was not kept constant. A431 and AD13 alone were injected at 1 × 106 cells/animal. The 1:2 ratio animals received 1 × 106 A431 and 2 × 106 AD13, whereas the 1:4 and 1:8 ratio animals were injected with 0.25 × 106 A431 and 1 × 106 AD13, or 0.2 × 106 A431 and 1 × 106 AD13, respectively. Each point represents the mean ± S.E. (n = 5). B, in these experiments, the number of A431 cells was kept constant at 0.25 × 106/animal in the co-injection. Wild-type A431 cells (n = 9) include animals injected with either 0.25 × 106 or 1 × 106 cells, and the results are pooled as the groups grew identically. The values represent the mean ± S.E. (n = 3 for each group except for A431 controls, where n = 9). C, morphology of the tumors from A. Notice the invasion of skeletal muscle (i) and prominent tumor angiogenesis (ii). In contrast, the co-injected tumors show no invasion of the subcutaneous structures (iii) and evidence of cytodifferentiation with tumor cells growing as islands surrounded by a collagenous matrix (iv) and showing evidence of polarization (v) and abundant deposition of cytokeratin (vi). D, Coomassie-stained SDS-polyacrylamide gel electrophoresis of pooled triplicates (60 μg/lane) from tumor cell lysates as indicated. The molecular mass in kDa of prestained size markers is indicated on the left. E, Western immunoblotting of samples identical to D using anti-phosphotyrosine antibodies. Note the reduction of tyrosyl phosphorylation of the EGFR in the 1:4 and 1:8 co-injected tumor xenografts.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Transmission of extracellular signals often starts with binding of a growth factor to surface receptors that in many cases carry an intrinsic tyrosine kinase activity (19Schlesinger J. Ullrich A. Neuron. 1992; 9: 383-391Abstract Full Text PDF PubMed Scopus (1295) Google Scholar, 20Riese D.J., II Stern D.F. Bioessays. 1998; 20: 41-48Crossref PubMed Scopus (698) Google Scholar). For example, EGF stimulates the formation of homo- and heterodimers with other members of the family of receptor tyrosine kinase, a process that generally leads to growth stimulation (21van der Geer P. Hunter T. Lindberg R.A. Annu. Rev. Cell Biol. 1994; 10: 251-337Crossref PubMed Scopus (1252) Google Scholar). However, the proliferation of tumor cells bearing high levels of EGFR, such as A431 squamous (22Fan Z. Lu Y. Wu X. DeBlasio A. Koff A. Mendelsohn J. J. Cell Biol. 1995; 131: 235-242Crossref PubMed Scopus (132) Google Scholar) or MDA468 mammary (23Filmus J. Trent J.M. Pollak M.N. Buick R.N. Mol. Cell. Biol. 1987; 7: 251-257Crossref PubMed Scopus (98) Google Scholar) carcinoma cells, is stimulated by picomolar but paradoxically suppressed by nanomolar concentrations of EGF, presumably via down-regulation of the EGFR (24Carpenter G. J. Cell Biol. 1999; 146: 697-702Crossref PubMed Scopus (247) Google Scholar). To investigate whether decorin might induce growth suppression by activating a similar pathway, we established a number of A431 clones expressing the full-length human decorin driven by the potent cytomegalovirus promoter. All of the decorin-expressing clones (Fig. 1,A and B) exhibited increased levels of endogenous p21 (Fig. 1 C) and became growth-retarded (Fig.1 D), with a proportional increase in cells arrested in G1 (Fig. 1 E). The addition of either recombinant decorin or decorin protein core (1 μm) caused growth inhibition in wild-type A431 cells, an effect mediated by the EGFR insofar as AG1478 (1 μm), a tyrphostin that specifically inhibits the EGFR kinase (25Levitzki A. Gazit A. Science. 1995; 267: 1782-1788Crossref PubMed Scopus (1628) Google Scholar), abrogated the cytostatic effects induced by either decorin or EGF (not shown). Three decorin-expressing clones (AD13, AD14, and AD15) were studied in depth. While the steady state levels of EGFR declined by ∼40% (42 ± 8%, n = 5), the degree of EGFR activation, as measured by its phosphorylation status, declined by ∼95% (Fig.2, A and B). When the blots were exposed for a short time (2 s), there was no detectable EGFR phosphorylation in the decorin-expressing clones. Only when the blots were saturated (after a 6-s exposure) could tyrosyl phosphorylation of the EGFR be detected (Fig. 2 A). The involvement of a nonspecific phosphatase was ruled out by experiments in which even relatively high concentrations of Na3VO4 (100 μm) caused no significant change in the state of EGFR phosphorylation (not shown). Moreover, the steady state levels of EGFR mRNA were not significantly altered in the decorin-expressing clones (Fig.2 C), although there was clonal variability in EGFR expression. When 125I-EGF binding was conducted, the number of binding sites/cell was reduced in all of the decorin-expressing clones to levels comparable with those obtained with immunoblotting (Fig.2 D). Specifically, the binding sites/cell were 2.4 × 106 in A431 cells, in contrast to the AD13, AD14, and AD15, which expressed 1.5, 1.1, and 1.2 × 106 binding sites/cell, respectively. Interestingly, the affinity constants determined by Scatchard analysis were not significantly different among the various cell lines (K d = 15–21 nm). These experiments were repeated five times with comparable results. To further prove that the affinity of the EGFR for EGF was not significantly affected by decorin, quiescent cells were exposed for 10 min to various concentrations of EGF. In agreement with the data presented above, the decorin-expressing cells showed reduced levels of EGFR protein (Fig. 2 E, upper panel) and, in the absence of exogenous EGF, displayed no detectable EGFR phosphorylation (Fig. 2 E, lower panel). However, the activation of the remaining EGFRs was not markedly altered. When the blots were quantified by scanning densitometry, the degree of EGFR phosphorylation upon EGF challenge followed a pattern similar to the controls (Fig. 2 F) although somewhat attenuated at submaximal (10 ng/ml) concentrations of EGF. Similar results were obtained with a 5-min exposure to EGF (not shown). Thus, decorin induces a substantial reduction in the number of EGFRs without affecting EGFR mRNA steady-state levels. However, decorin causes an even greater suppression of EGFR phosphorylation. A potential mechanism for the down-regulation of EGFR includes an increased rate of intracellular dephosphorylation after ligand activation (19Schlesinger J. Ullrich A. Neuron. 1992; 9: 383-391Abstract Full Text PDF PubMed Scopus (1295) Google Scholar, 24Carpenter G. J. Cell Biol. 1999; 146: 697-702Crossref PubMed Scopus (247) Google Scholar, 26Weiss F.U. Daub H. Ullrich A. Curr. Opin. Genet. Dev. 1997; 7: 80-86Crossref PubMed Scopus (106) Google Scholar). To address this issue, we exposed quiescent (incubated in serum-free medium for 24 h) A431 or AD13 cells to EGF (40 ng/ml) for 5 min and then chased the cells for 1–40 min in the same medium supplemented with or without AG1478 (2 μm) to block EGFR kinase. While the control samples, treated with Me2SO vector alone, showed no appreciable EGFR dephosphorylation within the 40-min chase, the AG1478-treated samples showed a very rapid disappearance of EGFR tyrosyl phosphorylation (Fig.3 A). Although quantitatively different, the half-life of EGFR tyrosyl phosphorylation was essentially the same in both A431 and AD13 cells,t 12 ∼ 40 ± 10 s (Fig. 3 B). Notably, the EGF-induced tyrosyl phosphorylation of c-Cbl and the 85-kDa subunit of phosphatidylinositol 3-kinase, both known to be substrates for EGFR kinase, decayed even faster than EGFR (Fig.3 A). Because in these experiments the medium was not changed before adding EGF or AG1478, we conclude that the accumulated decorin synthesized in 24–25 h does not significantly affect the rate of EGFR dephosphorylation following ligand-induced phosphorylation of the EGFR. A potential problem in interpreting data from stably transfected cells is the plausible integration of the transgene into a locus that may inadvertently activate or suppress a gene involved in the EGFR signaling pathway. To address this issue, we established co-culture experiments in which either A431 or the decorin-secreting clones (AD13, AD14, or AD15) were cultured in the top chamber of a microporous (3-μm diameter) well, while the bottom wells contained A431 cells. Thus, any soluble molecules, but not cells, would be able to diffuse through the membrane and affect the behavior of the target A431 cells. After 5 days in culture in complete medium containing 10% serum, there was a significant inhibition of A431 cell growth only when the cells were co-cultured in the presence of the decorin-expressing clones at various ratios (Fig. 4,A and B). Concurrent with these changes, there was a marked suppression of EGFR tyrosyl phosphorylation (Fig. 4,C and D). In a time course experiment, decorin-mediated EGFR phosphorylation peaked at ∼ 2 h and declined to very low levels at 6–8 h (not shown). Collectively, these results indicate that secreted decorin can induce the same biochemical changes as the endogenous transgene and demonstrate a role for decorin as a paracrine regulator of tumor cell growth and EGFR kinase activity. One of the downstream effectors of the EGFR is phospholipase Cγ that binds through its SH2 domains to the activated EGFR and becomes tyrosine-phosphorylated. Phospholipase Cγ catalyzes the formation of inositol 1,4,5-triphosphate that in turn mobilizes Ca2+ from internal stores, leading to elevations of [Ca2+]c, a signal that plays fundamental roles in the control of cellular growth and differentiation (27Berridge M. Lipp P. Bootman M. Curr. Biol. 1999; 9: R157-R159Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar, 28Clapham D.E. Cell. 1995; 80: 259-268Abstract Full Text PDF PubMed Scopus (2272) Google Scholar). It has also been shown that down-regulation of EGFR results in attenuated EGF-activated [Ca2+]c signals (12Patel S. Santra M. McQuillan D.J. Iozzo R.V. Thomas A.P. J. Biol. Chem. 1998; 273: 3121-3124Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar,29Carpenter G. Annu. Rev. Biochem. 1987; 56: 881-914Crossref PubMed Scopus (1055) Google Scholar). To test if the EGFR-linked [Ca2+]c signaling could be involved in the changes in expression and phosphorylation of the EGFR described above, we monitored [Ca2+]c in intact, individual cells loaded with the ratiometric fluorescent Ca2+ indicator fura2 (18Csordás G. Thomas A.P. Hajnóczky G. EMBO J. 1999; 18: 96-108Crossref PubMed Scopus (454) Google Scholar). The resting [Ca2+]c did not vary between the wild-type and decorin-expressing cells (48 ± 11 nm in A431, 44 ± 5 nm in AD13, and 49 ± 5 nm in AD14 cells, respectively). The addition of a maximal dose of EGF (100 ng/ml) to A431 cells evoked a large [Ca2+]c increase essentially in every cell (green-red shift,images i–iii in Fig.5 A), and the [Ca2+]c signal displayed a rapid upstroke followed by a slow decay (Fig. 5 A, panel iv). In contrast, the decorin-expressing clones showed either no elevation or a slow and small elevation of [Ca2+]c (images i–iii in Fig. 5, B and C). Time course traces of the mean [Ca2+]c changes in AD13 and AD14 cells also showed the attenuated [Ca2+]c signal in response to maximal EGF (Fig. 5, A–C, panels iv). To further investigate the mechanisms underlying the impaired [Ca2+]c signaling in the decorin-expressing cells, we monitored the [Ca2+]c responses evoked by submaximal and maximal doses of EGF and by ATP that elicits Ca2+ mobilization without activation of EGFR or phospholipase Cγ. A suboptimal dose of EGF (10 ng/ml) elicited [Ca2+]c elevations that were 70 ± 11 and 58 ± 16% smaller in AD13 and AD14 cells, respectively, than A431 cells (n = 6, p < 0.05 for both, Fig.6). The extent of suppression of the elevations evoked by maximal EGF (100 ng/ml) was 50 ± 14 and 66 ± 10% in AD13 and AD14 cells, respectively (n= 4, p < 0.02 and <0.01, respectively). In contrast, ATP (200 μm) evoked [Ca2+]cincreases that were not significantly different in the wild-type and decorin-expressing clones (AD13, 87 ± 18%; AD14, 129 ± 19% of A431; n = 10). These data show impairment of the EGF-activated calcium signaling pathway in the decorin-expressing cells. Because no attenuation of the ATP-induced calcium signals was found in the decorin-expressing cells, changes in the EGFR or a factor proximal to inositol 1,4,5-triphosphate formation should account for the abnormal EGF-activated calcium signaling. To test whether decorin could act as an autocrine and paracrine inhibitor of in vivo tumor growth, A431 or AD13 cells were injected into nude mice either alone or in various combinations. Notably, the decorin-expressing AD13 cells did not form any tumors (Fig.7 A) even after 3 months of observation following the end point of the experiment. In contrast, the A431 cells and the 1:2 ratio generated tumors essentially with similar kinetics. However, the 1:4 and 1:8 tumors were significantly smaller than controls after 4 weeks of xenograft growth. In a second set of experiments (Fig. 7 B), we utilized ratios in which the number of co-injected tumor cells was kept proportional to the wild-type cells. The A431-injected mice (0.25 and 1.0 × 106 cells) exhibited identical rates of tumor growth and are shown as a single line (Fig. 7 B). At no time did the 1:4 (0.25; 1.0 × 106 A431/AD13 cells), the 1:8 (0.25; 2.0 × 106 A431/AD13 cells) mixtures or the AD13-injected mice show any tumor growth. These animals were allowed to live for an additional 3 months with no signs of tumor. All of the A431-generated tumor xenografts revealed extensive invasion of the deep fascia and subcutaneous skeletal muscles (Fig.7 C, i), together with copious neovascularization (Fig. 7 C, ii). In contrast, the 1:4 ratio showed very sharp margins and no infiltration of the deeper soft tissues (Fig.7 C, iii). In addition, the 1:8 ratio revealed evidence of cytodifferentiation characterized by the formation of tumor nests surrounded by a dense collagenous matrix (Fig. 7 C,iv), polarized epithelial cells (Fig. 7 C,v), and extensive keratin formation (Fig. 7 C,vi). Western immunoblotting of pooled tumor cell lysates from three xenografts revealed that at similar protein concentration (Fig. 7 D) there was a significant down-regulation of EGFR phosphorylation in the 1:4 and 1:8 xenografts (Fig. 7 E). However, no human decorin was detected in the tumor xenografts using sensitive immunoblotting assays and purified decorin as positive controls (not shown). We estimated that the decorin levels in the 1:4 and 1:8 tumor xenografts were ≤10 ng/50 μg of total cell protein. This is probably due to the poor survival of the decorin-expressing cells, since the AD13 alone never generated tumors in several independent experiments using different tumor cell inocula. Thus, the initial exposure to decorin might have been sufficient to down-regulate the EGFR for a protracted time in the A431 cells. These data substantiate the co-culture experiments described above and further demonstrate that tumor cells genetically engineered ex vivo to express decorin can suppress the growth of EGFR-overexpressing tumor cells in both a paracrine and autocrine fashion. Overexpression of ErbB receptor tyrosine kinase correlates wit

Perlecan is a modular heparan sulfate proteoglycan that is an intrinsic constituent of all basement membranes and extracellular matrices. Because of its strategic position and unique structure, perlecan has been implicated in modulating the activity of various growth factors required for normal development and tissue homeostasis. To gain insights into the potential function of perlecan in vivo, we examined the spatiotemporal distribution of its mRNA and protein core during murine embryogenesis. We utilized a new affinity-purified antibody that recognizes specifically the protein core of perlecan together with an in situ RT-PCR approach to perform a systematic analysis of perlecan expression and deposition during murine ontogeny. Perlecan appeared early (E10.5) in tissues of vasculogenesis including heart, pericardium, and major blood vessels. Its early expression coincided with the development of the cardiovascular system. Subsequently (E11–13), the greatest deposition of perlecan occurred within the developing cartilage, especially the cartilage undergoing endochondral ossification, where it remained elevated throughout all the developmental stages, and up to adulthood. Interestingly, the mRNA levels of perlecan were always higher in all the vascularized tissues, principally within endothelial cells, while chondrocytes displayed relatively low mRNA levels. This suggests a higher biosynthesis and turnover rates in the blood vessels vis-à-vis those of cartilaginous and other mesenchymal tissues. During later stages of development (E13–17.5) perlecan mRNA levels progressively increased and its expression correlated with the onset of tissue differentiation of various parenchymal organs including the developing kidneys, lungs, liver, spleen, and gastrointestinal tract. The central nervous system showed no perlecan expression with the exception of the calvaria and choroid plexus. Collectively, the results indicate that perlecan may play crucial roles not only in vasculogenesis but also in the maturation and maintenance of differentiated tissues, including cartilage. Dev. Dyn. 1997;210:130–145. © 1997 Wiley-Liss, Inc.

Perlecan, a ubiquitous heparan sulfate proteoglycan, possesses angiogenic and growth-promoting attributes primarily by acting as a coreceptor for basic fibroblast growth factor (FGF-2). In this report we blocked perlecan expression by using either constitutive CMV-driven or doxycycline- inducible antisense constructs. Growth of colon carcinoma cells was markedly attenuated upon obliteration of perlecan gene expression and these effects correlated with reduced responsiveness to and affinity for mitogenic keratinocyte growth factor (FGF-7). Exogenous perlecan effectively reconstituted the activity of FGF-7 in the perlecan-deficient cells. Moreover, soluble FGF-7 specifically bound immobilized perlecan in a heparan sulfate-independent manner. In both tumor xenografts induced by human colon carcinoma cells and tumor allografts induced by highly invasive mouse melanoma cells, perlecan suppression caused substantial inhibition of tumor growth and neovascularization. Thus, perlecan is a potent inducer of tumor growth and angiogenesis in vivo and therapeutic interventions targeting this key modulator of tumor progression may improve cancer treatment.

Perlecan, a multidomain heparan sulfate proteoglycan (PG), is an intrinsic component of basement membranes and extracellular matrices. We used a prokaryotic expression vector to generate fusion proteins encoding various domains of human perlecan protein core and these recombinant proteins were used as immunogens to produce mouse anti-human monoclonal antibodies (MAb). One MAb, designated 7B5, was characterized by Western blotting and ELISA and was shown to react specifically with the laminin-like region of perlecan (Domain III) but not with two other fusion proteins encoding Domain II or V. This perlecan epitope was detected by immunoenzymatic staining in the basement membranes of human tissues including pituitary gland, skin, breast, thymus, prostate, colon, liver, pancreas, spleen, heart, and lung. All vascular basement membranes tested contained this gene product. In addition, sinusoidal vessels of liver, spleen, lymph nodes, and pituitary gland expressed high levels of perlecan in the subendothelial region. In situ hybridization, using as probe the same human cDNA-encoding Domain III, localized perlecan mRNA to specific cell types within the tissues and demonstrated that in skin, perlecan appears to be synthesized exclusively by connective tissue cells in the dermal layer. The availability of MAb against precise regions of human perlecan will allow the investigation of this gene product in normal and diseased states.

The connective tissue stroma of malignant tumors is a newly formed tissue that supports the growth and progression of neoplastic cells. Proteoglycans are intrinsic components of this complex structure and molecular changes in this class of macromolecules can significantly affect behavioral properties of transformed cells. We report that human colon carcinoma contained increased levels of a chondroitin sulfate proteoglycan that exhibited an altered glycosaminoglycan structure in which 0- and 6-sulfated units, as detected by specific monoclonal antibodies, predominated. Proteoglycans with such epitopes were localized primarily to the connective tissue stroma surrounding the tumor cells but not to the tumor cells themselves or the native, non-cancerous connective tissue. Analysis of mRNA encoding PG-40, the main chondroitin sulfate proteoglycan of colon tissue, revealed a 7-fold increase in the two transcripts encoding this gene product. This increase was evident whether the data were normalized to total RNA content or beta-actin mRNA levels. The altered steady state levels of PG-40 mRNA did not correlate with any significant gene amplification or rearrangement of PG-40 in human colon cancer. However, when genomic DNA was tested for degree of methylation, the colon carcinoma tissue showed a marked hypomethylation of PG-40 gene locus, a finding that has been associated with increased gene activation. Interestingly, PG-40 gene was also hypomethylated in cultured colon fibroblasts, which express PG-40, but not in colon carcinoma cells which do not express this gene. These results indicate that specific proteoglycan changes occur in colon carcinoma and that these alterations are the product of stromal cells that are topologically associated with and functionally respondent to the growing malignant cells. This is the first evidence that enhanced PG-40 expression in a human malignant tissue is associated with a hypomethylated gene and suggests that the control of PG-40 gene expression may represent an important factor in the progression of colon carcinoma.

After 24 h of continuous labeling with radioactive precursors, a high molecular weight heparan sulfate proteoglycan (HS-PG) was isolated from both the medium and cell layer of human colon carcinoma cells (WiDr) in culture. The medium HS-PG eluted from a diethylaminoethyl anion exchange column with 0.45-0.50 M NaCl, had an average density of 1.46-1.49 g/ml on dissociative CsCl density-gradient ultracentrifugation, and eluted from Sepharose CL-2B with a Kav = 0.57. This proteoglycan had an estimated Mr of congruent to 8.5 X 10(5), with glycosaminoglycan chains of Mr = 3 X 10(4) which were all susceptible to HNO2 deaminative cleavage. Deglycosylation of the HS-PG with polyhydrogen fluoride resulted in a 3H-core protein with Mr congruent to 2.4 X 10(5). The cell layer contained a population of HS-PG with characteristics almost identical to that released into the medium but with a larger Mr = 9.5 X 10(5). Furthermore, an intracellular pool contained smaller heparan sulfate chains (Mr congruent to 1 X 10(4)) which were mostly devoid of protein core. In pulse chase experiments, only the large cell-associated HS-PG was released (approximately 58%) into the medium as intact proteoglycan and/or internalized and degraded (approximately 42%), with a t1/2 = 6 h. However, the small intracellular component was never released into the medium and was degraded at a much slower rate. When the cells were subjected to mild proteolytic treatment, only the large cell-associated HS-PG, but none of the small component, was displaced. Addition of exogenous heparin did not displace any HS-PG into the medium. Both light and electron microscopic immunocytochemistry revealed that the cell surface reacted with antibody against an HS-PG isolated from a basement membrane-producing tumor. Electron microscopic histochemistry using ruthenium red and/or cuprolinic blue revealed numerous 10-50-nm diam granules and 70-220-nm-long electron-dense filaments, respectively, on the surface of the tumor cells. The results indicate that colon carcinoma cells synthesize HS-PGs with distinct structural and metabolic characteristics: a large secretory pool with high turnover, which appears to be synthesized as an integral membrane component and localized primarily at the cell surface, and a small nonsecretory pool with low turnover localized predominantly within the cell interior. This culture system offers an opportunity to investigate in detail the mechanisms involved in the regulation of proteoglycan metabolism, and in the establishment of the neoplastic phenotype.

Inflammation and autophagy have emerged as prominent issues in the context of proteoglycan signaling. In particular, two small, leucine‐rich proteoglycans, biglycan and decorin, play pivotal roles in the regulation of these vital cellular pathways and, as such, are intrinsically involved in cancer initiation and progression. In this minireview, we will address novel functions of biglycan and decorin in inflammation and autophagy, and analyze new emerging signaling events triggered by these proteoglycans, which directly or indirectly modulate these processes. We will critically discuss the dual role of proteoglycan‐driven inflammation and autophagy in tumor biology, and delineate the potential mechanisms through which soluble extracellular matrix constituents affect the microenvironment associated with inflammatory and neoplastic diseases.

Previous studies have shown that decorin expression is significantly reduced in colorectal cancer tissues and cancer cells, and genetic deletion of the decorin gene is sufficient to cause intestinal tumor formation in mice, resulting from a downregulation of p21, p27(kip1) and E-cadherin and an upregulation of β-catenin signaling [Bi,X. et al. (2008) Genetic deficiency of decorin causes intestinal tumor formation through disruption of intestinal cell maturation. Carcinogenesis, 29, 1435-1440]. However, the regulation of E-cadherin by decorin and its implication in cancer formation and metastasis is largely unknown. Using a decorin knockout mouse model (Dcn(-/-) mice) and manipulated expression of decorin in human colorectal cancer cells, we found that E-cadherin, a protein that regulates cell-cell adhesion, epithelial-mesenchymal transition and metastasis, was almost completely lost in Dcn(-/-) mouse intestine, and loss of decorin and E-cadherin accelerated colon cancer cell growth and invasion in Dcn(-/-) mice. However, increasing decorin expression in colorectal cancer cells attenuated cancer cell malignancy, including inhibition of cancer cell proliferation, promotion of apoptosis and importantly, attenuation of cancer cell migration. All these changes were linked to the regulation of E-cadherin by decorin. Moreover, overexpression of decorin upregulated E-cadherin through increasing of E-cadherin protein stability as E-cadherin messenger RNA and promoter activity were not affected. Co-immunoprecipitation assay showed a physical binding between decorin and E-cadherin proteins. Taken together, our results provide direct evidence that decorin-mediated inhibition of colorectal cancer growth and migration are through the interaction with and stabilization of E-cadherin.

Although the growth factor progranulin was discovered more than two decades ago, the functional receptor remains elusive. Here, we discovered that EphA2, a member of the large family of Ephrin receptor tyrosine kinases, is a functional signaling receptor for progranulin. Recombinant progranulin bound with high affinity to EphA2 in both solid phase and solution. Interaction of progranulin with EphA2 caused prolonged activation of the receptor, downstream stimulation of mitogen-activated protein kinase and Akt, and promotion of capillary morphogenesis. Furthermore, we found an autoregulatory mechanism of progranulin whereby a feed-forward loop occurred in an EphA2-dependent manner that was independent of the endocytic receptor sortilin. The discovery of a functional signaling receptor for progranulin offers a new avenue for understanding the underlying mode of action of progranulin in cancer progression, tumor angiogenesis, and perhaps neurodegenerative diseases.

Biglycan, a small leucine-rich proteoglycan, acts as a danger signal and is classically thought to promote macrophage recruitment via Toll-like receptors (TLR) 2 and 4. We have recently shown that biglycan signaling through TLR 2/4 and the CD14 co-receptor regulates inflammation, suggesting that TLR co-receptors may determine whether biglycan-TLR signaling is pro- or anti-inflammatory. Here, we sought to identify other co-receptors and characterize their impact on biglycan-TLR signaling. We found a marked increase in the number of autophagic macrophages in mice stably overexpressing soluble biglycan. In vitro, stimulation of murine macrophages with biglycan triggered autophagosome formation and enhanced the flux of autophagy markers. Soluble biglycan also promoted autophagy in human peripheral blood macrophages. Using macrophages from mice lacking TLR2 and/or TLR4, CD14, or CD44, we demonstrated that the pro-autophagy signal required TLR4 interaction with CD44, a receptor involved in adhesion, migration, lymphocyte activation, and angiogenesis. In vivo, transient overexpression of circulating biglycan at the onset of renal ischemia/reperfusion injury (IRI) enhanced M1 macrophage recruitment into the kidneys of Cd44+/+ and Cd44-/- mice but not Cd14-/- mice. The biglycan-CD44 interaction increased M1 autophagy and the number of renal M2 macrophages and reduced tubular damage following IRI. Thus, CD44 is a novel signaling co-receptor for biglycan, an interaction that is required for TLR4-CD44-dependent pro-autophagic activity in macrophages. Interfering with the interaction between biglycan and specific TLR co-receptors could represent a promising therapeutic intervention to curtail kidney inflammation and damage.

The αvβ3 integrin has been shown to promote aggressive phenotypes in many types of cancers, including prostate cancer. We show that GFP-labeled αvβ3 derived from cancer cells circulates in the blood and is detected in distant lesions in NOD scid gamma (NSG) mice. We, therefore, hypothesized that αvβ3 travels through exosomes and tested its levels in pools of vesicles, which we designate extracellular vesicles highly enriched in exosomes (ExVs), and in exosomes isolated from the plasma of prostate cancer patients. Here, we show that the αvβ3 integrin is found in patient blood exosomes purified by sucrose or iodixanol density gradients. In addition, we provide evidence that the αvβ3 integrin is transferred through ExVs isolated from prostate cancer patient plasma to β3-negative recipient cells. We also demonstrate the intracellular localization of β3-GFP transferred via cancer cell-derived ExVs. We show that the ExVs present in plasma from prostate cancer patients contain higher levels of αvβ3 and CD9 as compared to plasma ExVs from age-matched subjects who are not affected by cancer. Furthermore, using PSMA antibody-bead mediated immunocapture, we show that the αvβ3 integrin is expressed in a subset of exosomes characterized by PSMA, CD9, CD63, and an epithelial-specific marker, Trop-2. Finally, we present evidence that the levels of αvβ3, CD63, and CD9 remain unaltered in ExVs isolated from the blood of prostate cancer patients treated with enzalutamide. Our results suggest that detecting exosomal αvβ3 integrin in prostate cancer patients could be a clinically useful and non-invasive biomarker to follow prostate cancer progression. Moreover, the ability of αvβ3 integrin to be transferred from ExVs to recipient cells provides a strong rationale for further investigating the role of αvβ3 integrin in the pathogenesis of prostate cancer and as a potential therapeutic target.

The tumor microenvironment is becoming a crucial factor in determining the aggressiveness of neoplastic cells. The glycosaminoglycan hyaluronan is one of the principal constituents of both the tumor stroma and the cancer cell surfaces, and its accumulation can dramatically influence patient survival. Hyaluronan functions are dictated by its ability to interact with several signaling receptors that often activate pro-angiogenic and pro-tumorigenic intracellular pathways. Although hyaluronan is a linear, non-sulfated polysaccharide, and thus lacks the ability of the other sulfated glycosaminoglycans to bind and modulate growth factors, it compensates for this by the ability to form hyaluronan fragments characterized by a remarkable variability in length. Here, we will focus on the role of both high and low molecular weight hyaluronan in controlling the hallmarks of cancer cells, including cell proliferation, migration, metabolism, inflammation, and angiogenesis. We will critically assess the multilayered regulation of HAS2, the most critical hyaluronan synthase, and its role in cancer growth, metabolism, and therapy.

Joint biomechanical functions rely on the integrity of cartilage extracellular matrix. Understanding the molecular activities that govern cartilage matrix assembly is critical for developing effective cartilage regeneration strategies. This study elucidated the role of decorin, a small leucine-rich proteoglycan, in the structure and biomechanical functions of cartilage. In decorin-null cartilage, we discovered a substantial reduction of aggrecan content, the major proteoglycan of cartilage matrix, and mild changes in collagen fibril nanostructure. This loss of aggrecan resulted in significantly impaired biomechanical properties of cartilage, including decreased modulus, elevated hydraulic permeability, and reduced energy dissipation capabilities. At the cellular level, we found that decorin functions to increase the retention of aggrecan in the neo-matrix of chondrocytes, rather than to directly influence the biosynthesis of aggrecan. At the molecular level, we demonstrated that decorin significantly increases the adhesion between aggrecan and aggrecan molecules and between aggrecan molecules and collagen II fibrils. We hypothesize that decorin plays a crucial structural role in mediating the matrix integrity and biomechanical functions of cartilage by providing physical linkages to increase the adhesion and assembly of aggrecan molecules at the nanoscale.

Autophagy, a fundamental and evolutionarily-conserved eukaryotic pathway, coordinates a complex balancing act for achieving both nutrient and energetic requirements for proper cellular function and homeostasis. We have discovered that soluble proteoglycans evoke autophagy in endothelial cells and mitophagy in breast carcinoma cells by directly interacting with receptor tyrosine kinases, including VEGF receptor 2 and Met. Under these circumstances, autophagic regulation is considered “non-canonical” and is epitomized by the bioactivity of the small leucine-rich proteoglycan, decorin. Soluble matrix-derived cues being transduced downstream of receptor engagement converge upon a newly-discovered nexus of autophagic machinery consisting of Peg3 for endothelial cell autophagy and mitostatin for tumor cell mitophagy. In this thematic mini-review, we will provide an overview of decorin-mediated autophagy and mitophagy and propose that regulating intracellular catabolism is the underlying molecular basis for the versatility of decorin as a potent oncosuppressive agent.

The malignant phenotype of various cancers is linked to enhanced expression of hyaluronan, a pro-angiogenic glycosaminoglycan whose expression is suppressed by 4-methylumbelliferone (4-MU), a non-toxic oral agent used as a dietary supplement to improve health and combat prostate cancer. In this study, we investigated the role of 4-MU in mammary carcinoma cells with distinct malignant phenotypes and estrogen receptor (ER) status, a major prognostic factor in the clinical management of breast cancers. We focused on two breast cancer cell lines, the low metastatic and ERα+ MCF-7 cells, and the highly-aggressive and ERα− MDA-MB-231 cells. Treatment with 4-MU caused a dose-dependent decrease of hyaluronan accumulation in the extracellular matrix as well as within the breast cancer cells, most prevalent in cells lacking ERα. This decrease in hyaluronan was accompanied by suppression of Hyaluronan Synthase 2 (HAS2), the major enzyme responsible for the synthesis of hyaluronan, and by induction of hyaluronidases (HYALs) -1 and -2. Moreover, 4-MU induced intense phenotypic changes and substantial loss of CD44, a major hyaluronan receptor, from cell protrusions. Importantly, 4-MU evoked differential effects depending on the absence or presence of ERα. Only the ERα+ cells showed signs of apoptosis, as determined by cleaved PARP-1, and anoikis as shown by concurrent loss of E-cadherin and β-catenin. Interestingly, 4-MU significantly reduced migration, adhesion and invasion of ERα− breast cancer cells, and concurrently reduced the expression and activity of several matrix degrading enzymes and pro-inflammatory molecules with tumor-promoting functions. Collectively, our findings suggest that 4-MU could represent a novel therapeutic for specific breast cancer subtypes with regard to their ER status via suppression of hyaluronan synthesis and regulation of HAS2, CD44, matrix-degrading enzymes and inflammatory mediators.

The tumor microenvironment encompasses a complex cellular network that includes cancer-associated fibroblasts, inflammatory cells, neo-vessels, and an extracellular matrix enriched in angiogenic growth factors. Decorin is one of the main components of the tumor stroma, but it is not expressed by cancer cells. Lack of this proteoglycan correlates with down-regulation of E-cadherin and induction of β-catenin signaling. In this study, we investigated the role of a decorin-deficient tumor microenvironment in colon carcinoma progression and metastasis. We utilized an established model of colitis-associated cancer by administering Azoxymethane/Dextran sodium sulfate to adult wild-type and Dcn-/- mice. We discovered that after 12 weeks, all the animals developed intestinal tumors independently of their genotype. However, the number of intestinal neoplasms was significantly higher in the Dcn-/- microenvironment vis-à-vis wild-type mice. Mechanistically, we found that under unchallenged basal conditions, the intestinal epithelium of the Dcn-/- mice showed a significant increase in the protein levels of epithelial-mesenchymal transition associated factors including Snail, Slug, Twist, and MMP2. In comparison, in the colitis-associated cancer evoked in the Dcn-/- mice, we found that intercellular adhesion molecule 1 (ICAM-1) was also significantly increased, in parallel with epithelial-mesenchymal transition signaling pathway-related factors. Furthermore, a combined Celecoxib/decorin treatment revealed a promising therapeutic efficacy in treating human colorectal cancer cells, in decorin-deficient animals. Collectively, our results shed light on colorectal cancer progression and provide a protein-based therapy, i.e., treatment using recombinant decorin, to target the tumor microenvironment.

Decorin, a small dermatan-sulfate proteoglycan, participates in extracellular matrix assembly and influences directly and indirectly cell behavior via interactions with signaling membrane receptors and transforming growth factor (TGF)-β. We have therefore compared the development of tubulointerstitial kidney fibrosis in wild-type (WT) and decorin−/− mice in the model of unilateral ureteral obstruction. Without obstruction, kidneys from decorin−/− mice did not differ in any aspect from their WT counterparts. However, already 12 hours after obstruction decorin−/− animals showed lower levels of p27KIP1 and soon thereafter a more pronounced up-regulation and activation of initiator and effector caspases followed by enhanced apoptosis of tubular epithelial cells. Later, a higher increase of TGF-β1 became apparent. After 7 days, there was an up to 15-fold transient up-regulation of the related proteoglycan biglycan, which was mainly caused by the appearance of biglycan-expressing mononuclear cells. Other small proteoglycans showed no similar response. Because of enhanced degradation of type I collagen, end-stage kidneys from decorin−/− animals were more atrophic than WT kidneys. These data suggest that decorin exerts beneficial effects on tubulointerstitial fibrosis, primarily by influencing the expression of a key cyclin-dependent kinase inhibitor and by limiting the degree of apoptosis, mononuclear cell infiltration, tubular atrophy, and expression of TGF-β1. Decorin, a small dermatan-sulfate proteoglycan, participates in extracellular matrix assembly and influences directly and indirectly cell behavior via interactions with signaling membrane receptors and transforming growth factor (TGF)-β. We have therefore compared the development of tubulointerstitial kidney fibrosis in wild-type (WT) and decorin−/− mice in the model of unilateral ureteral obstruction. Without obstruction, kidneys from decorin−/− mice did not differ in any aspect from their WT counterparts. However, already 12 hours after obstruction decorin−/− animals showed lower levels of p27KIP1 and soon thereafter a more pronounced up-regulation and activation of initiator and effector caspases followed by enhanced apoptosis of tubular epithelial cells. Later, a higher increase of TGF-β1 became apparent. After 7 days, there was an up to 15-fold transient up-regulation of the related proteoglycan biglycan, which was mainly caused by the appearance of biglycan-expressing mononuclear cells. Other small proteoglycans showed no similar response. Because of enhanced degradation of type I collagen, end-stage kidneys from decorin−/− animals were more atrophic than WT kidneys. These data suggest that decorin exerts beneficial effects on tubulointerstitial fibrosis, primarily by influencing the expression of a key cyclin-dependent kinase inhibitor and by limiting the degree of apoptosis, mononuclear cell infiltration, tubular atrophy, and expression of TGF-β1. Glomerulosclerosis and tubulointerstitial fibrosis are common pathological features of most end-stage kidneys irrespective of the underlying etiology.1Eddy AA Molecular insights into renal interstitial fibrosis.J Am Soc Nephrol. 1996; 7: 2495-2508Crossref PubMed Google Scholar, 2Norman JT Fine LG Progressive renal disease: fibroblasts, extracellular matrix, and integrins.Exp Nephrol. 1999; 7: 167-177Crossref PubMed Scopus (65) Google Scholar The importance of tubulointerstitial fibrosis is underscored by the striking correlation between the morphological severity of matrix deposition and the progression of renal insufficiency culminating in eventual organ failure.3Müller GA Zeisberg M Strutz F The importance of tubulointerstitial damage in progressive renal disease.Nephrol Dial Transplant Suppl. 2000; 6: S76-S77Crossref Scopus (51) Google Scholar Infiltration of blood mononuclear cells, proliferation of interstitial mesenchymal cells, and apoptosis of tubular epithelial cells are considered to be important processes influencing renal inflammation and fibrosis.1Eddy AA Molecular insights into renal interstitial fibrosis.J Am Soc Nephrol. 1996; 7: 2495-2508Crossref PubMed Google Scholar, 2Norman JT Fine LG Progressive renal disease: fibroblasts, extracellular matrix, and integrins.Exp Nephrol. 1999; 7: 167-177Crossref PubMed Scopus (65) Google Scholar, 3Müller GA Zeisberg M Strutz F The importance of tubulointerstitial damage in progressive renal disease.Nephrol Dial Transplant Suppl. 2000; 6: S76-S77Crossref Scopus (51) Google Scholar Among the many profibrotic factors transforming growth factor (TGF)-β1 plays a pivotal role in the development of the disease.4Border WA Noble NA Targeting TGF-β for treatment of disease.Nat Med. 1995; 1: 1000-1001Crossref PubMed Scopus (73) Google Scholar In light of this complexity, the experimental model of unilateral ureteral obstruction (UUO) has been widely used to study the pathogenesis of tubulointerstitial fibrosis5Diamond JR Ricardo SR Klahr S Mechanisms of interstitial fibrosis in obstructive nephropathy.Semin Nephrol. 1998; 18: 594-602PubMed Google Scholar, 6Klahr S Morrissey JJ The role of growth factors, cytokines, and vasoactive compounds in obstructive nephropathy.Semin Nephrol. 1998; 18: 622-632PubMed Google Scholar because the evolution of fibrosis after ureteral obstruction is highly reproducible and reflects the sequence of pathogenetic events in an accelerated manner. As in human interstitial fibrosis there is rapid infiltration by mononuclear cells that finally leads to hypocellular fibrotic scarring and tubular atrophy without primary glomerular damage.7Diamond JR Macrophages and progressive renal disease in experimental hydronephrosis.Am J Kidney Dis. 1995; 26: 133-140Abstract Full Text PDF PubMed Scopus (87) Google Scholar Although important insights into the mechanisms of interstitial fibrosis have been obtained in these investigations, long-term studies are scarce, and some aspects of tissue remodeling have not yet been addressed at all. In this investigation we were interested in the sequence of pathogenetic events and in the mechanisms leading to fibrosis in the absence of the small leucine-rich proteoglycan (SLRP)8Iozzo RV Matrix proteoglycans: from molecular design to cellular function.Annu Rev Biochem. 1998; 67: 609-652Crossref PubMed Scopus (1359) Google Scholar, 9Iozzo RV The biology of the small leucine-rich proteoglycans. Functional network of interactive proteins.J Biol Chem. 1999; 274: 18843-18846Crossref PubMed Scopus (581) Google Scholar decorin in obstructive nephropathy for the following reasons. First, decorin, and other SLRPs (eg, biglycan and fibromodulin) have been shown to form complexes with TGF-β10Hildebrand A Romaris M Rasmussen LM Heinegård D Twardzik DR Border WA Ruoslahti E Interaction of the small interstitial proteoglycans biglycan, decorin and fibromodulin with transforming growth factor-β.Biochem J. 1994; 302: 527-534Crossref PubMed Scopus (879) Google Scholar leading to inhibition and/or sequestration of the cytokine within the extracellular matrix.11Border WA Noble NA Yamamoto T Harper JR Yamaguchi Y Pierschbacher MD Ruoslahti E Natural inhibitor of transforming growth factor-β protects against scarring in experimental kidney disease.Nature. 1992; 360: 361-364Crossref PubMed Scopus (946) Google Scholar, 12Markmann A Hausser H Schönherr E Kresse H Influence of decorin on transforming growth factor-β-mediated collagen retraction and biglycan induction.Matrix Biol. 2000; 19: 631-636Crossref PubMed Scopus (73) Google Scholar, 13Schaefer L Raslik I Gröne HJ Schönherr E Macakova K Ugorcakova J Budny S Schaefer RM Kresse H Small proteoglycans in human diabetic nephropathy: discrepancy between glomerular expression and protein accumulation of decorin, biglycan, lumican and fibromodulin.FASEB J. 2001; 15: 559-561Crossref PubMed Scopus (119) Google Scholar, 14Massague J Chen YG Controlling TGF-beta signaling.Genes Dev. 2000; 14: 627-644Crossref PubMed Google Scholar Second, decorin, biglycan, and fibromodulin as well as a fourth member of this family, lumican, interact with fibrillar collagens,15Schönherr E Witsch-Prehm P Harrach B Robenek H Rauterberg J Kresse H Interaction of biglycan with type I collagen.J Biol Chem. 1995; 270: 2776-2783Crossref PubMed Scopus (330) Google Scholar, 16Scott JE Haigh M Identification of specific binding sites for keratan sulphate proteoglycans and chondroitin-dermatan sulphate proteoglycans on collagen fibrils in cornea by the use of cupromeronic blue in ‘critical-electrolyte-concentration’ techniques.Biochem J. 1998; 253: 607-610Google Scholar, 17Hedbom E Heinegård D Binding of fibromodulin and decorin to separate sites on fibrillar collagens.J Biol Chem. 1993; 268: 27307-27312Abstract Full Text PDF PubMed Google Scholar thereby modulating fibril formation and stability. Ablation of each of the four SLRP genes results in grossly disturbed type I collagen-containing fibrils18Danielson KG Baribault H Holmes DF Graham H Kadler KE Iozzo RV Targeted disruption of decorin leads to abnormal collagen fibril morphology and skin fragility.J Cell Biol. 1997; 136: 729-743Crossref PubMed Scopus (1199) Google Scholar, 19Chakravarti S Magnuson T Lass JH Jepsen KJ LaMantia C Carroll H Lumican regulates collagen fibril assembly: skin fragility and corneal opacity in the absence of lumican.J Cell Biol. 1998; 141: 1277-1286Crossref PubMed Scopus (598) Google Scholar, 20Svensson L Aszodi A Reinholt FP Fässler R Heinegård D Oldberg Å Fibromodulin-null mice have abnormal collagen fibrils, tissue organization, and altered lumican deposition in tendon.J Biol Chem. 1999; 274: 9636-9647Crossref PubMed Scopus (386) Google Scholar, 21Corsi A Bianco P Fisher LW Chen X-D Xu T Iozzo RV Danielson KG Gehron Robey P Young MF Biglycan and decorin deficiency interferes with collagen assembly and structure and results in diverse Ehlers-Danlos-like phenotypes.J Bone Miner Res. 1999; 14: 134AGoogle Scholar indicating the need for an orchestrated action of all four proteoglycans during fibril assembly. The presence of all four SLRPs in the kidney has been reported previously.22Schaefer L Gröne H-J Raslik I Robenek H Ugorcakova J Budny S Schaefer RM Kresse H Small proteoglycans of normal adult human kidney: distinct expression pattern of decorin, biglycan, fibromodulin and lumican.Kidney Int. 2000; 58: 1557-1568Crossref PubMed Scopus (87) Google Scholar Third, biglycan has been shown to stimulate growth and differentiation of monocytic lineage cells from various lymphatic organs23Kamo I Kikuchi A Nonaka I Yamada E Kondo J Haemopoietic activity associated with biglycan like proteoglycan.Biochem Biophys Res Commun. 1993; 195: 1119-1126Crossref PubMed Scopus (10) Google Scholar, 24Kikuchi A Tomoyasu H Kido I Takahashi K Tanaka A Nonaka I Iwakami N Kamo I Haemopoietic biglycan produced by brain cells stimulates growth of microglial cells.J Neuroimmunol. 2000; 106: 78-86Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar and may, hence, support acute inflammation in obstructive nephropathy. Fourth, decorin expression enables endothelial cells to escape apoptosis.25Schönherr E O'Connell BC Schittny J Robenek H Fastermann D Fisher LW Plenz G Vischer P Young MF Kresse H Paracrine or virus-mediated induction of decorin expression by endothelial cells contributes to tube formation and prevention of apoptosis in collagen lattices.Eur J Cell Biol. 1999; 78: 44-45Crossref PubMed Scopus (76) Google Scholar, 26Schönherr E Schaefer L O'Connell BC Kresse H Matrix metalloproteinase expression by endothelial cells in collagen lattices changes during co-culture with fibroblasts and upon induction of decorin expression.J Cell Physiol. 2001; 187: 37-47Crossref PubMed Scopus (34) Google Scholar Furthermore, decorin expression is followed by an up-regulation of cyclin-dependent kinase inhibitors,27DeLuca A Santra M Baldi A Giordano A Iozzo RV Decorin-induced growth suppression is associated with up-regulation of p21Cip1/Waf1, an inhibitor of cyclin-dependent kinases.J Biol Chem. 1996; 271: 18961-18965Crossref PubMed Scopus (226) Google Scholar probably via interaction with an epidermal growth factor-receptor family member,28Moscatello DK Santra M Mann DM McQuillan DJ Wong AJ Iozzo RV Decorin suppresses tumor cell growth by activating the epidermal growth factor receptor.J Clin Invest. 1998; 101: 406-412Crossref PubMed Scopus (246) Google Scholar and is therefore also involved in cell cycle control. Here we show that the course of obstructive nephropathy becomes greatly altered in mice with an inactivated decorin gene because of a decrease in p27KIP1 expression, caspase induction, and tissue remodeling affecting primarily tubular epithelia. Differences between normal and decorin−/− mice in the extent of apoptosis, in TGF-β levels, and the number of biglycan-expressing macrophages appear as major determinants for this type of end-stage kidney disease. All animal work was done in accordance with the German Animal Protection Law. Decorin-deficient mice were generated as described previously.18Danielson KG Baribault H Holmes DF Graham H Kadler KE Iozzo RV Targeted disruption of decorin leads to abnormal collagen fibril morphology and skin fragility.J Cell Biol. 1997; 136: 729-743Crossref PubMed Scopus (1199) Google Scholar The kidneys studied were from wild-type (WT) (n = 43), decorin−/− (Dcn−/−) (n = 78), and decorin+/− (Dcn+/−) (n = 4) mice of the same inbred strain background.18Danielson KG Baribault H Holmes DF Graham H Kadler KE Iozzo RV Targeted disruption of decorin leads to abnormal collagen fibril morphology and skin fragility.J Cell Biol. 1997; 136: 729-743Crossref PubMed Scopus (1199) Google Scholar Obstruction of the left ureter was performed in 2-month-old male mice. The contralateral and sham-operated kidney served as control. Kidneys (at least n = 4 per group) were analyzed up to 70 days after ligation. Total RNA was extracted from whole kidneys as described.29Schaefer L Hausser H Altenburger M Ugorcakova J August C Fisher LW Schaefer RM Kresse H Decorin, biglycan and their endocytosis receptor in rat renal cortex.Kidney Int. 1998; 54: 1529-1541Crossref PubMed Scopus (59) Google Scholar The cDNA probes for decorin and biglycan were those used previously in the rat.29Schaefer L Hausser H Altenburger M Ugorcakova J August C Fisher LW Schaefer RM Kresse H Decorin, biglycan and their endocytosis receptor in rat renal cortex.Kidney Int. 1998; 54: 1529-1541Crossref PubMed Scopus (59) Google Scholar cDNA probes for rat fibromodulin and lumican, which are also homologous to the respective murine sequences, were generated by reverse transcriptase-polymerase chain reaction (PCR) from total rat tail RNA with Super Script II reverse transcriptase (Life Technologies, Eggenstein, Germany) and random hexanucleotide cDNA primers. Double-stranded cDNAs were generated by using primers that contained additionally appropriate sequences with restriction sites for Sma I and Hin dIII, respectively. For fibromodulin the primer pair was 5′-TCCCCCGGGGATGCAGTGGGCCTCCATC-3′, and 5′-GCTCCAGATCTCGATGAGGCT-3′, thus yielding a rat fibromodulin cDNA encompassing bp 54 to 1184 (GenBank X82152). For lumican, the respective primer pair was 5′-TCCCCCGGGGATGAATGTATGTACGTTCAC-3′ and 5′-CCCAAGCTTTAATTAACCGTGATTTCA-3′ yielding lumican cDNA between bp 46 to 1062 (GenBank X84039). Endonuclease-treated PCR products were cloned into pGEM 3Z (Promega, Madison, WI) and verified by DNA sequencing. Mouse caspase-3 and caspase-8 were generated with the primer pairs 5′-TGTCATCTCGCTCTGGTACG-3′, 5′-AGCATGGACACAATACACGG-3′ (245 to 869 bp, GenBank NM009810) and 5′-CCGAGAGGAGATGGTGAGAG-3′, 5′-TCGGTAGGAAACGCAGTTCT-3′ (292 to 1291 bp, GenBank AJ007749), respectively, and cloned into pCRII (Invitrogen, Groningen, The Netherlands). cDNAs for α1(I) collagen, TGF-β1, and GAPDH were from ATCC (Rockville, MD). Northern blots were performed and analyzed as described previously.13Schaefer L Raslik I Gröne HJ Schönherr E Macakova K Ugorcakova J Budny S Schaefer RM Kresse H Small proteoglycans in human diabetic nephropathy: discrepancy between glomerular expression and protein accumulation of decorin, biglycan, lumican and fibromodulin.FASEB J. 2001; 15: 559-561Crossref PubMed Scopus (119) Google Scholar In situ hybridization of renal sections from WT and Dcn−/− mice was performed with the sense and antisense probes in parallel and under the same conditions.13Schaefer L Raslik I Gröne HJ Schönherr E Macakova K Ugorcakova J Budny S Schaefer RM Kresse H Small proteoglycans in human diabetic nephropathy: discrepancy between glomerular expression and protein accumulation of decorin, biglycan, lumican and fibromodulin.FASEB J. 2001; 15: 559-561Crossref PubMed Scopus (119) Google Scholar cDNA synthesis from total RNA extracted from whole kidneys and amplification were performed as described previously13Schaefer L Raslik I Gröne HJ Schönherr E Macakova K Ugorcakova J Budny S Schaefer RM Kresse H Small proteoglycans in human diabetic nephropathy: discrepancy between glomerular expression and protein accumulation of decorin, biglycan, lumican and fibromodulin.FASEB J. 2001; 15: 559-561Crossref PubMed Scopus (119) Google Scholar using the primers for the sequences of p21CIP1/WAF1: 5′-CTGCCCAAGCTCTACCTTCC-3′ and 5′-CAGGTCCACATGGTCTTCCT-3′ (123 bp); p27KIP1: 5′-CCGGCTAACTCTGAGGACAC-3′ and 5′-AGAAGAATCGTCGGTTGCAG-3′ (120 bp); GAPDH: 5′-GTCAGTGGTGGACCTGACCT-3′ and 5′-ACCTGGTGCTCAGTGTGCC-3′ (123 bp). Melting curves and agarose gel electrophoresis were used to verify the quality of the PCR products. The data obtained were referred to standard curves where plasmids with authentic cDNAs were analyzed analogously. Finally, the values were normalized to the results of GAPDH-reverse transcriptase-PCR. Statistical analysis was performed with Prism, version 3 (GraphPad Software, San Diego, CA). Serial sections (2 to 6 μm) of paraffin-embedded samples were stained with periodic acid-Schiff reaction (PAS) and processed for immunohistochemical studies or in situ hybridization. Morphometric evaluation of tubulointerstitial damage (defined as the occurrence of dilated or collapsed tubules with flattened and vacuolated epithelia), glomerular volumes, interstitial fibrosis, and infiltration of mononuclear cells was performed by a computer-aided image analysis system.30Scheuer H Gwinner W Hohbach J Gröne EF Brandes RP Malle E Olbricht CJ Walli AK Gröne HJ Oxidant stress in hyperlipidemia-induced renal damage.Am J Physiol. 2000; 278: F63-F74PubMed Google Scholar Paraffin sections were stained by immunoperoxidase or APAAP techniques.29Schaefer L Hausser H Altenburger M Ugorcakova J August C Fisher LW Schaefer RM Kresse H Decorin, biglycan and their endocytosis receptor in rat renal cortex.Kidney Int. 1998; 54: 1529-1541Crossref PubMed Scopus (59) Google Scholar Primary antibodies included LF-113 (a rabbit anti-murine decorin antiserum31Fisher LW Stubbs JT Young MF Antisera and cDNA probes to human and certain animal model bone matrix noncollagenous proteins.Acta Orthop Scand. 1995; 266: S61-S65Google Scholar kindly provided by Dr. L.W. Fisher, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, MD), a chicken antibody against the Ser67 to Gln78 peptide of rat biglycan,29Schaefer L Hausser H Altenburger M Ugorcakova J August C Fisher LW Schaefer RM Kresse H Decorin, biglycan and their endocytosis receptor in rat renal cortex.Kidney Int. 1998; 54: 1529-1541Crossref PubMed Scopus (59) Google Scholar rabbit anti-rat type I collagen (Biogenesis, Berlin, Germany) and the marker for murine monocytes/macrophages: rat anti-mouse F4/80 (Serotec, Eching, Germany). After blocking endogenous peroxidase and incubation with respective antibodies, sections were developed with the Diaminobenzidine Substrate Kit (Vector Laboratories, Burlingame, CA). Antibodies against proliferating cell nuclear antigen were visualized with the EPOS-horseradish peroxidase-coupled system (DAKO, Hamburg, Germany). Counterstaining was with methyl green. Mouse anti-fibronectin extradomain A (FN-EDA+), clone OST-9 (kindly provided by Dr. L. Zardi, Genova, Italy), was conjugated with digoxigenin. The active form of caspase-3, the proform of caspase-8, and its active p20 subunit were visualized with digoxigenin-conjugated affinity-purified antibodies (R&D Systems, Wiesbaden, and Santa Cruz, Heidelberg, Germany, respectively) in paraffin and frozen sections. Early apoptosis was evaluated in frozen sections by a digoxigenin-conjugated monoclonal antibody to single-stranded DNA (F7-26: Alexis, Grünberg, Germany). Antibody-treated tissue sections were incubated with alkaline phosphatase-conjugated Fab fragments of anti-digoxigenin antibodies.29Schaefer L Hausser H Altenburger M Ugorcakova J August C Fisher LW Schaefer RM Kresse H Decorin, biglycan and their endocytosis receptor in rat renal cortex.Kidney Int. 1998; 54: 1529-1541Crossref PubMed Scopus (59) Google Scholar The specificity of immunostaining was tested by omitting the primary antibody, by using nonimmune serum/unspecific IgG, and by preabsorption of antisera with antigens (decorin, biglycan). To evaluate individual kidneys, 20 randomly selected nonoverlapping fields of renal cortex were examined under higher magnification (×400) and the number of biglycan-positive cells, proliferating cell nuclear antigen-labeled nuclei, and apoptotic nuclei in tubular and interstitial cells were evaluated. Mean values of at least four kidneys per group were averaged. Morphometrical evaluation of FN-EDA+ was performed as described previously.32Kaneto H Morrissey J McCracken R Reyes A Klahr S Enalapril reduces collagen type IV synthesis and expansion of the interstitium in the obstructed rat kidney.Kidney Int. 1994; 45: 1637-1647Crossref PubMed Scopus (217) Google Scholar In brief, a grid containing 117 (13 × 9) sampling points was superimposed on images of cortical high-power fields (×400) and FN-EDA+ deposits were counted and expressed as percentage of all sampling points. All morphometric evaluations were performed by a blinded observer. For the characterization of biglycan-positive cells double-labeling of 6-μm frozen sections was performed with antibodies against biglycan (1:500) and F4/80 (1:10) for identification of macrophages. Unspecific staining was blocked with phosphate-buffered saline/1% bovine serum albumin/20% goat serum. For visualization 1:200 dilutions of Texas Red-conjugated donkey anti-chicken IgG and fluorescein-conjugated goat anti-rat IgG (Dianova, Hamburg, Germany) were used. Laser scan microscopy was performed with a Nikon confocal microscope PCM 2000 (Nikon, Duesseldorf, Germany). Nonspecific staining was determined by the use of secondary antibodies alone. Caspase-3 activity was determined in homogenates of sham-operated, obstructed, and contralateral kidneys by the Caspase-3/CPP32 Colorimetric Assay Kit (BioCat, Heidelberg, Germany). In this assay, active caspase-3 cleaves the chromophore p-nitroanilide (p NA) from the labeled substrate DEVD-p NA. Enzyme activity was measured as described previously.33Truong LD Choi YJ Tsao CC Ayala G Sheikh-Hamad D Nassar G Suki WN Renal cell apoptosis in chronic obstructive uropathy: the roles of caspases.Kidney Int. 2001; 60: 924-934Crossref PubMed Scopus (78) Google Scholar Briefly, whole kidneys were homogenized in 2 ml of cell lysis buffer at 4°C for 15 minutes. After centrifugation at 10,000 × g, proteins were measured in the supernatants (cytosolic extractions) and adjusted to a concentration of 200 μg/50 μl. After incubation with substrate for 2 hours at 37°C, the optical density was determined using a microplate reader at 405 nm. Data were calculated as the average of duplicates for each sample and per whole kidney. Mean values of three kidneys per group were averaged. Thin section electron microscopy was performed as described.15Schönherr E Witsch-Prehm P Harrach B Robenek H Rauterberg J Kresse H Interaction of biglycan with type I collagen.J Biol Chem. 1995; 270: 2776-2783Crossref PubMed Scopus (330) Google Scholar Serum urea and creatinine levels were measured using a Hitachi 747 autoanalyzer and urinary and tissue protein by the BCA Protein Assay Reagent (Pierce, Rockford, IL). Western blots were performed and quantified as described previously13Schaefer L Raslik I Gröne HJ Schönherr E Macakova K Ugorcakova J Budny S Schaefer RM Kresse H Small proteoglycans in human diabetic nephropathy: discrepancy between glomerular expression and protein accumulation of decorin, biglycan, lumican and fibromodulin.FASEB J. 2001; 15: 559-561Crossref PubMed Scopus (119) Google Scholar using rabbit anti-p21CIP1/WAF1, anti-p27KIP1, and anti-β-tubulin as primary antibodies (all from Santa Cruz). Type I collagen was determined after exhaustive pepsin digestion of whole minced kidneys followed by 4 to 12.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing and nonreducing conditions and quantification (IQ Solutions Image Quant Software: Molecular Dynamics, Uppsala, Sweden) of Coomassie Blue-stained bands of α1(I)- and α2(I)-chains of collagen I.34Bruckner P Hörler I Mendler M Houze Y Winterhalter KH Eich-Bender SG Spycher MA Induction and prevention of chondrocyte hypertrophy in culture.J Cell Biol. 1989; 109: 2537-2545Crossref PubMed Scopus (163) Google Scholar A high- and low-molecular weight mixture of proteins (Life Technologies) and pepsin-digested purified type I, III, and IV collagens were used as standards. For Western blotting and type I collagen quantification the results from three kidneys per group were averaged. Results are expressed as means ± SEM. Statistical analysis was performed by the unpaired Student's t-test. Significance was accepted at the 5% level. UUO was performed in WT and Dcn−/− animals, and the development of hydronephrosis was followed throughout a period of 70 days (Figure 1A). No macroscopical differences in the development of hydronephrosis between WT and Dcn−/− mice were evident for up to 35 days after UUO. However, thereafter (42, 53, and 70 days) obstructed kidneys from Dcn−/− mice became progressively smaller in size and weight. After aspiration of fluid from the dilated pyelon the weight of Dcn−/− kidneys was 123 ± 11 mg versus 201 ± 17 mg in WT animals at day 53 (n = 4, P < 0.05) and 87 ± 9 mg versus 152 ± 14 mg at day 70 (n = 6, P < 0.05). These differences have to be interpreted in light of the finding that weights of normal kidneys were comparable in age- and sex-matched WT and Dcn−/− mice. No differences were observed in body weight or in the weight of contralateral kidneys between both groups at any time point. PAS-stained sections of nonligated kidneys from 43 WT and 78 Dcn−/− mice were similar in terms of renal morphology and matrix deposition. There were no genotype-specific differences in survival, serum urea and creatinine levels, or urinary protein excretion both in unaffected mice as well as in animals after UUO throughout the whole experimental period. Already at day 7 quantitative histomorphometry of PAS-stained sections (Figure 1, B and C) from obstructed kidneys revealed a significantly higher percentage (P < 0.05) of damaged tubules in Dcn−/− (58 ± 10%, n = 6) than in WT mice (20 ± 6%, n = 4). During the subsequent course of the disorder this difference increased further, and at day 53 tubules with segment-specific differentiation were no longer recognizable in decorin-deficient mice whereas in obstructed kidneys from WT animals such tubules could still be occasionally detected (Figure 1, D and E). Another striking difference in end-stage fibrosis concerned the extent of infiltration by mononuclear cells. At day 53 an area covering 34 ± 3% of the tissue sections was occupied by infiltrating mononuclear cells, frequently in the form of lymphoid follicles, in Dcn−/− animals (Figure 1, D and E) whereas the corresponding value in WT animals was 2.3 ± 0.4% (n = 4 for each group, P < 0.05). Similar data were obtained from kidneys on day 70. The majority of these cells were positive for monocyte/macrophage markers. At days 53 and 70, the volumes of glomeruli in obstructed kidneys from WT were 1.8-fold to 2.9-fold higher as compared to Dcn−/− animals. Early tubular damage and enhanced infiltration of macrophages were the two most prominent histomorphological differences observed in obstructed kidneys from Dcn−/− as compared to WT mice. In contrast, semiquantitative evaluation of interstitial fibrosis showed no difference in the extent of fibrosis in obstructed kidneys from WT and Dcn−/− animals. In addition, there were no differences in PAS-stained sections from contralateral and sham-operated kidneys in either genotype. The earliest detectable difference between WT and Dcn−/− kidneys concerned the expression of p27KIP1. Overexpression of p27KIP1 in obstructed kidneys from WT both on the mRNA (Figure 2A) and protein level (Figure 2, C and D) was detected at 12 hours, whereas in Dcn−/− kidneys p27KIP1 started to increase at 36 hours after UUO. Lower levels of p27KIP1 were detected in obstructed Dcn−/− than in WT kidneys until day 4. In later stages of UUO levels of p27KIP1 did not differ between WT and Dcn−/− kidneys. In agreement with earlier reports,6Klahr S Morrissey JJ The role of growth factors, cytokines, and vasoactive compounds in obstructive nephropathy.Semin Nephrol. 1998; 18: 622-632PubMed Google Scholar p21CIP1/WAF1 mRNA (Figure 2B) was up-regulated in obstructed kidneys starting at 36 hours. However, no genotype-specific differences were observed (Figure 2; B, C, and E) at any experimental time point (12 hours to 70 days). Because a lack of p27KIP1 has been shown to enhance tubular epithelial cell apoptosis in UUO35Ophascharoensuk V Fero ML Hughes J Roberts JM Shankland SJ The cyclin-dependent kinase inhibitor p27Kip1 safeguards against inflammatory

Versican is a modular proteoglycan involved in the control of cellular growth and differentiation.To understand versican gene regulation and transcriptional control, we have isolated genomic clones spanning the entire gene locus including 5'-and 3"flanking sequences.Versican was encoded by 15 exons encompassing over 90 kilobase pairs of continuous DNA.The exon organization corresponded to the protein subdomains encoded by homologous proteins, with a remarkable conservation of exon size and intron phase.W e discovered an additional exon just proximal to the glycosaminoglycanbinding region that was identical to a recently identified splice variant of versican (Dours-Zimmermann, M. T., and Zimmermann, D. R. (1994) J. BioZ.Chern.269,32992-32998).The versican promoter harbored a typical TATA box located approximately 16 base pairs upstream of the transcription start site and binding sites for a number of transcription factors involved in regulated gene expression.This promoter was shown to be highly functional in transiently transfected cells of both mesenchymal and epithelial origin.Stepwise 5' deletions identified a strong enhancer element between -209 and -445 base pairs and a strong negative element between -445 and -632 base pairs.This study provides the molecular basis for discerning the transcriptional control of the versican gene and offers the opportunity to investigate genetic disorders linked to this important human gene.~ .

Without new blood vessels, neoplasms cannot expand beyond a few millimeters, the point at which the diffusion of nutrients and the disposal of waste products become rate-limiting. Regulation of angiogenesis thus must be controlled at multiple levels. For instance, the VEGF family of heparin-binding proteins and their primary receptors, VEGFR-1 and VEGFR-2 (KDR), products of the flt-1 and the flk-1 gene, respectively, are required for angioblast differentiation and vasculogenesis, and specific VEGF isoforms play distinct roles in promoting endothelial growth and migration during angiogenesis. In addition, angiogenesis is profoundly affected by several members of the FGF family and their four receptors, and indeed, supplementing the media of endothelial cell cultures with basic FGF (FGF2) and heparin is now well established as a means to obtain optimal growth, migration, and capillary morphogenesis. In addition to producing proangiogenic factors, tumor cells also directly or indirectly generate negative angiogenic stimuli. The ultimate growth rate of the tumors is thus a fine balance between positive and negative angiogenic cues.

Microbial adhesion to the host tissue represents an early, critical step in the pathogenesis of most infectious diseases. BORRELIA: burgdorferi, the causative agent of Lyme disease (LD), expresses two surface-exposed decorin-binding adhesins, DbpA and DbpB. A decorin-deficient (Dcn(-/-)) mouse was recently developed and found to have a relatively mild phenotype. We have now examined the process of experimental LD in Dcn(-/-) mice using both needle inoculation and tick transmission of spirochetes. When exposed to low doses of the infective agent, Dcn(-/-) mice had fewer Borrelia-positive cultures from most tissues analyzed than did Dcn(+/+) or Dcn(+/-) mice. When the infection dose was increased, similar differences were not observed in most tissues but were seen in bacterial colonization of joints and the extent of Borreila-induced arthritis. Quantitative PCR demonstrated that joints harvested from Dcn(-/-) mice had diminished Borrelia numbers compared with issues harvested from Dcn(+/+) controls. Histological examination also revealed a low incidence and severity of arthritis in Dcn(-/-) mice. Conversely, no differences in the numbers of Borreila-positive skin cultures were observed among the different genotypes regardless of the infection dose. These differences, which were observed regardless of genetic background of the mice (BALB/c or C3H/HeN) or method of infection, demonstrate the importance of decorin in the pathogenesis of LD.

Decorin, a leucine-rich proteoglycan with ubiquitous tissue distribution, may play essential biological roles during inflammation and cancer growth through its ability to bind extracellular matrix constituents and growth factors. In this study, we demonstrate that decorin gene expression is greatly enhanced after normal diploid fibroblasts reach confluency and cease to proliferate. Elevation of decorin mRNA steady state levels was maintained for up to 16 days posteonfluency. In vitro transcription analyses indicated enhanced transcriptional activity in quiescent fibroblasts when compared to cells harvested in their logarithmic phase of growth. This phenotypic trait was reversed by the exogenous addition of tumor necrosis factor-α (TNF-α). Furthermore, transforming growth faetor-β (TGF-β) down-regulated deeorin gene expression in an additive manner with TNF-α. Transient cell transfection assays using plasmid constructs harboring the decorin promoter linked to the chloramphenicol acetyltransferase reporter gene demonstrated a dose-dependent transcriptional repression by TNF-α. These findings were further corroborated by in vitro transcription experiments using nuclear extracts from control and TNF-α-treated quiescent fibroblasts. In contrast, the decorin promoter constructs failed to respond to TGF-β, thus suggesting either post-transcriptional regulation by this growth factor or lack of TGF-β-responsive elements. Further experiments with 5′ deletion constructs showed two TNF-α response elements, one residing within the 5-untranslated region (exon Ib), the other one between residues –188 and –140 of the decorin promoter. Collectively, our results indicate that TNF-α, through its ability to transcriptionally inhibit decorin gene expression in growth-arrested cells, may be a key modulator of the biological functions of this proteoglycan. Decorin, a leucine-rich proteoglycan with ubiquitous tissue distribution, may play essential biological roles during inflammation and cancer growth through its ability to bind extracellular matrix constituents and growth factors. In this study, we demonstrate that decorin gene expression is greatly enhanced after normal diploid fibroblasts reach confluency and cease to proliferate. Elevation of decorin mRNA steady state levels was maintained for up to 16 days posteonfluency. In vitro transcription analyses indicated enhanced transcriptional activity in quiescent fibroblasts when compared to cells harvested in their logarithmic phase of growth. This phenotypic trait was reversed by the exogenous addition of tumor necrosis factor-α (TNF-α). Furthermore, transforming growth faetor-β (TGF-β) down-regulated deeorin gene expression in an additive manner with TNF-α. Transient cell transfection assays using plasmid constructs harboring the decorin promoter linked to the chloramphenicol acetyltransferase reporter gene demonstrated a dose-dependent transcriptional repression by TNF-α. These findings were further corroborated by in vitro transcription experiments using nuclear extracts from control and TNF-α-treated quiescent fibroblasts. In contrast, the decorin promoter constructs failed to respond to TGF-β, thus suggesting either post-transcriptional regulation by this growth factor or lack of TGF-β-responsive elements. Further experiments with 5′ deletion constructs showed two TNF-α response elements, one residing within the 5-untranslated region (exon Ib), the other one between residues –188 and –140 of the decorin promoter. Collectively, our results indicate that TNF-α, through its ability to transcriptionally inhibit decorin gene expression in growth-arrested cells, may be a key modulator of the biological functions of this proteoglycan. Decorin, also known as DS-PGII or PG-40, is a leucine-rich proteoglycan with ubiquitous tissue distribution and may play important biological roles through its ability to bind other extracellular matrix proteins and certain growth factors (Gallagher, 1989Gallagher J.T. Curr. Opin. Cell Biol. 1989; 1: 1201-1218Crossref PubMed Scopus (211) Google Scholar; Kresse et al., 1993Kresse H. Hausser H. Schönherr E. Experientia. 1993; 49: 403-416Crossref PubMed Scopus (124) Google Scholar; Iozzo and Cohen, 1993Iozzo R.V. Cohen I. Experientia. 1993; 49: 447-455Crossref PubMed Scopus (104) Google Scholar). Specifically, decorin is involved in the regulation of fundamental biological functions, such as matrix assembly, cell attachment, migration, and proliferation. These functions are thought to be mediated by its ability to bind collagen types I, II (Vogel et al., 1984Vogel K.G. Paulsson M. Heinegârd D. Biochem. J. 1984; 223: 587-597Crossref PubMed Scopus (701) Google Scholar, and VI (Bidanset et al., 1992Bidanset D.J. LeBaron R. Rosenberg L. Murphy-Ullrich J.E. Hook M. J. Cell Biol. 1992; 118: 1523-1531Crossref PubMed Scopus (90) Google Scholar, fibronectin (Winnemöller et al., 1991Winnemöller M. Schmidt G. Kresse H. Eur. J. Cell Biol. 1991; 54: 10-17PubMed Google Scholar; Schmidt et al., 1991Schmidt G. Hausser H. Kresse H. Biochem. J. 1991; 280: 411-414Crossref PubMed Scopus (86) Google Scholar, and throm-bospondin (Winnemöller et al., 1992Winnemöller M. Schön P. Vischer P. Kresse H. Eur. J. Cell Biol. 1992; 59: 47-55PubMed Google Scholar. These binding properties of decorin, which are mediated by the protein core of the molecule, imply an important role for the protein component of decorin in the structural organization and assembly of the extracellular matrix (Gallagher, 1989Gallagher J.T. Curr. Opin. Cell Biol. 1989; 1: 1201-1218Crossref PubMed Scopus (211) Google Scholar; Kresse et al., 1993Kresse H. Hausser H. Schönherr E. Experientia. 1993; 49: 403-416Crossref PubMed Scopus (124) Google Scholar. Furthermore, decorin may be involved in the control of cell proliferation and matrix assembly through its ability to bind and neutralize transforming growth faetor-β (TGF-β) 1The abbreviations used are: TGF-β, transforming growth factor-β; TK, thymidine kinase; TNF-α, tumor necrosis factor-α; TNFRE, TNF response element; CAT, chloramphenicol acetyltransferase; IL, interleukin; DMEM, Dulbecco's modified Eagle's medium; FCS, fetal calf serum; kb, kilobase(s); bp, base pair(s).1The abbreviations used are: TGF-β, transforming growth factor-β; TK, thymidine kinase; TNF-α, tumor necrosis factor-α; TNFRE, TNF response element; CAT, chloramphenicol acetyltransferase; IL, interleukin; DMEM, Dulbecco's modified Eagle's medium; FCS, fetal calf serum; kb, kilobase(s); bp, base pair(s). (Yamaguchi et al., 1990Yamaguchi Y. Mann D.M. Ruoslahti E. Nature. 1990; 346: 281-284Crossref PubMed Scopus (1285) Google Scholar. This affinity of decorin for TGF-β could lead to the removal of this potent growth factor from the cellular microenvironment, thereby neutralizing its biological activity. This property has been demonstrated in an experimental animal model of glomerulonephritis in which infusion of recombinant decorin prevents the fibrosis of renal glomeruli induced by TGF-β (Border et al., 1992Border W.A. Noble N.A. Yamamoto T. Harper J.R. Yamaguchi Y. Pierschbacher M.D. Ruoslahti E. Nature. 1992; 360: 361-364Crossref PubMed Scopus (924) Google Scholar. Also, decorin may play a role in the development of cancer, as evidenced by the altered decorin gene expression in the stroma of human colon carcinomas (Iozzo, 1985Iozzo R.V. J. Biol. Chem. 1985; 260: 7464-7473Abstract Full Text PDF PubMed Google Scholar; Adany et al., 1990Adany R. Heimer R. Caterson B. Sorrell J.M. Iozzo R.V. J. Biol. Chem. 1990; 265: 11389-11396Abstract Full Text PDF PubMed Google Scholar, suggesting that decorin could mediate signaling between neoplastic cells and the adjacent stromal elements (Iozzo and Cohen, 1993Iozzo R.V. Cohen I. Experientia. 1993; 49: 447-455Crossref PubMed Scopus (104) Google Scholar).Our recent cloning of the decorin gene in both human (Danielson et al., 1993Danielson K.G. Fazzio A. Cohen I. Cannizzaro L.A. Eichstetter I. Iozzo R.V. Genomics. 1993; 15: 146-160Crossref PubMed Scopus (89) Google Scholar and murine (Scholzen et al., 1994Scholzen T. Solursh M. Susuki S. Reiter R. Morgan J.L. Buchberg A.M. Siracusa L.D. Iozzo R.V. J. Biol. Chem. 1994; 269: 28270-28281Abstract Full Text PDF PubMed Google Scholar and the development of various decorin promoter/CAT reporter gene constructs (Santra et al., 1994Santra M. Danielson K.G. Iozzo R.V. J. Biol. Chem. 1994; 269: 579-587Abstract Full Text PDF PubMed Google Scholar have allowed us to investigate the molecular mechanisms regulating decorin gene expression during growth and quiescence of normal diploid human skin cells. Our results indicate a novel transcriptional activation of the decorin gene associated with confluency-induced quiescence of human dermal fibroblasts or when HeLa cells are rendered quiescent by serum deprivation. We also provide evidence for a transcriptional repression of decorin gene expression by TNF-α, through down-regulation of the decorin promoter activity. These results raise the possibility of a delicately balanced regulation of the transcriptional activity of the decorin gene during important phases of the cell cycle.MATERIALS AND METHODSCell CulturesHuman dermal fibroblast cultures, established from tissue specimens obtained from adult individuals during surgical procedures or from neonatal foreskins, were utilized in passages 3–8. The cell cultures were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum (FCS), 2 mM glutamine, 100 units/ml penicillin, and 50 μg/ml streptomycin.Cytokines/Growth FactorsHuman recombinant TGF-β2 was a generous gift from Dr. David Olsen, Celtrix Laboratories, Santa Clara, CA. Human recombinant TNF-α was purchased from Boehringer Mannheim.Northern AnalysesTotal RNA was isolated using standard procedures (Chirgwin et al., 1979Chirgwin J.M. Przybyla A.E. MacDonald R.J. Rutter W.J. Biochemistry. 1979; 18: 5294-5299Crossref PubMed Scopus (16619) Google Scholar; Chomczynski and Sacchi, 1987Chomczynski P. Sacchi N. Anal. Biochem. 1987; 162: 156-159Crossref PubMed Scopus (62983) Google Scholar) and analyzed by Northern hybridization with 32P-labeled cDNA probes (Sambrook et al., 1989Sambrook J. Frisch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual.2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989Google Scholar. The [32PIcDNA-mRNA hybrids were visualized by autoradiography, and the steady state levels of mRNA were quantitated by scanning densitometry using a He-Ne laser scanner at 633 nm (LKB Produkter, Bromma, Sweden).cDNAs and Plasmid ConstructsThe following cDNAs were used for Northern hybridizations to detect specific mRNA transcripts: a 1.8-kb human decorin cDNA (kindly provided by Dr. Tom Krusius, University of Helsinki, Finland) (Krusius and Ruoslahti, 1986Krusius T. Ruoslahti E. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 7683-7687Crossref PubMed Scopus (412) Google Scholar) or a 411-bp polymerase chain reaction fragment developed in our laboratory and described previously (Santra et al., 1994Santra M. Danielson K.G. Iozzo R.V. J. Biol. Chem. 1994; 269: 579-587Abstract Full Text PDF PubMed Google Scholar; a l.-kb biglycan cDNA, kindly provided by Dr. Larry W. Fisher, NIH (Fisher et al., 1989Fisher L.W. Termine J.D. Young M.F. J. Biol. Chem. 1989; 264: 4571-4576Abstract Full Text PDF PubMed Google Scholar; and a 1.3-kb rat glyceraldehyde-3-phosphate dehydrogenase used as a control (Fort et al., 1985Fort P. Marty L. Piechaczyk M. El Sabrouty S. Dani C. Jeanteur P. Blanchard J.-M. Nucleic Acids Res. 1985; 13: 1431-1442Crossref PubMed Scopus (1970) Google Scholar.To study the transcriptional regulation of decorin gene expression, transient transfection experiments were performed with various 5′ deletion constructs derived from pUCDEC-10/CAT, a plasmid containing ∼1 kb of decorin promoter linked to the CAT reporter gene cloned into pUCCAT vector. Preparation of these deletion constructs is described in detail elsewhere (Santra et al., 1994Santra M. Danielson K.G. Iozzo R.V. J. Biol. Chem. 1994; 269: 579-587Abstract Full Text PDF PubMed Google Scholar. Also, a synthetic oligonucleotide spanning the region from –188 to –140 of the decorin promoter, which is fundamental for TNF-α response (see text) was cloned as a Hindlll/Sall fragment upstream of the thymidine kinase (TK) promoter linked to the CAT gene, generating the plasmid pDEC-188–140TK/CAT used in transfection experiments to investigate the function of the elements comprised within this region of the decorin promoter. The plasmid pTK/CAT was used as a control.Transient Transfection and CAT AssaysTransient transfections of human foreskin fibroblasts were performed by the calcium phosphate/DNA co-precipitation method, as described previously (Santra et al., 1994Santra M. Danielson K.G. Iozzo R.V. J. Biol. Chem. 1994; 269: 579-587Abstract Full Text PDF PubMed Google Scholar. Briefly, the cells were transfected with 10 or 20 μg of DNA mixed with 5 μg of the pRSV-β-galactosidase plasmid DNA in order to monitor transfection efficiencies. After glycerol shock, the cells were placed in DMEM containing 1% FCS, 4 h prior to the addition of growth factors and cytokines. In experiments without growth factors and cytokines, the cells were placed in DMEM containing 10% FCS. After an additional 40 h of incubation, the cells were rinsed twice with phosphate-buffered saline, harvested by scraping, and lysed by three cycles of freeze-thawing in 100 ml of 0.25 M Tris-HCl, pH 7.8. The β-galactosid-ase activities were measured according to standard protocols (Sambrook et al., 1989Sambrook J. Frisch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual.2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989Google Scholar. Aliquots corresponding to identical β-galacto-sidase activity were used for each CAT assay with [14C]chloramphenicol as substrate (Gorman et al., 1982Gorman C.M. Moffat L.F. Howard B.H. Mol. Cell. Biol. 1982; 2: 1044-1051Crossref PubMed Scopus (5288) Google Scholar using thin layer chromatography. Following autoradiography, the plates were cut and counted by liquid scintillation to quantify the acetylated [14C]chloramphenicol.In Vitro Transcription AssaysA polymerase chain reaction-generated 1008-bp fragment containing exon Ib, and ∼1 kb of the 5′-flanking sequence was subcloned into pBluescript-KS+ as a SmaI/SacI insert (Santra et al., 1994Santra M. Danielson K.G. Iozzo R.V. J. Biol. Chem. 1994; 269: 579-587Abstract Full Text PDF PubMed Google Scholar. Further deletions were prepared by restriction enzyme digestion. Detailed procedures for preparation and characteristics of the generated constructs are provided in the legend to Fig. 2 and in the text under “Results.” Plasmid DNA constructs were purified by affinity chromatography on DNA purification columns (QIAGEN Inc., Chatsworth, CA), according to the manufacturer's protocol and digested with EcoRI in order to linearize the plasmids at the 3′ end of exon lb. Fibroblast nuclear extracts were prepared according to previously described methods (Stein et al., 1989Stein B. Rahmsdorf H.J. Steffen A. Liftin M. Herrlich P. Mol. Cell. Biol. 1989; 9: 5169-5181Crossref PubMed Scopus (452) Google Scholar; Andrews and Faller, 1991Andrews N.C. Faller D.V. Nucleic Acids Res. 1991; 192499Crossref PubMed Scopus (2209) Google Scholar). In vitro transcription products were generated from this linearized DNA (1 μg) in reactions containing either fibroblast nuclear extracts or HeLa cell nuclear protein extracts (Promega, in vitro transcription grade), according to the method of the manufacturer, and analyzed on a denaturing 8% Polyacrylamide gel, as described previously (Santra et al., 1994Santra M. Danielson K.G. Iozzo R.V. J. Biol. Chem. 1994; 269: 579-587Abstract Full Text PDF PubMed Google Scholar.Gel Mobility Shift AssaysFor gel retardation assays, nuclear extracts were prepared according to either the method of Andrews and Faller, 1991Andrews N.C. Faller D.V. Nucleic Acids Res. 1991; 192499Crossref PubMed Scopus (2209) Google Scholar or to that of Stein et al., 1989Stein B. Rahmsdorf H.J. Steffen A. Liftin M. Herrlich P. Mol. Cell. Biol. 1989; 9: 5169-5181Crossref PubMed Scopus (452) Google Scholar. For DNA binding assays, two double-stranded oligomers containing the potential TNFREs identified in our study were generated: a 54-bp fragment containing the region –188 to –140 of the decorin promoter: 5′-AGCTTAGTGATGGT-CAATTGAGTCATTTGTGTGCAAAATATTGTGCAAGGCCCG-3′ and a 22-bp fragment corresponding to a sequence present in the exon lb of the decorin gene, sharing high homology with the osteocalcin gene TNFRE (underlined): 5′-TTGCCTGGATGAGCCAGGGGAC-31. The end-labeled oligomer (∼7 × 104 cpm) was incubated with 3–12 μg of protein extracts for 30 min on ice in 20 μl of binding reaction buffer (12 mM HEPES, pH 7.9, 4 mM Tris, pH 7.9, 60 mM KCl, 1 IHM EDTA, 12% glycerol), in the presence of 2–4 μg of poly(dI-dC), as described previously (Dignam et al., 1983Dignam J.D. Martin P.L. Shastry B.S. Roeder R.G. Methods Enzymol. 1983; 101: 582-598Crossref PubMed Scopus (744) Google Scholar. For competition experiments, a 20- to 500-fold molar excess of DNA was added to the binding reaction. Details of the competition assays are provided in the legends to the figures and the corresponding text under “Results.” DNA-protein complexes were separated from unbound oligomers on 4 or 5% acrylamide gels in 0.4 × TBE. The gels were fixed for 30 min in 30% methanol, 10% acetic acid, dried, and exposed to x-ray films at –70 °C.RESULTSGrowth Arrest Induces Decorin Gene ExpressionModulation of cellular growth is often associated with the expression of key regulatory genes. To investigate whether the growth phase of normal diploid cells had any effect on decorin gene expression, three experimental approaches were taken. First, RNA was isolated from fibroblast cultures at different time points before and after reaching confluency and hybridized under high stringency with a decorin cDNA probe. Significant expression of decorin was noted in exponentially growing fibroblasts (Fig. 1, lanes 1–3). However, decorin expression was dramatically enhanced (∼8-fold above level in proliferating fibroblast cultures) 8 days after fibroblast cultures reached confluency and had stopped proliferating (lane 4), and this enhancement (∼4fold) persisted for up to 16 days postconfluency (lane 5).FIG. 1Effect of growth state on decorin mRNA levels in human dermal fibroblasts in culture. Total RNA was extracted from fibroblast cultures growing in logarithmic phase for 1, 2, and 3 days (lanes 1–3, respectively) or 8 and 16 days after reaching confluency (lanes 4 and 5, respectively). RNA (10 μg/lane) was analyzed by Northern hybridization with a decorin cDNA under stringent conditions, allowing the detection of closely migrating transcripts of 1.6 and 1.9 kb, which encode human decorin (upperpanel). Ethidium bromide staining of ribosomal RNA (rRNA) prior to transfer in shown in the lower panel.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Secondly, to understand the mechanisms leading to increased decorin expression in growth-arrested cells and to determine whether the elevation of decorin mRNA levels observed in confluent fibroblast cultures was due to transcriptional activation of the decorin gene, nuclear proteins were extracted from both proliferating and 5-day postconfluent fibroblast cultures. The nuclear extracts were examined for their ability to drive the transcription of the decorin gene using various 5′ deletion constructs of the decorin promoter (Fig. 2A). Fig. 2B depicts an autoradiogram of a denaturing 89< Polyacrylamide gel electrophoresis of in vitro transcription products generated from a 983-bp decorin promoter construct with nuclear extracts from both proliferating and confluent fibroblasts. The transcription reactions generated an ∼ 100-bp RNA whose synthesis was elevated when transcription was driven by the nuclear extracts from confluent fibroblasts (Fig. 2B, lane 4 versus lane 3). Transcription products generated from quiescent HeLa cell nuclear extracts are shown in lane 2 (B).To identify the promoter region responsible for enhanced decorin expression in nonproliferating fibroblasts, we performed parallel in vitro transcription reactions using DNA constructs containing three 5′ deletions of the decorin promoter (Fig. 2C). The amounts of transcription products generated with nuclear extracts from either proliferating (C, lanes 1–3) or confluent (lanes 6–8) fibroblast cultures were not dependent on the deletion segments of the decorin gene 5′-flanking region. Increased transcription in nuclear extracts from confluent fibroblast cultures was specific for the decorin promoter because neither SV40 promoter-driven nor cytomegalovirus promoter-driven transcription of the decorin gene was affected by the growth state of fibroblasts (lanes 4, 5, 9, and 11, respectively). Taken together, these results indicate that up-regulation of decorin gene expression during quiescence is transcriptionally regulated and involves a region comprised between position –140 and the transcription start site.Thirdly, to further ascertain that the elevation of decorin mRNA levels in confluent fibroblasts was due to increased decorin promoter activity, we performed transient cell transfection experiments with pUCDEC-140/CAT, which contains 140 bp of decorin promoter linked to the CAT reporter gene, together with pRSV-j3-galactosidase construct, utilizing either proliferating or confluent fibroblast cultures. The CAT activity, reflecting decorin promoter activity, was significantly elevated in confluent cultures of fibroblasts (Fig. 3, lane 2) as compared to that in proliferating fibroblast cultures (Fig. 3, lane 1), after normalization of the CAT activity by β-galactosidase activity in the same cell extracts, so as to correct for differences in transfection efficiency. Interestingly, induction of decorin promoter activity was also observed in HeLa cells rendered quiescent by serum deprivation (Fig. 3, lane 4 versus lane 3). These findings indicate that elevated decorin gene expression correlates with growth arrest.FIG. 3Enhancement of decorin promoter activity upon cell growth arrest. Fibroblast cultures, either proliferating or confluent (lanes 1 and 2, respectively) and HeLa cell cultures, either exponentially growing in DMEM containing 10% fetal calf serum or rendered quiescent by serum-starvation (lanes 3 and 4, respectively) were co-transfected with a minimal decorin promoter (-140 relative to the transcriptional start site +D/CAT construct, together with pRSV-β-galactosidase by the calcium phosphate/DNA coprecipitation procedure, as described under “Materials and Methods.” After transfection, the cultures were either incubated for 72 h in DMEM containing 10% FCS in order to maintain them in their logarithmic phase of growth (lanes 1 and 3) or allowed to grow in DMEM containing 10% FCS for 24 h, replaced with serum-free medium for another 48 h in order to force them into a quiescent state. The cell extracts were assayed for CAT activity using 1HC [chloramphenicol as a substrate by thin layer chromatography after normalization for β-galactosidase activity. The autoradiogram of a representative experiment is shown. C, [14C]chloramphenicol; AC, acetylated [14C]chloramphenicol.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Collectively, these data indicate that the elevated decorin mRNA levels present in confluent fibroblast cultures result from transcriptional activation of the decorin gene. Furthermore, it appears that the proximal 140-bp segment of the decorin promoter is sufficient to allow transcriptional activation of the gene upon quiescence.Elevated Decorin Gene Expression in Confluent Fibroblast Cultures Is Reduced by TNF-αIn a set of experiments, we investigated whether the growth state of fibroblasts could influence the response of the decorin gene to cytokine modulation. For this purpose, fibroblast cultures, either proliferating (Fig. 4, lanes 1 and 2) or 3 days (lanes 3 and 4) or 6 days (lanes 5 and 6) following confluency, were cultured for 24 h in the absence (-) or in the presence (+) of TNF-α (10 ng/ml). Total RNA was extracted and subjected to Northern analysis. The results confirmed the previous finding of an up-regulation of decorin gene expression after fibroblasts reach confluency (lanes 3 and 5 versus lane 1). Interestingly, TNF-α was a potent inhibitor of decorin gene expression in confluent fibroblast cultures (lanes 4 and 6 versus lanes 3 and 5, respectively), whereas it had no effect on proliferating cells (lane 2 versus lane 1). This selective effect of TNF-α was specific for decorin, as demonstrated by unaltered glyceraldehyde-3-phosphate dehydrogenase mRNA levels in the same experiments. Quantitation of the autoradiograms by scanning densitometry and correction of the values against glyceraldehyde-3-phosphate dehydrogenase mRNA levels in the same RNA preparations revealed that TNF-α inhibited decorin gene expression in confluent fibroblast cultures by approximately 60%, at both 3 and 6 days after confluency, whereas it had no effect on the decorin transcript levels when cells were still proliferating.FIG. 4Effect of TNF-α on decorin mRNA levels in human dermal fibroblasts in culture. Fibroblast cultures, in logarithmic growth phase (lanes 1 and 2), 3 days (lanes 3 and 4), and 6 days (lanes 5 and 6) after reaching confluency, were incubated in DMEM containing 10%, fetal calf serum, without (–) or with (+) TNF-α (10 ng/ml) for 24 h. Total RNA was extracted and analyzed by Northern hybridization with a decorin cDNA. A glyceraldehyde-3-phosphate dehydrogenase IGAPDH) cDNA was used as a control probe to ensure the specificity of the TNF-α effect on decorin gene expression.View Large Image Figure ViewerDownload Hi-res image Download (PPT)To investigate whether the inhibitory effect of TNF-α on decorin mRNA levels occurred at the transcriptional level, we performed in vitro transcription assays utilizing nuclear extracts from either proliferating or confluent fibroblast cultures incubated with or without TNF-α (Fig. 5). In vitro transcription using the 662-bp decorin promoter template together with nuclear extracts from proliferating fibroblast cultures generated small amounts of transcripts (Fig. 5, lane 2). Consistent with the results observed at the mRNA level (see Fig. 4), TNF-α treatment did not alter the transcriptional activity of proliferating fibroblasts (Fig. 5, lane 3 versus lane 2). In contrast, as expected from the data of the experiments presented in Fig. 3, nuclear extracts from confluent fibroblast cultures exhibited elevated transcriptional activity as compared to that from proliferating cultures (lanes 4 and 6). This elevated activity was dramatically reduced when the cell cultures were treated for 24 h with TNF-α (lanes 5 and 7 versus lanes 4 and 6), indicating transcriptional repression of decorin gene expression.FIG. 5Effect of TNF-α on decorin promoter transcription, as measured by in vitro transcription assay. Experimental conditions were the same as those described in Fig. 4. The arrow indicates the exon lb transcripts generated by in vitro transcription from the –661-bp decorin promoter construct utilizing nuclear extracts from proliferating (lanes 2 and 3), 3 days (lanes 4 and 5), and 6 days (lanes 6 and 7) postconfluency fibroblast cultures, treated with (+) or without (-) TNF-α (10 ng/ml). Lane I shows radioactive DNA molecular mass markers whose sizes (bp) are indicated on the left of the panel.View Large Image Figure ViewerDownload Hi-res image Download (PPT)TGF-β and TNF-α Separately and Additively Inhibit Decorin Gene ExpressionTGF-β has been previously shown to inhibit fibroblast decorin gene expression (Kähäri et al., 1991Kähäri V.-M. Larjava H. Uitto J. J. Biol. Chem. 1991; 266: 10608-10615Abstract Full Text PDF PubMed Google Scholar; Vogel and Hernandez, 1992Vogel K.G. Hernandez D.J. Eur. J. Cell Biol. 1992; 59: 304-313PubMed Google Scholar) and to antagonize the effect of TNF-α on the expression on various extracellular matrix-related genes (Kähäri et al., 1990Kähäri V.-M. Chen Y.Q. Su M.W. Ramirez F. Uitto J. J. Clin. Invest. 1990; 86: 1489-1495Crossref PubMed Scopus (167) Google Scholar; Armendariz-Borunda et al., 1992Armendariz-Borunda J. Katayama K. Seyer J.M. J. Biol. Chem. 1992; 267: 14316-14321Abstract Full Text PDF PubMed Google Scholar; Mauviel and Uitto, 1993Mauviel A. Uitto J. Wounds. 1993; 5: 137-152Google Scholar; Mauviel et al., 1993aMauviel A. Chen Y.Q. Dong W. Evans C.H. Uitto J. Curr. Biol. 1993; 3: 822-831Abstract Full Text PDF PubMed Scopus (50) Google Scholar. We therefore examined whether TGF-β and TNF-α utilize the same mechanism(s) to inhibit decorin gene expression. To this end, confluent fibroblast cultures, which exhibited maximal levels of decorin mRNA transcripts, were incubated for 24 h with TNF-α (10 ng/ml) or TGF-β (5 ng/ml), either alone or in combination. Total RNA was then extracted and analyzed for decorin gene expression. The two cytokines, tested separately, inhibited decorin

There is mounting evidence that decorin inhibits the growth of various tumor cell lines when either over-expressed in vitro or provided as a recombinant protein. The mechanism of action is primarily via a protracted inactivation of the epidermal growth factor receptor (EGFR) tyrosine kinase. In this study we explored the possibility of retarding the growth of tumor xenografts by decorin gene delivery into the growing neoplastic tissues. We demonstrate that transient transgene expression of replication-deficient adenovirus-containing decorin causes a significant growth inhibition of colon and squamous carcinoma tumor xenografts. These cytostatic effects were achieved with relatively low viral titers and correlated with a reduced proliferative index and an attenuation of the EGFR phosphorylation in vivo. Thus, decorin gene therapy helps in retarding the growth of human tumors in immunocompromised animals and could represent a new independent or adjunctive therapeutic modality against cancer.

The small leucine-rich proteoglycan decorin has been demonstrated to be a key regulator of collagen fibrillogenesis; decorin deficiencies lead to irregularly shaped collagen fibrils and weakened material behavior in postnatal murine connective tissues. In an in vitro investigation of the contributions of decorin to tissue organization and material behavior, model tissues were engineered by seeding embryonic fibroblasts, harvested from 12.5-13.5 days gestational aged decorin null (Dcn(-/-)) or wild-type mice, within type I collagen gels. The resulting three-dimensional collagen matrices were cultured for 4 weeks under static tension. The collagen matrices seeded with Dcn(-/-) cells exhibited greater contraction, cell density, ultimate tensile strength, and elastic modulus than those seeded with wild-type cells. Ultrastructurally, the matrices seeded with Dcn(-/-) cells contained a greater density of collagen. The decorin-null tissues contained more biglycan than control tissues, suggesting that this related proteoglycan compensated for the absence of decorin. The effect of transforming growth factor-beta (TGF-beta), which is normally sequestered by decorin, was also investigated in this study. The addition of TGF-beta1 to the matrices seeded with wild-type cells improved their contraction and mechanical strength, whereas blocking TGF-beta1 in the Dcn(-/-) cell-seeded matrices significantly reduced the collagen gel contraction. These results indicate that the inhibitory interaction between decorin and TGF-beta1 significantly influenced the matrix organization and material behavior of these in vitro model tissues.

The Lyme disease spirochete, Borrelia burgdorferi, is an extracellular microbe that causes persistent infection despite the development of strong immune responses against the bacterium. B. burgdorferi expresses several ligand-binding lipoproteins, including the decorin-binding proteins (Dbps) A and B, which may mediate attachment to decorin, a major component of the host extracellular matrix during murine infection. We show that B. burgdorferi was better protected in the joints and skin, two tissues with a higher decorin expression, than in the urinary bladder and heart, two tissues with a lower decorin expression, during chronic infection of wild-type mice. Targeted disruption of decorin alone completely abolished the protective niche in chronically infected decorin-deficient mice but did not affect the spirochete burden during early infection. The nature of protection appeared to be specific because the spirochetes with higher outer surface protein C expression were not protected while the protective niche seemed to favor the spirochetes with a higher dbpA expression during chronic infection. These data suggest that spirochetal DbpA may interact with host decorin during infection and such interactions could be a mechanism that B. burgdorferi uses to evade humoral immunity and establish chronic infection. The Lyme disease spirochete, Borrelia burgdorferi, is an extracellular microbe that causes persistent infection despite the development of strong immune responses against the bacterium. B. burgdorferi expresses several ligand-binding lipoproteins, including the decorin-binding proteins (Dbps) A and B, which may mediate attachment to decorin, a major component of the host extracellular matrix during murine infection. We show that B. burgdorferi was better protected in the joints and skin, two tissues with a higher decorin expression, than in the urinary bladder and heart, two tissues with a lower decorin expression, during chronic infection of wild-type mice. Targeted disruption of decorin alone completely abolished the protective niche in chronically infected decorin-deficient mice but did not affect the spirochete burden during early infection. The nature of protection appeared to be specific because the spirochetes with higher outer surface protein C expression were not protected while the protective niche seemed to favor the spirochetes with a higher dbpA expression during chronic infection. These data suggest that spirochetal DbpA may interact with host decorin during infection and such interactions could be a mechanism that B. burgdorferi uses to evade humoral immunity and establish chronic infection. Borrelia burgdorferi, the Lyme disease spirochete, causes persistent mammalian infection despite the development of vigorous immune responses against the pathogen.1Steere AC Lyme disease.N Engl J Med. 2001; 345: 115-125Crossref PubMed Scopus (1059) Google Scholar, 2Seiler KP Weis JJ Immunity to Lyme disease: protection, pathology and persistence.Curr Opin Immunol. 1996; 8: 503-509Crossref PubMed Scopus (51) Google Scholar This bacterium expresses lipoproteins to form an antigenic layer that protects itself from, and directly interacts with, the environment.3Haake DA Spirochaetal lipoproteins and pathogenesis.Microbiology. 2000; 146: 1491-1504Crossref PubMed Scopus (150) Google Scholar Lipoproteins may stimulate innate responses via Toll-like receptors 1 and 2, enhancing adaptive immune responses to B. burgdorferi.4Wooten RM Ma Y Yoder RA Brown JP Weis JH Zachary JF Kirschning CJ Weis JJ Toll-like receptor 2 is required for innate, but not acquired, host defense to Borrelia burgdorferi.J Immunol. 2002; 168: 348-355PubMed Google Scholar, 5Alexopoulou L Thomas V Schnare M Lobet Y Anguita J Schoen RT Medzhitov R Fikrig E Flavell RA Hyporesponsiveness to vaccination with Borrelia burgdorferi OspA in humans and in TLR1- and TLR2-deficient mice.Nat Med. 2002; 8: 878-884Crossref PubMed Scopus (374) Google Scholar Antibodies to many lipoproteins such as outer surface proteins (Osp) A, B, and C and decorin-binding protein (Dbp) A can effectively protect the mammalian host from an initial B. burgdorferi infection,6Fikrig E Barthold SW Kantor FS Flavell RA Protection of mice against the Lyme disease agent by immunizing with recombinant OspA.Science. 1990; 250: 553-556Crossref PubMed Scopus (361) Google Scholar, 7Gilmore Jr, RD Kappel KJ Dolan MC Burkot TR Johnson BJ Outer surface protein C (OspC), but not P39, is a protective immunogen against a tick-transmitted Borrelia burgdorferi challenge: evidence for a conformational protective epitope in OspC.Infect Immun. 1996; 64: 2234-2239Crossref PubMed Google Scholar, 8Hagman KE Lahdenne P Popova TG Porcella SF Akins DR Radolf JD Norgard MV Decorin-binding protein of Borrelia burgdorferi is encoded within a two-gene operon and is protective in the murine model of Lyme borreliosis.Infect Immun. 1998; 66: 2674-2683Crossref PubMed Google Scholar, 9Feng S Hodzic E Stevenson B Barthold SW Humoral immunity to Borrelia burgdorferi N40 decorin binding proteins during infection of laboratory mice.Infect Immun. 1998; 66: 2827-2835Crossref PubMed Google Scholar but none of them are able to clear an established infection. Little is known about how this extracellular bacterium survives in the extremely hostile environment of chronic mammalian infection after specific immune responses have developed. A broad array of spirochetal surface lipoproteins have been shown in vitro to have interactions with mammalian host antigens. These include some of OspE-related lipoproteins (Erps) and BBA68 that acquire complement inhibitor factor H and/or factor H-like protein 1,10Hellwage J Meri T Heikkila T Alitalo A Panelius J Lahdenne P Seppala IJ Meri S The complement regulator factor H binds to the surface protein OspE of Borrelia burgdorferi.J Biol Chem. 2001; 276: 8427-8435Crossref PubMed Scopus (295) Google Scholar, 11Stevenson B El-Hage N Hines MA Miller JC Babb K Differential binding of host complement inhibitor factor H by Borrelia burgdorferi Erp surface proteins: a possible mechanism underlying the expansive host range of Lyme disease spirochetes.Infect Immun. 2002; 70: 491-497Crossref PubMed Scopus (185) Google Scholar, 12Kraiczy P Hellwage J Skerka C Becker H Kirschfink M Simon MM Brade V Zipfel PF Wallich R Complement resistance of Borrelia burgdorferi correlates with the expression of BbCRASP-1, a novel linear plasmid-encoded surface protein that interacts with human factor H and FHL-1 and is unrelated to Erp proteins.J Biol Chem. 2004; 279: 2421-2429Crossref PubMed Scopus (191) Google Scholar DbpA and DbpB,13Guo BP Brown EL Dorward DW Rosenberg LC Hook M Decorin-binding adhesins from Borrelia burgdorferi.Mol Microbiol. 1998; 30: 711-723Crossref PubMed Scopus (223) Google Scholar, 14Brown EL Guo BP O'Neal P Hook M Adherence of Borrelia burgdorferi. Identification of critical lysine residues in DbpA required for decorin binding.J Biol Chem. 1999; 274: 26272-26278Crossref PubMed Scopus (42) Google Scholar, 15Pikas DS Brown EL Gurusiddappa S Lee LY Xu Y Hook M Decorin-binding sites in the adhesin DbpA from Borrelia burgdorferi: a synthetic peptide approach.J Biol Chem. 2003; 278: 30920-30926Crossref PubMed Scopus (25) Google Scholar and the fibronectin-binding protein BBK32.16Fikrig E Barthold SW Sun W Feng W Telford III, SR Flavell RA Borrelia burgdorferi P35 and P37 proteins, expressed in vivo, elicit protective immunity.Immunity. 1997; 6: 531-539Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar, 17Probert WS Johnson BJ Identification of a 47 kDa fibronectin-binding protein expressed by Borrelia burgdorferi isolate B31.Mol Microbiol. 1998; 30: 1003-1015Crossref PubMed Scopus (227) Google Scholar Decorin and fibronectin are both major components of the host extracellular matrix (ECM).18Patti JM Allen BL McGavin MJ Hook M MSCRAMM-mediated adherence of microorganisms to host tissues.Annu Rev Microbiol. 1994; 48: 585-617Crossref PubMed Scopus (938) Google Scholar In addition, at least two integral outer membrane proteins P66 and Bgp (Borrelia glycosaminoglycan-binding protein) have been identified as spirochetal adhesins that mediate interactions of B. burgdorferi with the ECM and host cells.19Bunikis J Noppa L Bergstrom S Molecular analysis of a 66-kDa protein associated with the outer membrane of Lyme disease Borrelia.FEMS Microbiol Lett. 1995; 131: 139-145Crossref PubMed Google Scholar, 20Coburn J Chege W Magoun L Bodary SC Leong JM Characterization of a candidate Borrelia burgdorferi beta3-chain integrin ligand identified using a phage display library.Mol Microbiol. 1999; 34: 926-940Crossref PubMed Scopus (103) Google Scholar, 21Defoe G Coburn J Delineation of Borrelia burgdorferi p66 sequences required for integrin alpha(IIb)beta(3) recognition.Infect Immun. 2001; 69: 3455-3459Crossref PubMed Scopus (44) Google Scholar, 22Parveen N Leong JM Identification of a candidate glycosaminoglycan-binding adhesin of the Lyme disease spirochete Borrelia burgdorferi.Mol Microbiol. 2000; 35: 1220-1234Crossref PubMed Scopus (121) Google Scholar, 23Parveen N Robbins D Leong JM Strain variation in glycosaminoglycan recognition influences cell-type-specific binding by Lyme disease spirochetes.Infect Immun. 1999; 67: 1743-1749PubMed Google Scholar Spirochetes in tissue specimens taken from infected mice and patients with Lyme disease are often associated with collagenous connective tissues and vessel walls.24Barthold SW Persing DH Armstrong AL Peeples RA Kinetics of Borrelia burgdorferi dissemination and evolution of disease after intradermal inoculation of mice.Am J Pathol. 1991; 139: 263-273PubMed Google Scholar, 25Barthold SW de Souza MS Janotka JL Smith AL Persing DH Chronic Lyme borreliosis in the laboratory mouse.Am J Pathol. 1993; 143: 959-971PubMed Google Scholar, 26Nocton JJ Steere AC Lyme disease.Adv Intern Med. 1995; 40: 69-117PubMed Google Scholar These tissues are rich in ECM components. B. burgdorferi could reside in the ECM during a persistent infection. The interactions of the spirochetes with the ECM, mediated by Dbps, BBK32, and others, may therefore play critical roles in the persistence of B. burgdorferi in tissues. It has been speculated for decades that microbial pathogens may acquire host antigens to avoid immune clearance. DbpA is the best-characterized ligand-binding lipoprotein of B. burgdorferi. A recent study shows that a decorin deficiency reduces the susceptibility of laboratory mice to Lyme arthritis.27Brown EL Wooten RM Johnson BJ Iozzo RV Smith A Dolan MC Guo BP Weis JJ Hook M Resistance to Lyme disease in decorin-deficient mice.J Clin Invest. 2001; 107: 845-852Crossref PubMed Scopus (111) Google Scholar We used DbpA and decorin as a model system to explore the hypothesis that the interactions between a bacterial antigen and a host component may facilitate immune evasion. B. burgdorferi B31 clone 5A1128Purser JE Norris SJ Correlation between plasmid content and infectivity in Borrelia burgdorferi.Proc Natl Acad Sci USA. 2000; 97: 13865-13870Crossref PubMed Scopus (380) Google Scholar (a gift from Steven Norris, University of Texas, Houston, TX) was cultivated in Barbour-Stoenner-Kelly H complete medium at 33°C (Sigma Chemical Co., St. Louis, MO). BALB/c wild-type mice (Dcn+/+) and severe combined immunodeficient (SCID) mice on a BALB/c background were purchased from the Jackson Laboratories (Bar Harbor, ME). Decorin-deficient (Dcn−/−) mice on a BALB/c background were generated as previously described.27Brown EL Wooten RM Johnson BJ Iozzo RV Smith A Dolan MC Guo BP Weis JJ Hook M Resistance to Lyme disease in decorin-deficient mice.J Clin Invest. 2001; 107: 845-852Crossref PubMed Scopus (111) Google Scholar, 29Danielson KG Baribault H Holmes DF Graham H Kadler KE Iozzo RV Targeted disruption of decorin leads to abnormal collagen fibril morphology and skin fragility.J Cell Biol. 1997; 136: 729-743Crossref PubMed Scopus (1189) Google Scholar All mice were 4 to 8 weeks old when they were infected. Mice were given one single intradermal injection of 105 cultured spirochetes or inoculated with host-adapted spirochetes through a tissue transplant. Mice that were infected with cultured B. burgdorferi were sacrificed 2 months after infection. Urinary bladders, hearts, joints, and dorsal skins (not from the inoculation site) were harvested and immediately frozen in liquid nitrogen. Frozen samples were stored at −80°C until DNA and RNA were isolated. To prepare host-adapted spirochetes, donor mice were infected with cultured spirochetes for 3 months. Ear punches were taken and used to quantify spirochete burdens by quantitative polymerase chain reaction (qPCR) as described below. Donor ears were cut into small pieces. Each piece that that was estimated to contain ∼100 spirochetes was implanted into the dorsal skin of recipient mice as described previously.30Barthold SW Fikrig E Bockenstedt LK Persing DH Circumvention of outer surface protein A immunity by host-adapted Borrelia burgdorferi.Infect Immun. 1995; 63: 2255-2261PubMed Google Scholar The donor and recipient mouse strains were identical. Recipient mice were sacrificed either 2 weeks or 2 months after infection. Urinary bladders, hearts, joints, and skins were collected and stored as described above. To prepare anti-Borrelia sera, BALB/c mice were infected with cultured B31 5A11 spirochetes as described above. Blood was drawn between 2 and 4 months after infection and sera were isolated, pooled, and stored at −20°C. Ten SCID mice were infected with cultured spirochetes for 2 months as described above and each subcutaneously received six doses of 100-μl anti-Borrelia sera at intervals of 2 days. All animals were sacrificed 3 days after the last passive immunization. Hearts, joints, and skins were collected and stored as described above. Frozen bladder, heart, joint, and skin samples were transferred in liquid nitrogen and ground thoroughly with a mortar and pestle. An appropriate amount of ground tissue was transferred in a 500-μl polypropylene PCR tube for DNA preparation using the DNeasy mini kit (Qiagen Inc., Valencia, CA). RNA was isolated from the remaining tissue using TRIzol reagent (Invitrogen Life Technologies, Carlsbad, CA). To ensure that there was no DNA contamination, RNA preparations were first digested in solution with RNase-free DNase I (Life Technologies, Inc., Gaithersburg, MD) at 37°C for 2 hours; and then loaded to the RNeasy mini columns and further treated with RNase-free DNase I (Qiagen) for an additional 20 minutes at room temperature. Doubly digested samples were repurified and analyzed for potential DNA contamination by PCR amplification of the flaB gene. The DNA-free RNA preparation was first annealed with the reverse oligonucleotide primers of flaB, dbpA, ospC, decorin, and actin genes (Table 1) at 65°C, 60°C, 55°C, 50°C, and 45°C each for 1 minute, in the presence of reverse transcription buffer (Invitrogen). dNTPs and SuperScript II RNase H− reverse transcriptase (Invitrogen) were added and reverse transcription was conducted at 42°C for 1 hour and then inactivated at 95°C for 5 minutes following the manufacturer's instructions.Table 1qPCR Primers and ProbesGenePrimerProbeflaBForward5′-GAGTTTCTGGTAAGATTAATGCTC-3′5′-AGAGGTTTGTCACAAGCTTCTAGAAATACTTCAAAGGC-3′Reverse5′-CATTTAAATTCCCTTCTGTTGTCTGA-3′dbpAForward5′-CTTAAACTAACTATACTTGTTAAC-3′5′-TTATATCATGTGGACTAACAGGAGCAAC-3′Reverse5′-AATGTCTTTAGCGCTTCGTTC-3′ospCForward5′-TACGGATTCTAATGCGGTTTTAC-3′5′-TGAAGCGTTGCTGTCATCTATAGATGAAATTGCTGCT-3′Reverse5′-GTGATTATTTTCGGTATCCAAACCA-3′actin*Used for actin cDNA quantification.Forward5′-CCATGTACCCAGGCATTGC-3′5′-TGCAGAAGGAGATCACAGCCCTAGCACC-3′Reverse5′-CCAGACTGAGTACTTGCGTTC-3′actin†Used for actin genomic DNA quantification.Forward5′-CATCATGAAGTGTGACGTTGAC-3′5′-GTATGCCAATACAGTGCTGTCTGGTGGTACCAC-3′Reverse5′-GCATCCTGTCAGCAATGCC-3′decorinForward5′-TGTCATCTTCGAGTGGTGCA-3′5′-CACCCGACACAACCTTGCTAGACCTGC-3′Reverse5′-GAGGTTTGAATGCCTCTGGA-3′* Used for actin cDNA quantification.† Used for actin genomic DNA quantification. Open table in a new tab flab, dbpA, and ospC DNA concentration standards were prepared from cultured B. burgdorferi. B31 5A11 spirochetes were grown to stationary phase in BSK-H complete medium, counted in a Petroff-Hausser Counter (Hausser Scientific Partnership, Horsham, PA) and harvested by centrifugation at 13,000 rpm for 10 minutes. Pellets were digested with proteinase K (Qiagen) at 55°C for 2 hours, inactivated at 95°C for 10 minutes, and diluted to 100 to 105 spirochete DNA copies/μl. To generate an actin DNA concentration standard for cDNA and DNA quantification, primers (forward, 5′-TGAGCGGTTCCGGTGTCC-3′; reverse, 5′-CAGTGAGGCCAGAATGGA-3′) were designed to amplify a 292-bp internal fragment of the actin gene by PCR using murine DNA as a template. To prepare a decorin DNA concentration standard for cDNA quantification, mouse cDNA was prepared as described above and PCR amplified to generate a 205-bp internal fragment of decorin cDNA using the primers (forward, 5′-GGACAAAGTGCCCTGGGA-3′; reverse, 5′-GTCCTTCAGGTTCTTGAAGGC-3′). Taq polymerase was purchased from Roche Diagnostics Co. (Indianapolis, IN). The PCR conditions used were: 95°C for 5 minutes; 95°C for 40 seconds, 50°C for 1 minute, 72°C for 40 seconds, 50 cycles; 72°C for 10 minutes. PCR products were purified using the Quick PCR Product Purification kit (Qiagen). Purity was examined by agarose gel electrophoresis. DNA concentrations were determined by measuring the optical density at 260-nm wavelength, converted to copy number, and diluted at 102 to 107 DNA copies/μl. qPCR analyses were performed using the iCycler (Bio-Rad Laboratories, Hercules, CA). The Platinum TaqDNA Polymerase High Fidelity kit was purchased from Invitrogen. The sequences of primers and internal probes of flaB, dbpA, ospC, decorin, and actin genes were listed in Table 1. TaqMan TAMRA probes were ordered from Applied Biosystems (Foster City, CA). Amplification was performed in a 50-μl final volume reaction mix in individual wells of a 96-well iCycler iQ PCR plate (Bio-Rad). Twelve wells of each plate were assigned for DNA standards at six different concentrations in duplicate. Each cDNA or DNA sample was amplified in duplicate for flaB, dbpA, ospC, decorin, or actin genes. A PCR program with the following parameters was used: 95°C for 5 minutes; 95°C for 30 seconds, 60°C for 1 minute, 50 cycles. The mean cDNA copy numbers for flaB, dbpA, decorin, and actin transcripts of each cDNA pool and mean DNA copy numbers for flaB and actin genes of each DNA sample were automatically calculated from duplicate wells using the iCycler software. Tissue spirochete burdens were calculated as flaB DNA copy numbers per 106 actin DNA copies. Mouse decorin expression levels were presented as decorin mRNA copy numbers per 103 actin mRNA transcripts. Spirochetal dbpA expression levels were expressed as dbpA mRNA copy numbers per 103 flaB mRNA transcripts. Data were analyzed using Microsoft Excel (Redmond, WA) software. A two-tailed Student's t-test was used to analyze qPCR and quantitative reverse transcriptase-PCR data. A P value <0.05 was considered to be a significant difference. To define a protective niche for B. burgdorferi to resist immune clearance, the tissue spirochete burden was first investigated in infected SCID mice. Ten SCID mice were infected with cultured B. burgdorferi for 2 months and sacrificed. DNA samples were prepared from the urinary bladder, heart, joints, and skin and quantified for the tissue spirochete burden. These tissues were investigated for different reasons: the bladder has been shown to be a consistent source of B. burgdorferi and the skin is the most commonly examined biopsy site because it represents the tissue of initial entry. Finally, both the heart and joints are major sites of inflammation in the murine model of Lyme disease. The highest spirochete burden was detected in the heart, which was 48%, 123%, and 210% higher than the joints (P = 0.01), skin (P = 6.9 × 10−6), and bladder (P = 1.7 × 10−7), respectively (Figure 1A). The second highest spirochete burden was found in the joints, which was 34% and 52% higher than in the skin (P = 0.05) and bladder (P = 0.004), respectively. There was no significant difference in the spirochetal burden between the skin and bladder tissues (P = 0.12). We next examined the spirochete burden in wild-type mice infected in a similar manner. The tissue spirochete burden analysis revealed that the joint and skin tissues provided the better protection for B. burgdorferi to resist immune clearance (Figure 1B). The spirochete burden in the joint tissue was 5.4 and 7.0 times higher than those of heart (P = 5.9 × 10−4) and bladder tissues (P = 9.5 × 10−4), respectively; and the skin bacterial burden was 6.0 and 4.6 times higher than those of the heart (P = 1.6 × 10−4) and bladder tissues (P = 3.1 × 10−4). There were no significant differences in the tissue spirochete burden between the heart and bladder tissues (P = 0.34). The better protection in the joint and skin tissues was further demonstrated by a passive immunization study. Transferred antibodies more effectively reduced the tissue spirochete burden in the bladder and heart than the joints and skin (Figure 1C). The spirochete burden in the joint was 5.4 and 6.3 times higher than those of the heart (P = 1.1 × 10−5) and bladder tissues (P = 5.4 × 10−6), respectively. The bacterial burden in the skin tissue was 2.6 and 3.0 times higher than those of the heart (P = 8.7 × 10−3) and bladder tissues (P = 3.9 × 10−3). There was no significant difference in the tissue spirochetal burden between the heart and bladder tissues (P = 0.57). It is well documented that some of the tissues with the highest decorin expression are the skin, cartilage, and tendon.31Choi HU Johnson TL Pal S Tang LH Rosenberg L Neame PJ Characterization of the dermatan sulfate proteoglycans, DS-PGI and DS-PGII, from bovine articular cartilage and skin isolated by octyl-Sepharose chromatography.J Biol Chem. 1989; 264: 2876-2884Abstract Full Text PDF PubMed Google Scholar, 32Vogel KG Evanko SP Proteoglycans of fetal bovine tendon.J Biol Chem. 1987; 262: 13607-13613Abstract Full Text PDF PubMed Google Scholar To establish a correlation between decorin levels and Borrelia persistence, we analyzed relative decorin expression in the bladder, heart, joint, and skin tissues. Total RNA was isolated and converted to cDNA and quantified for actin and decorin mRNA copy numbers. Decorin transcription in the joint tissue was 163% and 68% higher than bladder (P = 5.2 × 10−6) and heart tissues (P = 5.7 × 10−4), respectively (Figure 2). The expression levels in the skin were higher than those of all of the three tissues. These data are fully consistent with a large body of published evidence indicating high levels of decorin expression in the skin.29Danielson KG Baribault H Holmes DF Graham H Kadler KE Iozzo RV Targeted disruption of decorin leads to abnormal collagen fibril morphology and skin fragility.J Cell Biol. 1997; 136: 729-743Crossref PubMed Scopus (1189) Google Scholar B. burgdorferi was more sensitive to immune clearance in both bladder and heart tissues than the joint and skin (Figure 1, B and C), and the decorin expression levels in these two former tissues were significantly lower, suggesting that better protection of B. burgdorferi against immune clearance may require a threshold level of decorin expression. To demonstrate that decorin is the key component in establishing a protective niche, Dcn−/− mice were used to confirm this hypothesis. Dcn+/+ and Dcn−/− mice were infected with cultured B. burgdorferi for 2 months and sacrificed after the anti-Borrelia immune response was fully developed. The bacterial burdens in the bladder, heart, joints, and skin were analyzed by qPCR. As shown in Figure 3, significant differences in the spirochete burdens between Dcn+/+ and Dcn−/− mice were noted only in the joints and skin (P = 0.0006 and 0.004, respectively), the two tissues with the highest decorin expression but not in the bladder or heart tissues (P = 0.78 and 0.80). The average spirochete burdens in the joints and skin of Dcn+/+ mice were 7.8 and 2.9 times higher than those in the same tissues in Dcn−/− mice. In contrast, the spirochete burdens in all of the four tissues of Dcn−/− mice and in the bladder and heart tissues of Dcn+/+ mice were similar (P = 0.10 to 0.64). These data indicate that a decorin deficiency completely diminished the protective niche in the joint and skin tissues of Dcn−/− mice, further defining a key role for decorin in the formation of a protective niche against immune clearance of B. burgdorferi during chronic infection. We next investigated whether decorin deficiency affected the gene expression of B. burgdorferi. dbpA mRNA transcripts were examined in the bladder, heart, joint, and skin tissues from both Dcn+/+ and Dcn−/− mice at 2 months after infection, a time point when the immune response against the spirochete had fully developed. No significant differences in dbpA expression were found in the bladder (P = 0.69), heart (P = 0.61), or skin (P = 0.69) between Dcn+/+ and Dcn−/− mice (Figure 4). However, higher levels of dbpA expression were observed in the joints of Dcn+/+ mice compared to Dcn−/− mice (P = 0.005). The dbpA expression level in the joint tissue of Dcn+/+ mice was 68% higher than the same tissue in Dcn−/− mice (Figure 4). This correlation between elevated dbpA expression in tissues containing elevated decorin expression eg, joints, indicated that a threshold level of decorin might be required to protect the spirochetes in this tissue, presumably against anti-Borrelia antibodies. Alternatively, the reduced dbpA expression observed in the joints of Dcn−/− mice could be a consequence related to the lack of interaction between DbpA and decorin because this interaction alone might influence dbpA expression. To rule out this possibility, dbpA expression was also examined in the joints of mice that had been infected just for 2 weeks. No significant differences in dbpA expression were noted between Dcn+/+ and Dcn−/− mice (data not shown), indicating that the presence of decorin did not affect dbpA expression before the maturation of immune responses. Taken together, these data suggest that the interactions between the DbpAs and decorin may give an advantage to DbpA-expressing spirochetes by preventing immune-mediated clearance of B. burgdorferi from high decorin-expressing tissues during chronic infection. After mammalian host infection, B. burgdorferi first replicates at the inoculation site and then rapidly disseminates and colonizes remote tissues. Infection also induces specific immune responses that effectively diminish tissue spirochete burdens. Our data demonstrated that deletion of decorin alone significantly reduced the spirochete burdens in chronically infected mice (Figure 3). This could be a function of impaired colonization by B. burgdorferi relating to the lack of host decorin and independent of immune pressure or the presence of decorin provided the bacteria with a protective niche against immune clearance, either by preventing Borrelia-specific antibodies from recognizing their targets or by making the bacteria unrecognizable to innate immune mechanisms. To distinguish between these two mechanisms of action, we examined tissue spirochete burdens before the anti-B. burgdorferi response was fully developed. Thirty Dcn−/− and 30 Dcn+/+ mice were infected with host-adapted spirochetes from Borrelia-infected donor mice. Half of the mice were sacrificed at 2 weeks after inoculation. The spirochete burdens in the bladder, heart, joints, and skin were quantified by qPCR and are presented in Figure 5A. No significant differences in the spirochete burdens between decorin-deficient and control mice were detected in the bladder (P = 0.52), heart (P = 0.45), joints (P = 0.44), or skin (P = 0.50). These data indicate that the influence of decorin deficiency on the spirochete burden is insignificant before the maturation of the anti-B. burgdorferi antibody response. The remaining mice were allowed to fully develop an immune response against the spirochetes and sacrificed 2 months after infection. Quantification of spirochetes in various tissues confirmed the role of decorin in the protection of B. burgdorferi against immune clearance. The differences in the tissue spirochete burdens in the joints (P = 0.003) and skin (P = 0.02) between Dcn−/− and Dcn+/+ mice were 4.3 and 3.1 times, respectively. In contrast, bladder (P = 0.48) and heart tissues (P = 0.73) had similar spirochete numbers regardless of decorin genotype (Figure 5B), a result consistent with the data obtained from mice infected with cultured spirochetes (Figure 3). The crucial role for decorin in the establishment of the protective niche could result from two mechanisms. First, decorin, which has been shown to play a role in collagen fibril formation and ECM integrity, could nonspecifically affect the transportation of immunoglobulin molecules into the tissues. Second, decorin may bind to DbpA in such a manner as to specifically interfere with the interactions of anti-Borrelia antibody. To test the hypothesis, we examined whether the protective niche could protect ospC-expressing spirochetes from clearance. SCID, Dcn−/−, and Dcn+/+ mice were infected intradermally and sacrificed 2 months after infection. RNA samples were prepared from the bladder, heart, joint, and skin specimens, converted to cDNA and quantified for ospC and flaB mRNA copy numbers. In the absence of immune selection pressure, B. burgdorferi exhibited differential ospC expression in the four tissues (Figure 6A). B. burgdorferi more actively transcribed ospC in the heart a

The complete intron-exon organization of the gene encoding human perlecan (HSPG2), the major heparan sulfate proteoglycan of basement membranes, has been elucidated, and specific exons have been assigned to coding sequences for the modular domains of the protein core. The gene was composed of 94 exons, spanning > 120 kbp of genomic DNA. The exon arrangement was analyzed vis-à-vis the modular structure of the perlecan, which harbors protein domains homologous to the low density lipoprotein receptor, laminin, epidermal growth factor, and neural cell adhesion molecule. The exon size and the intron phases were highly conserved when compared to the corresponding domains of the homologous genes, suggesting that most of this modular proteoglycan has evolved from a common ancestor by gene duplication or exon shuffling. The 5' flanking region revealed a structural organization characteristic of housekeeping and growth control-related genes. It lacked canonical TATA or CAAT boxes, but it contained several GC boxes with binding sites for the transcription factors SP1 and ETF. Consistent with the lack of a TATA element, the perlecan gene contained multiple transcription initiation sites distributed over 80 bp of genomic DNA. These results offer insights into the evolution of this chimeric molecule and provide the molecular basis for understanding the transcriptional control of this important gene.

Decorin, a proteoglycan known to interact with collagen and growth factors, may play key roles during ontogenesis, tissue remodeling, and cancer. We have deciphered the complete protein sequence of the murine decorin by cDNA cloning, elucidated its gene structure and chromosomal location, and investigated its expression in the developing embryo. The decorin protein and the gene were highly conserved vis a vis the human counterpart; however, the murine gene lacked a leader exon, exon Ib, which was found only in the human. Using interspecific backcrossing, we assigned the gene to chromosome 10 just proximally to the Steel gene locus. In situ hybridization studies of developing mouse embryos showed a distinct pattern of expression with a progressive increase of decorin mRNA during ontogenesis. At early stages (day 11 postconception), decorin was detectable only in the floor plate region. Subsequently (days 13-16 postconception), decorin expression was especially prominent in the meninges and mesothelial linings of pericardium, pleura, and coelomic cavity, as well as in the dermis and subepithelial layers of the intestine and urinary bladder. In contrast, the major parenchymal organs were only weakly positive for decorin mRNA. These findings suggest that decorin may play a role in epithelial/mesenchymal interactions during organ development and shaping.

Epidermolysis bullosa (EB) is a group of genodermatoses characterized by the fragility of skin.Previous studies on the dys- trophic (scarring) forms of EB have suggested abnormalities in anchoring fibrils, morphologically recognizable attachment structures that provide stability to the association ofthe cutaneous basement membrane to the underlying dermis.Since type VII collagen is the major component ofthe anchoring fibrils, we examined the genetic linkage of dominant dystrophic EB (EBDD) and the type VII collagen gene (COL7A1) locus, which we have recently mapped to chromosome 3p, in three large kindreds with abnormal anchoring fibrils.Strong genetic linkage of EBDD and COL7A1 loci was demonstrated with the maximum logarithm ofodds (LOD) score of 8.77 ata =0.This linkage was further confirmed with two additional markers in this region of the short arm of chromosome 3, and these analyses allowed further refinement of the map locus of COL7A1.Since there were no recombinants between the COL7A1 and EBDD loci, our findings suggest that type VII collagen is the candidate gene that may harbor the mutations responsible for the EB phenotype in these three families.(J.Clin.Invest.

The subcellular localization of human skin chymase to mast cell granules was established by immunoelectron microscopy, and binding of chymase to the area of the dermo-epidermal junction, a basement membrane, was demonstrated immunocytochemically in cryosections incubated with purified proteinase prior to immunolabeling. Because heparin and heparan sulfate proteoglycans are major constituents of mast cell granules and basement membranes, respectively, the ability of chymase to bind to glycosaminoglycans (GAG) was investigated. Among a variety of GAGs, only binding of chymase to heparin and heparan sulfate appears physiologically significant. Binding was ionic strength-dependent, involved amino groups on the proteinase, and correlated with increasing GAG sulfate content, indicating a predominantly electrostatic association. Interaction with heparin was observed in solutions containing up to 0.5 M NaCl, and interaction with heparan sulfate was observed in solutions containing up to 0.3 M NaCl. Binding of heparin did not detectably affect catalysis of peptide substrates, but may reduce accessibility of proteinase to protein substrates. Measurements among a series of serine class proteinases indicated that heparin binding was a more common property of mast cell proteinases than proteinases stored in other secretory granules. Binding of chymase to heparin is likely to have a storage as well as a structural role within the mast cell granule, whereas binding of chymase to heparan sulfate may have physiological significance after degranulation.

A hallmark of systemic sclerosis (SSc) is the development of tissue fibrosis.Excessive production of several connective tis- sue components normally present in the dermis, including type I, III, V, and VI collagens as well as fibronectin and proteogly- cans, is a consistent finding in the skin of SSc patients.Type VII collagen is a major constituent of anchoring fibrils, present in the skin at the dermal-epidermal basement membrane zone.TGF-ft has been shown to upregulate the expression of the type VII collagen gene.In this study, we assessed the expression of type VII collagen and TGF-,6 in the skin of patients with SSc.Indirect immunofluorescence showed an abundance of type VII collagen in the patients' skin, including the dermis.Ultrastruc- tural analysis of SSc skin revealed an abundance of fibrillar material, possibly representing type VII collagen.The in- creased expression of type VII collagen epitopes was accompanied by the elevated expression of immunodetectable TGF-ftl and TGF-,82.Dermal fibroblasts cultured from the affected indi- viduals showed a statistically significant (P < 0.02) increase in the expression of type VII collagen at the mRNA level, as de- tected by reverse transcription-PCR with a mutated cDNA as an internal standard, and increased deposition of the protein as assessed by indirect immunofluorescence.Thus, type VII colla- gen is abundantly present in SSc patients' dermis, a location not characteristic of its normal distribution, and its aberrant ex- pression may relate to the presence of TGF-,8 in the same topo- graphic distribution.The presence of type VII collagen in the dermis may contribute to the tightly bound and indurated ap- pearance of the affected skin in SSc patients.(

The biosynthesis of proteoglycans in short term organ culture of human colon and colon carcinoma was studied. Proteoglycans, labeled with [35S]sulfate and [3H]serine, were extracted with either 4 M or 0.5 M guanidine HCl in the presence of protease inhibitors and sequentially purified by associative and dissociative CsCl density gradient ultracentrifugation. Normal colon synthesized two polydisperse classes of proteoglycans: a large heparan sulfate-containing monomer, with a Kav of 0.48 on Sepharose CL-2B and a small dermatan sulfate-containing monomer with a Kav of 0.65. A portion (25%) of the proteoglycans was found as aggregate when chromatographed under associative conditions, and the larger monomers interacted with hyaluronic acid to an extent greater than the smaller proteoglycans. Following papain or alkali treatment, the free glycosaminoglycan side chains of both monomers eluted as a single broad peak (Kav = 0.5) from Sepharose CL-6B, with an estimated Mr of 20 X 10(3). In contrast, colon carcinoma synthesized only one proteoglycan monomer, which aggregated to a limited extent (12%). This proteoglycan population, with a Kav of 0.7 on CL-2B, contained chondroitin sulfate as the major glycosaminoglycan (greater than 81%), with small amounts of dermatan sulfate. The glycosaminoglycans had an estimated Mr of 9 X 10(3), and the disaccharides released by chondroitinase ABC consisted of 32% 4-sulfate and 68% 6-sulfate. Electron microscopy of mixed proteoglycancytochrome c monolayers from the associative fractions of normal and neoplastic colon revealed aggregated complexes which were similar in over structure, although smaller than the proteoglycan aggregates from cartilage.

Background: The antiangiogenic approach to controlling cancer requires a better understanding of angiogenesis and the discovery of new compounds that modulate this key biological process. Here we investigated the role of endorepellin, an angiostatic protein fragment that is derived from the C-terminus of perlecan, a heparan sulfate proteoglycan, in controlling tumor angiogenesis in vivo. Methods: We administered human recombinant endorepellin systemically to mice bearing orthotopic squamous carcinoma xenografts or syngeneic Lewis lung carcinoma tumors. We monitored tumor growth, angiogenesis, metabolism, hypoxia, and mitotic index by using quantitative immunohistochemistry and positron emission tomography scan imaging. In addition, we determined the localization of injected endorepellin using near-infrared labeling and immunohistochemistry of frozen tumor sections. Finally, we isolated tumor-derived endothelial cells and tested whether endorepellin could interact with these cells and disrupt in vitro capillary morphogenesis. All statistical tests were two-sided. Results: Endorepellin specifically targeted the tumor vasculature as determined by immunohistochemical analysis and accumulated in the tumor perivascular zones where it persisted for several days as discrete deposits. This led to inhibition of tumor angiogenesis (as measured by decreased CD31-positive cells, mean control = 1902 CD31-positive pixels, mean endorepellin treated = 343.9, difference between means = 1558, 95% confidence interval [CI] = 1296 to 1820, P <.001), enhanced tumor hypoxia, and a statistically significant decrease in tumor metabolism and mitotic index (as measured by decreased Ki67-positive cells, mean control Ki67 pixels = 5970, mean endorepellin-treated Ki67 pixels = 3644, difference between means = 2326, 95% CI = 1904 to 2749, P <.001) compared to untreated controls. Endorepellin was actively internalized by tumor-derived endothelial cells causing a redistribution of α2β1 integrin such that both proteins colocalized to punctate deposits in the perivascular region. Endorepellin treatment inhibited in vitro capillary morphogenesis of both normal and tumor-derived endothelia. Conclusions: Our results provide support for the hypothesis that endorepellin is an effective antitumor vasculature agent that could be used as a therapeutic modality to combat cancer.

Endorepellin, the C-terminal module of perlecan, has angiostatic activity. Here we provide definitive genetic and biochemical evidence that the functional endorepellin receptor is the α2β1 integrin. Notably, the specific endorepellin binding to the receptor was cation-independent and was mediated by the α2I domain. We show that the anti-angiogenic effects of endorepellin cannot occur in the absence of α2β1. Microvascular endothelial cells from α2β1-/- mice, but not those isolated from either wild-type or α1β1-/- mice, did not respond to endorepellin. Moreover, syngeneic Lewis lung carcinoma xenografts in α2β1-/- mice failed to respond to systemic delivery of endorepellin. In contrast, endorepellin inhibited tumor growth and angiogenesis in the wild-type mice expressing integrin α2β1. We conclude that the angiostatic effects of endorepellin in vivo are mediated by a specific interaction of endorepellin with the α2β1 integrin receptor. Endorepellin, the C-terminal module of perlecan, has angiostatic activity. Here we provide definitive genetic and biochemical evidence that the functional endorepellin receptor is the α2β1 integrin. Notably, the specific endorepellin binding to the receptor was cation-independent and was mediated by the α2I domain. We show that the anti-angiogenic effects of endorepellin cannot occur in the absence of α2β1. Microvascular endothelial cells from α2β1-/- mice, but not those isolated from either wild-type or α1β1-/- mice, did not respond to endorepellin. Moreover, syngeneic Lewis lung carcinoma xenografts in α2β1-/- mice failed to respond to systemic delivery of endorepellin. In contrast, endorepellin inhibited tumor growth and angiogenesis in the wild-type mice expressing integrin α2β1. We conclude that the angiostatic effects of endorepellin in vivo are mediated by a specific interaction of endorepellin with the α2β1 integrin receptor. The incorporation of new blood vessels into growing neoplasms is a prerequisite for tumor viability and progression. Accordingly, much attention has been invested in the search for and characterization of anti-angiogenic agents to enable regulated and inhibited tumor angiogenesis as part of cancer therapies (1Carmeliet P. Nature. 2005; 438: 932-936Crossref PubMed Scopus (2818) Google Scholar). The proteoglycan perlecan plays a key role in the angiogenic process, primarily by modulating the availability and activity of growth factors involved in angiogenesis such as fibroblast growth factor 2, VEGF, 3The abbreviations used are: VEGFvascular endothelial growth factorHUVEChuman umbilical vein endothelial cell(s)LLCLewis lung carcinomasiRNAshort interfering RNABSAbovine serum albuminPBSphosphate-buffered salineDAPI4′,6′-diamino-2-phenylindoleLGlaminin-like globular. 3The abbreviations used are: VEGFvascular endothelial growth factorHUVEChuman umbilical vein endothelial cell(s)LLCLewis lung carcinomasiRNAshort interfering RNABSAbovine serum albuminPBSphosphate-buffered salineDAPI4′,6′-diamino-2-phenylindoleLGlaminin-like globular. and platelet-derived growth factor (2Whitelock J.M. Graham L.D. Melrose J. Murdoch A.D. Iozzo R.V. Underwood P.A. Matrix Biol. 1999; 18: 163-178Crossref PubMed Scopus (120) Google Scholar, 3Hassell J.R. Yamada Y. Arikawa-Hirasawa E. Glycoconj. J. 2003; 19: 263-267Crossref Scopus (57) Google Scholar, 4Knox S.M. Whitelock J.M. Cell. Mol. Life Sci. 2006; 63: 2435-2445Crossref PubMed Scopus (108) Google Scholar, 5Whitelock J.M. Iozzo R.V. Chem. Rev. 2005; 105: 2745-2764Crossref PubMed Scopus (346) Google Scholar, 6Farach-Carson M.C. Carson D.D. Glycobiology. 2007; 17: 897-905Crossref PubMed Scopus (108) Google Scholar). The most C-terminal part of perlecan (domain V), named endorepellin, is a powerful angiogenic inhibitor (7Mongiat M. Sweeney S. San Antonio J.D. Fu J. Iozzo R.V. J. Biol. Chem. 2003; 278: 4238-4249Abstract Full Text Full Text PDF PubMed Scopus (285) Google Scholar). Endorepellin carries three laminin-like globular (LG) domains separated by epidermal growth factor-like repeats (8Iozzo R.V. Nat. Rev. Mol. Cell Biol. 2005; 6: 646-656Crossref PubMed Scopus (398) Google Scholar) and binds to numerous extracellular matrix proteins, growth factors, and receptors including collagen XVIII, fibulin-2, nidogen, fibroblast growth factor 7, fibroblast growth factor-binding protein, ECM1 (7Mongiat M. Sweeney S. San Antonio J.D. Fu J. Iozzo R.V. J. Biol. Chem. 2003; 278: 4238-4249Abstract Full Text Full Text PDF PubMed Scopus (285) Google Scholar, 9Brown J.C. Sasaki T. Göhring W. Yamada E. Timpl R. Eur. J. Biochem. 1997; 250: 39-46Crossref PubMed Scopus (145) Google Scholar, 10Mongiat M. Taylor K. Otto J. Aho S. Uitto J. Whitelock J. Iozzo R.V. J. Biol. Chem. 2000; 275: 7095-7100Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar, 11Mongiat M. Otto J. Oldershaw R. Ferrer F. Sato J.D. Iozzo R.V. J. Biol. Chem. 2001; 276: 10263-10271Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar, 12Mongiat M. Fu J. Oldershaw R. Greenhalgh R. Gown A. Iozzo R.V. J. Biol. Chem. 2003; 278: 17491-17499Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar), α-dystroglycan, and integrin α2β1 (9Brown J.C. Sasaki T. Göhring W. Yamada E. Timpl R. Eur. J. Biochem. 1997; 250: 39-46Crossref PubMed Scopus (145) Google Scholar, 13Talts J.F. Andac Z. Göhring W. Brancaccio A. Timpl R. EMBO J. 1999; 18: 863-870Crossref PubMed Scopus (398) Google Scholar, 14Bix G. Fu J. Gonzalez E. Macro L. Barker A. Campbell S. Zutter M.M. Santoro S.A. Kim J.K. Höök M. Reed C.C. Iozzo R.V. J. Cell Biol. 2004; 166: 97-109Crossref PubMed Scopus (232) Google Scholar, 15Laplante P. Raymond M-A. Labelle A. Abe J.-I. Iozzo R.V. Hebért M.-J. J. Biol. Chem. 2006; 281: 30383-30392Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, 16Bix G. Iozzo R.A. Woodall B. Burrows M. McQuillan A. Campbell S. Fields G.B. Iozzo R.V. Blood. 2007; 109: 3745-3748Crossref PubMed Scopus (50) Google Scholar). The endorepellin anti-angiogenic effect is parallel to several proteolytically released fragments from vascular basement membrane such as endostatin, the NC-1 domain of collagen type XVIII, and tumstatin, the NC-1 domain the of type IV collagen α3 chain (8Iozzo R.V. Nat. Rev. Mol. Cell Biol. 2005; 6: 646-656Crossref PubMed Scopus (398) Google Scholar, 17Nyberg P. Xie L. Kalluri R. Cancer Res. 2005; 65: 3967-3979Crossref PubMed Scopus (471) Google Scholar, 18Bix G. Iozzo R.V. Trends Cell Biol. 2005; 15: 52-60Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar). These fragments principally act on endothelial cells as “negative” ligands for specific integrin receptors. Endorepellin is a potent inhibitor in several angiogenesis assays such as endothelial cell migration, collagen-induced capillary morphogenesis, blood vessel recruitment into Matrigel plugs, and chicken chorioallontoic membrane (7Mongiat M. Sweeney S. San Antonio J.D. Fu J. Iozzo R.V. J. Biol. Chem. 2003; 278: 4238-4249Abstract Full Text Full Text PDF PubMed Scopus (285) Google Scholar, 19Gonzalez E.M. Reed C.C. Bix G. Fu J. Zhang Y. Gopalakrishnan B. Greenspan D.S. Iozzo R.V. J. Biol. Chem. 2005; 280: 7080-7087Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar). It also effectively retards in vivo tumor growth by specifically targeting tumor angiogenesis (20Bix G. Castello R. Burrows M. Zoeller J.J. Weech M. Iozzo R.A. Cardi C. Thakur M.T. Barker C.A. Camphausen K.C. Iozzo R.V. J. Natl. Cancer Inst. 2006; 98: 1634-1646Crossref PubMed Scopus (96) Google Scholar). We hypothesize that endorepellin takes effect via the LG3 domain binding to the integrin α2β1 causing actin disassembly and therefore affecting three key steps of angiogenesis: endothelial cell adhesion, migration, and morphogenesis. vascular endothelial growth factor human umbilical vein endothelial cell(s) Lewis lung carcinoma short interfering RNA bovine serum albumin phosphate-buffered saline 4′,6′-diamino-2-phenylindole laminin-like globular. vascular endothelial growth factor human umbilical vein endothelial cell(s) Lewis lung carcinoma short interfering RNA bovine serum albumin phosphate-buffered saline 4′,6′-diamino-2-phenylindole laminin-like globular. Here we have further investigated the endorepellin-α2β1 integrin interactions by using cell-free experiments with a soluble form of the α2β1 integrin, by in vitro assays of endothelial cells deficient in the integrin α1or α2 subunits, by siRNA knockdown of the integrin α2 subunit, and in syngeneic tumor xenografts growing in mice lacking the α2β1 integrin receptor. We report a novel cation-independent binding between endorepellin and integrin α2 I domain and show that integrin α2β1 is necessary for recruitment of endorepellin to the vasculature and for its anti-angiogenic properties both in vitro and in vivo. The presented studies increase both the knowledge about this specific angiogenesis inhibitor and angiogenesis in general. Cell Lines and Reagents—HUVEC at passages 1-6, HT1080, and LLC cells were cultured under standard conditions. Human recombinant endorepellin and LG3 harboring a His6 tag at their C termini were purified on a nickel-nitrilotriacetic acid resin column as previously described (7Mongiat M. Sweeney S. San Antonio J.D. Fu J. Iozzo R.V. J. Biol. Chem. 2003; 278: 4238-4249Abstract Full Text Full Text PDF PubMed Scopus (285) Google Scholar). The soluble ectodomain heterodimer of the α2β1 integrin was generated as previously described (21Eble J.A. Beermann B. Hinz H.J. Schmidt-Hederich A. J. Biol. Chem. 2001; 276: 12274-12284Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). The recombinant production of the oligo-His-tagged integrin α2 I domain was carried out similar to the glutathione S-transferase-tagged α2 I domain (22Eble J.A. Tuckwell D.S. Biochem. J. 2004; 376: 77-85Crossref Google Scholar). The binding activity of both integrin constructs was tested on collagen I and rhodocetin, a high affinity α2β1 integrin-specific antagonist (21Eble J.A. Beermann B. Hinz H.J. Schmidt-Hederich A. J. Biol. Chem. 2001; 276: 12274-12284Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). Binding Assays with Recombinant α2β1 Integrin and α2I Domain—A binding assay of soluble α2β1 integrin to immobilized collagen I or endorepellin in the presence of either 1 mm MnCl2, 1 mm MgCl2, and integrin-activating antibody 9EG7 or 10 mm EDTA was performed as previously published (21Eble J.A. Beermann B. Hinz H.J. Schmidt-Hederich A. J. Biol. Chem. 2001; 276: 12274-12284Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). For the reciprocal binding assays, recombinant α2β1 integrin ectodomain heterodimer or oligo-His-tagged integrin α2 I domain was coated onto a microtiter plate at 5 μgml-1 in Tris-buffered saline/magnesium/manganese buffer (2 mm MgCl2, 1 mm MnCl2, pH 7.4) at 4 °C overnight. After blocking with 1% BSA in Tris-buffered saline/magnesium/manganese buffer, the wells were incubated with endorepellin at the indicated concentrations or with 10 μgml-1 CB3[IV], both in blocking buffer. This soluble collagen IV fragment harbors the α2β1 integrin-binding site (23Kern A. Eble J. Golbik R. Kühn K. Eur. J. Biochem. 1993; 215: 151-159Crossref PubMed Scopus (181) Google Scholar, 24Eble J.A. Golbik R. Mann K. Kühn K. EMBO J. 1993; 12: 4795-4802Crossref PubMed Scopus (177) Google Scholar) and was used as positive control. After washing, bound integrin ligands were fixed with 2.5% glutaraldehyde in HEPES-buffered saline (50 mm HEPES/HCl, 150 mm NaCl, 2 mm MgCl2, 1 mm MnCl2, pH 7.4) for 10 min and quantified by enzyme-linked immunosorbent assay using polyclonal rabbit antibodies against endorepellin and CB3[IV] as primary antibodies and secondary alkaline phosphatase-coupled antibodies directed against rabbit immunoglobulins. The plates were read at 405 nm. Cell Adhesion and Actin Disassembly Assays—CC2-treated chamber slides (Nunc Inc., Roskilde, Denmark) were coated with 100 μgml-1 rat tail collagen I (BD Biosciences, Bedford, MA) at 4 °C overnight. The wells were washed with PBS, and 104 HUVEC were seeded. After 24 h, the medium was changed to serum-free M199 for 30 min prior to treatment with 150 nm endorepellin, 150 nm endorepellin, and 1 μm human recombinant integrin α2 I domain or 1 μm human recombinant integrin α2 I domain alone for 30 min at 37 °C. The medium was removed, and M199 containing the prior incubated reagents were added for 25 min at 37 °C. The nuclei and actin stress fibers were visualized by DAPI and fluorescein isothiocyanate-phalloidin staining, respectively (14Bix G. Fu J. Gonzalez E. Macro L. Barker A. Campbell S. Zutter M.M. Santoro S.A. Kim J.K. Höök M. Reed C.C. Iozzo R.V. J. Cell Biol. 2004; 166: 97-109Crossref PubMed Scopus (232) Google Scholar). For quantification of cell adhesion, the cells were counted by their DAPI staining. For quantification of actin stress fibers, 80-100 randomly selected cells of each group were analyzed. The assays were performed in triplicate. siRNA Transfection of HUVEC and HT1080 Cells—HUVEC/HT1080 were plated at subconfluent density on 12-well culture plates (Nunc). The cells were allowed to grow in complete medium until ∼70% confluence. Following washing, the cells were transfected with two validated siRNA constructs (Ambion Applied Biosystems, Foster City, CA), targeting different regions of the integrin α2 mRNA. siRNA1 (antisense 5′-3′, UCUGAAAGUUGUCUCCUCCtt) and siRNA2 (antisense 5′-3′, AACACUUCCUGUUGUUACCtt) target exons 10 and 24/25, respectively. The cells were transfected for 48-72 h using siPORT lipid transfection agent with the two siRNA, either alone or in combination at 125-150 nm. Migration Assay of α2 Knockdown Endothelial and Fibrosarcoma Cells—HUVEC and HT1080 cells were transfected with the optimal concentrations of siRNA, and the time span was analyzed by immunoblotting. HUVEC and HT1080 that were treated with siPORT alone were used as a control. Both control and α2 siRNA-treated cells were preincubated with various concentrations of endorepellin for 30 min prior to migration through a collagen I-coated polycarbonate membrane, with 8-μm pores in a 48-well Boyden chamber (Neuroprobe Inc., Gaithersburg, MD). About 8 × 103 HUVEC or HT1080 cells were loaded into the upper chamber and allowed to migrate at 37 °C with 5% CO2 for 4-6 h ± VEGF165 (R & D Systems, Minneapolis, MN) at a concentration of 10 ng ml-1 in the lower chambers as a chemo-attractant for HUVEC, and heat-inactivated medium conditioned by HT1080 as a chemo-attractant for HT1080. Immunofluorescence Analysis of α2β1 Knockdown Cells—HUVEC and HT1080 cultured for 24 h on lysine-treated eight-well slides coated with rat tail collagen I (100 μgml-1) (BD Biosciences) were transfected with the two validated α2 siRNA constructs (150 mm each). The cells were rinsed with PBS, fixed in 5% paraformaldehyde, and permeabilized with 0.1% Triton X-100 for 10 s. The cells were rinsed in PBS and then blocked with 5% BSA in PBS for 1 h. Polyclonal anti-α2 primary antibody (Santa Cruz Biotechnology, Santa Cruz, CA) was added to wells at a 1:50 dilution in 5% BSA/PBS for 1 h. Primary antibody was detected using a fluorescein isothiocyanate-conjugated rabbit anti IgG (1:200) (Santa Cruz Biotechnology). The nuclei were visualized using DAPI. All of the images were acquired on an Olympus BX51 microscope equipped with a SPOT camera (Diagnostic Instruments, Inc., Sterling Heights, MI). Genotyping of Integrin α1 and α2 Null Mice and Isolation of Lung Microvascular Endothelial Cells—Tail genomic DNA was subjected to PCR in 20 μl of reaction volume with 1.25 mm MgCl2 and 1 pmol ml-1 each primer using a mix of three primers. Primer sequences were as follows: α1β1+/+, 5′-ggttggtgactaaagttgatcc-3′; α1 β1-/-, 5′-ggaacttcctgactaggggagg-3′; α1 β1 common, 5′ttttcacactcatgaaatcttgttg-3′; α2 β1+/+, 5′-cttgtctaatggctattgc-3′; α2 β1-/-, 5′-tggcttttcttcctcctatgg-3′; and α2 β1common, 5′-aagttgctcgcttgctcta-3′. Lung microvascular endothelial cells were isolated from wild-type or integrin null mice as previously described (25Pozzi A. Moberg P.E. Miles L.A. Wagner S. Soloway P. Gardner H.A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 2202-2207Crossref PubMed Scopus (349) Google Scholar). Briefly, the lung vasculature was perfused with PBS, 2.5 mm EDTA followed by 0.25% trypsin, 2.5 mm EDTA via the right ventricle. The lungs were removed and incubated at 37 °C for 20 min. The visceral pleura was subsequently trimmed, and the perfusion was repeated. Primary endothelial cells were recovered and grown o in EGM-2-MV containing 5% fetal calf serum (Clonetics). The cells at passages 2-4 were used for the experiments. Syngeneic Tumor Xenografts and Quantification of Tumor Angiogenesis—Female integrin α2 β1-/- and α2 β1+/+ mice in C57Bl/6 background were injected subcutaneously with 106 LLC cells. On the day tumors became visible, the mice were randomized into two groups, and one group received intraperitoneal injections of human recombinant endorepellin, whereas the other received vehicle (PBS) alone (20Bix G. Castello R. Burrows M. Zoeller J.J. Weech M. Iozzo R.A. Cardi C. Thakur M.T. Barker C.A. Camphausen K.C. Iozzo R.V. J. Natl. Cancer Inst. 2006; 98: 1634-1646Crossref PubMed Scopus (96) Google Scholar). Tumor sectioning and immunostains were performed as previously described (20Bix G. Castello R. Burrows M. Zoeller J.J. Weech M. Iozzo R.A. Cardi C. Thakur M.T. Barker C.A. Camphausen K.C. Iozzo R.V. J. Natl. Cancer Inst. 2006; 98: 1634-1646Crossref PubMed Scopus (96) Google Scholar). The following antibodies were used: rat anti-mouse CD31 monoclonal antibody (BD, Biosciences, San Jose, CA) and mouse anti His6 monoclonal (ABM, Vancouver, Canada). Both antibodies were diluted 1:100. The epitopes were revealed by using fluorescein isothiocyanate-conjugated goat anti rat IgG and rhodamine-conjugated goat-anti mouse IgG (Santa Cruz Biotechnology) secondary antibodies. Sections were counter-stained with DAPI. Sections from three different tumors in each group were stained with CD31, and pictures were taken at 20× magnification to cover the entire tumor section with a fixed exposure of 500 ms. The acquired images (n = 30-50) were quantified using the Image J software package (National Institutes of Health, Bethesda, MD). All of the statistical analyses were carried out with SigmaStat for Windows version 3.10 (Systat Software, Inc., Port Richmond, CA). The results were compared by using the two-sided Student's t test, and the differences were considered statistically significant at p < 0.05. The Soluble α2β1 Integrin Ectodomain Interacts Specifically with Endorepellin in a Cation-independent Manner—To investigate the molecular interaction between endorepellin and the α2β1 integrin, we utilized a soluble heterodimeric α2β1 integrin. This soluble receptor consists of the ectodomain of both α2 and β1 integrin subunits noncovalently associated by the dimerizing motif of the two transcription factors Fos and Jun, respectively (21Eble J.A. Beermann B. Hinz H.J. Schmidt-Hederich A. J. Biol. Chem. 2001; 276: 12274-12284Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). This soluble integrin binds with high affinity collagen types I, II, and IV as the native integrin and has been successfully utilized in various protein/protein interaction assays (21Eble J.A. Beermann B. Hinz H.J. Schmidt-Hederich A. J. Biol. Chem. 2001; 276: 12274-12284Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). Using this approach, together with a battery of monoclonal antibodies against the α2 I domain, the precise mapping of the disintegrin rhodocetin, an RGD-independent ligand of the α2β1 integrin, on the α2 I domain was determined (22Eble J.A. Tuckwell D.S. Biochem. J. 2004; 376: 77-85Crossref Google Scholar). We conducted several binding studies using endorepellin and the soluble α2β1 integrin. Unexpectedly, when endorepellin was utilized as the immobilized ligand, there was no significant binding to the soluble α2β1 (Fig. 1A). Under identical experimental conditions, immobilized collagen I readily bound to the soluble α2β1 receptor, and this interaction was enhanced by the activating monoclonal antibody 9EG7 and abolished by the addition of EDTA (Fig. 1A). In contrast, when endorepellin was used as a soluble ligand, it significantly bound to the immobilized α2β1 (Fig. 1B). Surprisingly, binding levels were found to be identical in the presence or absence of EDTA. As an internal positive control, we used the CB3 fragment of collagen IV, which harbors the α2 I domain-binding site (23Kern A. Eble J. Golbik R. Kühn K. Eur. J. Biochem. 1993; 215: 151-159Crossref PubMed Scopus (181) Google Scholar, 24Eble J.A. Golbik R. Mann K. Kühn K. EMBO J. 1993; 12: 4795-4802Crossref PubMed Scopus (177) Google Scholar). The binding of CB3[IV] to immobilized α2β1 receptor was robust and significantly reduced by EDTA (Fig. 1B). When a range of concentrations of endorepellin was added to α2β1-coated plates in the presence of Mn2+ (1 mm) or EDTA (10 mm), binding curves were almost identical, confirming the cation-independent interaction (Fig. 1C). Next, we determined whether endorepellin/α2β1 interaction was mediated by the α2 I domain, the region of the integrin known to bind collagen (26Emsley J. Knight C.G. Farndale R.W. Barnes M.J. Liddington R.C. Cell. 2000; 101: 47-56Abstract Full Text Full Text PDF PubMed Scopus (842) Google Scholar). When increasing concentrations of soluble endorepellin were added to the microtiter plates at the concentrations shown (Fig. 1D), a saturable binding of endorepellin to the α2 I domain was observed, albeit at lower levels than the whole heterodimeric integrin ectodomain (Fig. 1D) in agreement with our previous studies (14Bix G. Fu J. Gonzalez E. Macro L. Barker A. Campbell S. Zutter M.M. Santoro S.A. Kim J.K. Höök M. Reed C.C. Iozzo R.V. J. Cell Biol. 2004; 166: 97-109Crossref PubMed Scopus (232) Google Scholar). Functional Block of Endorepellin Activity by Soluble α2β1 Integrin and Its α2 I Domain—Having established a physical interaction between the soluble α2β1 integrin and endorepellin, we next wished to prove whether this interaction would have a biological meaning using live endothelial cells. Utilizing a highly sensitive functional assay (14Bix G. Fu J. Gonzalez E. Macro L. Barker A. Campbell S. Zutter M.M. Santoro S.A. Kim J.K. Höök M. Reed C.C. Iozzo R.V. J. Cell Biol. 2004; 166: 97-109Crossref PubMed Scopus (232) Google Scholar), we found that actin stress fiber disassembly evoked by a short exposure to endorepellin could be almost completely abolished by preincubating endorepellin with either soluble α2β1orthe α2 I domain (Fig. 2, A-F). Notably, similar effects were obtained using the terminal LG module, LG3, of endorepellin (supplemental Fig. S1), which is the active binding site for the integrin (19Gonzalez E.M. Reed C.C. Bix G. Fu J. Zhang Y. Gopalakrishnan B. Greenspan D.S. Iozzo R.V. J. Biol. Chem. 2005; 280: 7080-7087Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar). To further corroborate these finding, we quantified the total number of attached cells, as an adhesion assay on collagen I. The number of adherent endothelial cells was drastically (p < 0.001) reduced when incubated with endorepellin (Fig. 2G). However, this effect was abrogated by the soluble ectodomain of the α2β1 integrin and significantly reduced (p < 0.05) by preincubation with the α2 I domain (Fig. 2G). Collectively, these findings prove that the physical interaction between endorepellin and soluble α2β1 integrin translates into a functional block of endorepellin angiostatic activity on endothelial cells. Integrin α2β1 Is Vital for Normal Migration of Endothelial and Fibrosarcoma Cells through Collagen I—To further investigate the functional role of α2β1integrin, we took a gene targeting approach using validated siRNA to knockdown the α2 subunit in HUVEC and HT1080, a cell line that depends on α2β1 as the main collagen I receptor (21Eble J.A. Beermann B. Hinz H.J. Schmidt-Hederich A. J. Biol. Chem. 2001; 276: 12274-12284Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). Immunoblotting of cell lysates revealed almost complete knockdown of the integrin expression in HUVEC (∼5% of control levels) when the cells were transfected with two siRNA constructs each at a concentration of 150 nm targeting different regions of the α2 mRNA (Fig. 3A). Similarly high levels of knockdown were also seen in HT1080 transfected at 50 and 100 nm concentrations (Fig. 3B). The results were confirmed by immunofluorescence analysis that showed an almost complete loss of the α2 signal in the transfected HUVEC and HT1080 as compared with controls (Fig. 3, C and D, respectively). Migration of HUVEC through collagen I was used as a test for functionality because this is a critical process required for angiogenesis in vivo (27Davis G.E. Senger D.R. Circ. Res. 2005; 97: 1093-1107Crossref PubMed Scopus (938) Google Scholar). Knockdown of the α2 subunit caused a profound inhibition of migration, especially at higher dosages (Fig. 3E). The inhibition of HUVEC migration in the cells deficient in α2β1 was equal to that observed by endorepellin treatment, suggesting that this receptor is crucial for migration of endothelial cells. This further stresses the importance of this integrin in the migratory ability of HUVECs in vitro. However, in vivo, the lack of α2β1 integrin (see below) did not delay the process of tumor angiogenesis, a phenomenon that has also been observed in mutant mice lacking other integrins (28Hynes R.O. Nat. Med. 2002; 8: 918-921Crossref PubMed Scopus (487) Google Scholar). Similarly, the HT1080 devoid of the α2β1 integrin showed a ∼75% inhibition of migration, and these levels of inhibition did not appreciably change by progressively increasing endorepellin concentration, in contrast to untreated controls (Fig. 3F). Collectively, these data point to a central role for the α2β1 integrin in regulating the migration of both HUVEC and HT1080 through collagen. Genetic Requirement of α2β1 for Endorepellin-mediated Angiostatic Activity—Next, we conducted in vitro angiogenic assays utilizing lung microvascular endothelial cells isolated from wild-type or mutant animals lacking either the α1 (25Pozzi A. Moberg P.E. Miles L.A. Wagner S. Soloway P. Gardner H.A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 2202-2207Crossref PubMed Scopus (349) Google Scholar) or the α2 (29Holtkötter O. Nieswandt B. Smyth N. Müller W. Hafner M. Schulte V. Krieg T. Eckes B. J. Biol. Chem. 2002; 277: 10789-10794Abstract Full Text Full Text PDF PubMed Scopus (236) Google Scholar) subunit and thus lacking functional α1β1 and α2β1integrin receptors. The genotype of the wild-type and the two genetically engineered mice is shown in Fig. 4A. Wild-type cells showed the highest levels of VEGF-driven migration through collagen I and displayed a complete inhibition of migration when preincubated with endorepellin (Fig. 4B). Under unchallenged experimental conditions, both α1β1-/- and the α2β1-/- endothelial cells showed a reduced migration as compared with wild-type cells (Fig. 4B). However, α2β1-/- endothelial cells showed a greater decline in migratory ability than the α1β1-/- cells (45% versus 30%). These data are in agreement with siRNA experiments shown above and suggest that the α2β1-/- integrin is a key receptor for VEGF-induced chemotactic migration, a main biological process in angiogenesis (27Davis G.E. Senger D.R. Circ. Res. 2005; 97: 1093-1107Crossref PubMed Scopus (938) Google Scholar, 30Senger D.R. Claffey K.P. Benes J.E. Perruzzi C.A. Sergiou A.P. Detmar M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 13612-13617Crossref PubMed Scopus (458) Google Scholar). Most importantly, only the wild-type and the α1β1-/- microvascular endothelial cells showed a complete inhibition of migration when preincubated with endorepellin, whereas the α2β1-/- endothelial cells were totally unresponsive (Fig. 4B). These data provide robust genetic evidence that the inhibition of migration caused by endorepellin requires the presence of the α2β1 integrin receptor. Next, we tested the wild-type and mutant endothelial cells in actin disassembly assays (14Bix G. Fu J. Gonzalez E. Macro L. Barker A. Campbell S. Zutter M.M. Santoro S.A. Kim J.K. Höök M. Reed C.C. Iozzo R.V. J. Cell Biol. 2004; 166: 97-109Crossref PubMed Scopus (232) Google Scholar). Although endothelial cells from wild-type and α1β1-/- mice responded well to endorepellin by showing rapid actin disassembly, the endothelial cells from α2β1-/- mice were totally unresponsive to endorepellin (Fig. 4C). Notably, the same drastic disassembly of stress fibers vis-à-vis their respective controls was elicited by recombinant LG3 (supplemental Fig. S2). These findings corroborate the migration data and further prove that the effects of endorepellin fail to occur in the absence of α2β1 integrin and are not mediated by the α1β1 integrin. Endorepellin Inhibits Tumor Growth and Angiogenesis and Localizes to the Tumor Vasculature Only in the Presence of the α2β1 Integrin Receptor—To prove that endorepellin in vivo activity is mediated by the α2β1 integrin, we utilized a syngeneic animal model in which LLC xenografts were generated in wild-type and α2β1-/- mice with an identical genetic (C57Bl/6) background. After the tumor xenografts became palpable, the animals were randomized and treated with intraperitoneal injections of recombinant endorepellin. Notably, the tumor xenografts growing in the α2β1-/- animals that were treated with endorepellin grew as well as those that were treated with vehicle alone (Fig. 5A). In contrast, the LLC xenografts growing in the wild-type background responded well to systemic endorepellin treatment (Fig. 5A and supplemental Fig. S3). In two additio

Collagen fiber realignment is one mechanism by which tendon responds to load. Re-alignment is altered when the structure of tendon is altered, such as in the natural process of aging or with alterations of matrix proteins, such as proteoglycan expression. While changes in re-alignment and mechanical properties have been investigated recently during development, they have not been studied in (1) aged tendons, or (2) in the absence of key proteoglycans. Collagen fiber re-alignment and the corresponding mechanical properties are quantified throughout tensile mechanical testing in both the insertion site and the midsubstance of mouse supraspinatus tendons in wild type (WT), decorin-null (Dcn(-/-)), and biglycan-null (Bgn(-/-)) mice at three different ages (90 days, 300 days, and 570 days). Percent relaxation was significantly decreased with age in the WT and Dcn(-/-) tendons, but not in the Bgn(-/-) tendons. Changes with age were found in the linear modulus at the insertion site where the 300 day group was greater than the 90 day and 570 day group in the Bgn(-/-) tendons and the 90 day group was smaller than the 300 day and 570 day groups in the Dcn(-/-) tendons. However, no changes in modulus were found across age in WT tendons were found. The midsubstance fibers of the WT and Bgn(-/-) tendons were initially less aligned with increasing age. The re-alignment was significantly altered with age in the WT tendons, with older groups responding to load later in the mechanical test. This was also seen in the Dcn(-/-) midsubstance and the Bgn(-/-) insertion, but not in the other locations. Although some studies have found changes in the WT mechanical properties with age, this study did not support those findings. However, it did show fiber re-alignment changes at both locations with age, suggesting a breakdown of tendon's ability to respond to load in later ages. In the proteoglycan-null tendons however, there were changes in the mechanical properties, accompanied only by location-dependent re-alignment changes, suggesting a site-specific role for these molecules in loading. Finally, changes in the mechanical properties did not occur in concert with changes in re-alignment, suggesting that typical mechanical property measurements alone are insufficient to describe how structural alterations affect tendon's response to load.

Endorepellin, the COOH-terminal domain of the heparan sulfate proteoglycan perlecan, inhibits several aspects of angiogenesis. We provide evidence for a novel biological axis that links a soluble fragment of perlecan protein core to the major cell surface receptor for collagen I, α2β1 integrin, and provide an initial investigation of the intracellular signaling events that lead to endorepellin antiangiogenic activity. The interaction between endorepellin and α2β1 integrin triggers a unique signaling pathway that causes an increase in the second messenger cAMP; activation of two proximal kinases, protein kinase A and focal adhesion kinase; transient activation of p38 mitogen-activated protein kinase and heat shock protein 27, followed by a rapid down-regulation of the latter two proteins; and ultimately disassembly of actin stress fibers and focal adhesions. The end result is a profound block of endothelial cell migration and angiogenesis. Because perlecan is present in both endothelial and smooth muscle cell basement membranes, proteolytic activity during the initial stages of angiogenesis could liberate antiangiogenic fragments from blood vessels' walls, including endorepellin.

The extracellular matrix (ECM) plays an active and dynamic role that both reflects and facilitates the functional requirements of a tissue. The mature ECM of the nonpregnant cervix is drastically reorganized during pregnancy to drive changes in tissue mechanics that ensure safe birth. In this study, our research on mice deficient in the proteoglycan decorin have led to the finding that progesterone and estrogen play distinct and complementary roles to orchestrate structural reorganization of both collagen and elastic fibers in the cervix during pregnancy. Abnormalities in collagen and elastic fiber structure and tissue mechanical function evident in the cervix of nonpregnant and early pregnant decorin-null mice transiently recover for the remainder of pregnancy only to return 1 month postpartum. Consistent with the hypothesis that pregnancy levels of progesterone and estrogen may regulate ECM organization and turnover, expressions of factors required for assembly and synthesis of collagen and elastic fibers are temporally regulated, and the ultrastructure of collagen fibrils and elastic fibers is markedly altered during pregnancy in wild-type mice. Finally, utilizing ovariectomized nonpregnant decorin-null mice, we demonstrate structural resolution of collagen and elastic fibers by progesterone or estrogen, respectively, and the potential for both ECM proteins to contribute to mechanical function. These investigations advance understanding of regulatory factors that drive specialized ECM organization and contribute to an understanding of the cervical remodeling process, which may provide insight into potential complications associated with preterm birth that impact 9.6% of live births in the United States.

Angiogenesis is a crucial process occurring under physiological and pathological conditions, including cancer. The development of blood vessels is tightly regulated by a plethora of cytokines, endothelial cell (EC) receptors and extracellular matrix (ECM) components. In this context, we have shown that Multimerin 2 (MMRN2), an ECM molecule specifically secreted by ECs, exerts angiostatic functions by binding VEGFA and other pro-angiogenic cytokines. Here, we demonstrate that during angiogenic stimuli MMRN2 mRNA levels significantly decrease. Furthermore, we provide evidence that MMRN2 is processed by matrix metalloproteinases (MMPs) including MMP-9 and, to a lesser degree, by MMP-2. This proteolytic cleavage correlates with an increased migration of ECs. Accordingly, MMRN2 down-regulation is associated with an increased number of EC pseudopodia at the migrating front and this effect is attenuated using specific MMP-9 inhibitors. The down-modulation of MMRN2 occurs also in the context of tumor-associated angiogenesis. Immunofluorescence performed on tumor sections indicate a broad co-localization of MMP-9 and MMRN2, suggesting that the molecule may be extensively remodeled during tumor angiogenesis. Given the altered expression in tumors and the key role of MMRN2 in blood vessel function, we postulate that analyses of its expression may serve as a marker to predict the efficacy of the treatments. In conclusion, these data further support the role of MMRN2 as a key molecule regulating EC function and sprouting angiogenesis.

The extracellular matrix is rapidly emerging as a prominent contributor to various fundamental processes of tumorigenesis. In particular, decorin, a member of the small leucine-rich proteoglycan gene family, is assuming a central role as a potent soluble tumor repressor. Decorin binds and antagonizes various receptor tyrosine kinases and inhibits downstream oncogenic signaling in several solid tumors. Among other functions, decorin evokes cell cycle arrest, apoptosis, and antimetastatic, and antiangiogenic programs. Recent work has revealed a paradigmatic shift in our understanding of the molecular mechanisms underlying its tumoricidal properties. Decorin adversely compromises the genetic signature of the tumor microenvironment and induces endothelial cell autophagy downstream of VEGFR2. Moreover, decorin selectively evokes destruction of tumor cell mitochondria downstream of Met through mitophagy. Acting as a partial agonist, decorin signals via proautophagic receptors and triggers procatabolic processes that parallel the classical tumoricidal properties of this multifaceted proteoglycan.

The glycoprotein CD93 has recently been recognized to play an important role in the regulation of the angiogenic process. Moreover, CD93 is highly expressed in the endothelial cells of tumor blood vessel and faintly expressed in the non-proliferating endothelium. Much evidence suggests that CD93 mediates adhesion in the endothelium. Here we identify Multimerin 2 (MMRN2), a pan-endothelial extracellular matrix protein, as a specific ligand for CD93. We found that CD93 and MMRN2 are co-expressed in the blood vessels of various human tumors. Moreover, disruption of the CD93-MMRN2 interaction reduced endothelial cell adhesion and migration, making the interaction of CD93 with MMRN2 an ideal target to block pathological angiogenesis. Model structures and docking studies served to envisage the region of CD93 and MMRN2 involved in the interaction. Site-directed mutagenesis identified different residue hotspots either directly or indirectly involved in the binding. We propose a molecular model in which the coiled-coil domain of MMRN2 is engaged by F238 of CD93. Altogether, these studies identify the key interaction surfaces of the CD93-MMRN2 complex and provide a framework for exploring how to inhibit angiogenesis by hindering the CD93-MMRN2 interaction.

We previously discovered that systemic delivery of decorin for treatment of breast carcinoma xenografts induces paternally expressed gene 3 (Peg3), an imprinted gene encoding a zinc finger transcription factor postulated to function as a tumor suppressor. Here we found that de novo expression of Peg3 increased Beclin 1 promoter activity and protein expression. This process required the full-length Peg3 as truncated mutants lacking either the N-terminal SCAN domain or the zinc fingers failed to translocate to the nucleus and promote Beclin 1 transcription. Importantly, overexpression of Peg3 in endothelial cells stimulated autophagy and concurrently inhibited endothelial cell migration and evasion from a 3D matrix. Mechanistically, we found that Peg3 induced the secretion of the powerful angiostatic glycoprotein Thrombospondin 1 independently of Beclin 1 transcriptional induction. Thus, we provide a new mechanism whereby Peg3 can simultaneously evoke autophagy in endothelial cells and attenuate angiogenesis. We previously discovered that systemic delivery of decorin for treatment of breast carcinoma xenografts induces paternally expressed gene 3 (Peg3), an imprinted gene encoding a zinc finger transcription factor postulated to function as a tumor suppressor. Here we found that de novo expression of Peg3 increased Beclin 1 promoter activity and protein expression. This process required the full-length Peg3 as truncated mutants lacking either the N-terminal SCAN domain or the zinc fingers failed to translocate to the nucleus and promote Beclin 1 transcription. Importantly, overexpression of Peg3 in endothelial cells stimulated autophagy and concurrently inhibited endothelial cell migration and evasion from a 3D matrix. Mechanistically, we found that Peg3 induced the secretion of the powerful angiostatic glycoprotein Thrombospondin 1 independently of Beclin 1 transcriptional induction. Thus, we provide a new mechanism whereby Peg3 can simultaneously evoke autophagy in endothelial cells and attenuate angiogenesis.

The insulin-like growth factor-I receptor (IGF-IR), plays a key role in regulating mammalian development and growth, and is frequently deregulated in cancer contributing to tumor initiation and progression. Discoidin domain receptor 1 (DDR1), a collagen receptor tyrosine-kinase, is as well frequently overexpressed in cancer and implicated in cancer progression. Thus, we investigated whether a functional cross-talk between the IGF-IR and DDR1 exists and plays any role in cancer progression.Using human breast cancer cells we found that DDR1 constitutively associated with the IGF-IR. However, this interaction was enhanced by IGF-I stimulation, which promoted rapid DDR1 tyrosine-phosphorylation and co-internalization with the IGF-IR. Significantly, DDR1 was critical for IGF-IR endocytosis and trafficking into early endosomes, IGF-IR protein expression and IGF-I intracellular signaling and biological effects, including cell proliferation, migration and colony formation. These biological responses were inhibited by DDR1 silencing and enhanced by DDR1 overexpression.Experiments in mouse fibroblasts co-transfected with the human IGF-IR and DDR1 gave similar results and indicated that, in the absence of IGF-IR, collagen-dependent phosphorylation of DDR1 is impaired.These results demonstrate a critical role of DDR1 in the regulation of IGF-IR action, and identify DDR1 as a novel important target for breast cancers that overexpress IGF-IR.

Serglycin is an intracellular proteoglycan that is expressed and constitutively secreted by numerous malignant cells, especially prominent in the highly-invasive, triple-negative MDA-MB-231 breast carcinoma cells. Notably, de novo expression of serglycin in low aggressive estrogen receptor α (ERα)-positive MCF7 breast cancer cells promotes an aggressive phenotype. In this study, we discovered that serglycin promoted epithelial to mesenchymal transition (EMT) in MCF7 cells as shown by increased expression of mesenchymal markers vimentin, fibronectin and EMT-related transcription factor Snail2. These phenotypic traits were also associated with the development of drug resistance toward various chemotherapy agents and induction of their proteolytic potential as shown by the increased expression of matrix metalloproteinases, including MMP-1, MMP-2, MMP-9, MT1-MMP and up-regulation of urokinase-type plasminogen activator. Knockdown of serglycin markedly reduced the expression of these proteolytic enzymes in MDA-MB-231 cells. In addition, serglycin expression was closely linked to a pro-inflammatory gene signature including the chemokine IL-8 in ERα-negative breast cancer cells and tumors. Notably, serglycin regulated the secretion of IL-8 in breast cancer cells independently of their ERα status and promoted their proliferation, migration and invasion by triggering IL-8/CXCR2 downstream signaling cascades including PI3K, Src and Rac activation. Thus, serglycin promotes the establishment of a pro-inflammatory milieu in breast cancer cells that evokes an invasive mesenchymal phenotype via autocrine activation of IL-8/CXCR2 signaling axis.

The complex and adaptive nature of malignant neoplasm constitute a major challenge for the development of effective anti-oncogenic therapies. Emerging evidence has uncovered the pivotal functions exerted by the small leucine-rich proteoglycans, decorin and biglycan, in affecting tumor growth and progression. In their soluble forms, decorin and biglycan act as powerful signaling molecules. By receptor-mediated signal transduction, both proteoglycans modulate key processes vital for tumor initiation and progression, such as autophagy, inflammation, cell-cycle, apoptosis, and angiogenesis. Despite of their structural homology, these two proteoglycans interact with distinct cell surface receptors and thus modulate distinct signaling pathways that ultimately affect cancer development. In this review, we summarize growing evidence for the complex roles of decorin and biglycan signaling in tumor biology and address potential novel therapeutic implications.

In cartilage tissue engineering, one key challenge is for regenerative tissue to recapitulate the biomechanical functions of native cartilage while maintaining normal mechanosensitive activities of chondrocytes. Thus, it is imperative to discern the micromechanobiological functions of the pericellular matrix, the ~ 2-4 µm-thick domain that is in immediate contact with chondrocytes. In this study, we discovered that decorin, a small leucine-rich proteoglycan, is a key determinant of cartilage pericellular matrix micromechanics and chondrocyte mechanotransduction in vivo. The pericellular matrix of decorin-null murine cartilage developed reduced content of aggrecan, the major chondroitin sulfate proteoglycan of cartilage and a mild increase in collagen II fibril diameter vis-à-vis wild-type controls. As a result, decorin-null pericellular matrix showed a significant reduction in micromodulus, which became progressively more pronounced with maturation. In alignment with the defects of pericellular matrix, decorin-null chondrocytes exhibited decreased intracellular calcium activities, [Ca2+]i, in both physiologic and osmotically evoked fluidic environments in situ, illustrating impaired chondrocyte mechanotransduction. Next, we compared [Ca2+]i activities of wild-type and decorin-null chondrocytes following enzymatic removal of chondroitin sulfate glycosaminoglycans. The results showed that decorin mediates chondrocyte mechanotransduction primarily through regulating the integrity of aggrecan network, and thus, aggrecan-endowed negative charge microenvironment in the pericellular matrix. Collectively, our results provide robust genetic and biomechanical evidence that decorin is an essential constituent of the native cartilage matrix, and suggest that modulating decorin activities could improve cartilage regeneration.

Decorin is a stromal-derived prototype member of the small leucine-rich proteoglycan gene family. In addition to its functions as a regulator of collagen fibrillogenesis and TGF-β activity soluble decorin acts as a pan-receptor tyrosine kinase (RTK) inhibitor. Decorin binds to various RTKs including EGFR HER2 HGFR/Met VEGFR2 TLR and IGFR. Although the molecular mechanism for the action of decorin on these receptors is not entirely elucidated overall decorin evokes transient activation of these receptors with suppression of downstream signaling cascades culminating in growth inhibition followed by their physical downregulation via caveosomal internalization and degradation. In the case of Met decorin leads to decreased β-catenin signaling pathway and growth suppression. As most of these RTKs are responsible for providing a growth advantage to cancer cells the result of decorin treatment is oncosuppression. Another decorin-driven mechanism to restrict cancer growth and dissemination is by impeding angiogenesis via vascular endothelial growth factor receptor 2 (VEGFR2) and the concurrent activation of protracted endothelial cell autophagy. In this review we will dissect the multiple roles of decorin in cancer biology and its potential use as a next-generation protein-based adjuvant therapy to combat cancer.

Decorin is a small leucine-rich proteoglycan that interacts with several matrix molecules, including various types of collagen and growth factors, and suppresses the growth of neoplastic cells by an epidermal growth factor (EGF) receptor-mediated pathway. Decorin is abundantly expressed in the periodontal connective tissues during development and tissue maintenance. In periodontal disease, which is one of the most common diseases in the human kind, the level of decorin is decreased in the periodontal connective tissue. Abnormal expression of decorin may also associate with certain inherited disorders that involve increased susceptibility to severe periodontal disease in the early childhood. Therefore, we investigated the periodontal tissues of mice with targeted disruption of the decorin gene. Gross and microscopic analyses showed that decorin-deficient mice appeared to have normal tooth development and eruption, and there were no signs of periodontal disease. However, electron microscopic analysis revealed abnormal morphology and organization of the collagen fibrils in the periodontal ligament. The number of periodontal ligament fibroblasts in the decorin-deficient mice was also increased about two-fold as compared with the wild-type mice. In cell culture, ectopic overexpression of decorin in NIH 3T3 fibroblasts or decorin added exogenously to periodontal fibroblasts suppressed cell growth. However, blocking the EGF receptor tyrosine kinase activity did not prevent the decorin-elicited growth suppression in periodontal fibroblasts. Additionally, decorin did not induce a marked increase in the relative expression of p21 mRNA in periodontal fibroblasts. Therefore, decorin appeared to regulate growth of normal periodontal fibroblasts by a mechanism distinct from that reported for neoplastic cells. The findings demonstrate that decorin plays a role in the organization of collagen fibrils and regulates cell proliferation in the periodontal ligament.

The mechanism by which alcohol injures the pancreas remains unknown. Recent investigations suggest a role for fatty acid ethyl ester (FAEE), a nonoxidative metabolite of ethanol, in the pathogenesis of alcohol pancreatitis. In this study, we characterized ethanol-induced injury in rats and evaluated the contribution of oxidative and nonoxidative ethanol metabolites in this form of acute pancreatitis. Pancreatic injury in rats was assessed by edema, intrapancreatic trypsinogen activation, and microscopy after infusing ethanol with or without inhibitors of oxidative ethanol metabolism. Plasma and tissue levels of FAEE and ethanol were measured and correlated with pancreatic injury. Ethanol infusion generated plasma and tissue FAEE and, in a dose-dependent fashion, induced a pancreas-specific injury consisting of edema, trypsinogen activation, and formation of vacuoles in the pancreatic acini. Inhibition of the oxidation of ethanol significantly increased both FAEE concentration in plasma and pancreas and worsened the pancreatitis-like injury. This study provides direct evidence that ethanol, through its nonoxidative metabolic pathway, can produce pancreas-specific toxicity in vivo and suggests that FAEE are responsible for the development of early pancreatic cell damage in acute alcohol-induced pancreatitis.

Heparan sulfate proteoglycans (HSPG) play a critical role in the formation of distinct fibroblast growth factor (FGF)-HS complexes, augmenting high-affinity binding and receptor activation. Perlecan, a secreted HSPG abundant in proliferating cells, is capable of inducing FGF-receptor interactions in vitro and angiogenesis in vivo. Stable and specific reduction of perlecan levels in mouse NIH 3T3 fibroblasts and human metastatic melanoma cells has been achieved by expression of antisense cDNA corresponding to the N-terminal and HS attachment domains of perlecan. Long-term perlecan downregulation is evidenced by reduced levels of perlecan mRNA and core protein as indicated by Northern blot analysis, immunoblots, and immunohistochemistry, using DNA probes and antibodies specific to mouse or human perlecan. The response of antisense perlecan-expressing cells to increasing concentrations of basic FGF (bFGF) is dramatically reduced in comparison to that in wild-type or vector-transfected cells, as measured by thymidine incorporation and rate of proliferation. Furthermore, receptor binding and affinity labeling of antisense perlecan-transfected cells with 125I-bFGF is markedly inhibited, indicating that eliminating perlecan expression results in reduced high-affinity bFGF binding. Both the binding and mitogenic response of antisense-perlecan-expressing clones to bFGF can be rescued by exogenous heparin or perlecan. These results support the notion that perlecan is a major accessory receptor for bFGF in mouse fibroblasts and human melanomas and point to the possible use of perlecan antisense constructs as specific modulators of bFGF-mediated responses.

Biosynthetic and proliferative characteristics of tubulointerstitial fibroblasts probed with paracrine cytokines.Fibroblasts in parenchymal organs potentially contribute extracellular matrix to local fibrogenic processes.This contribution, in some circumstances, may be initiated by cytokines disseminated from inflammatory lesions.Different populations of fibrobiasts, however, might respond distinctively to this cytokine bath depending on the microenvironment in which they reside.We have begun to explore this issue using syngeneic, low-passage fibroblasts cultured in serum-free media that were derived originally from the dermis (DFBs) and from tubulointerstitium (TFB5) of the kidney.Our findings indicate that, while fibroblasts from each compartment appear similar at the ultrastructural level, there are a variety of functional differences which distinguish their proliferative response, and their collagen secretory response (types I, III, IV, and V) following challenge with various doses of immune-relevant cytokines (TGF/3, EGF, IL-i, IL-2 and yIFN) in culture.DFB5, for example, express more surface EGF receptors than do TFBs, and, as a consequence, exhibit a more robust proliferative response to EGF in serum-free media.Unstimulated DFBs also secrete more collagen types I and III than TFBs, while unstimulated TFBs secrete more types IV and V.The expression of these collagens in TFBs was confirmed by Northern blot hybridization.When these sets of fibroblasts were further stimulated by cytokines, some of the cytokines not only differentially effect the secretion of various species of collagens within the same group of cells, but also between cells from populations which are anatomically distinct.DFBs, furthermore, at mid-level doses of cytokine, demonstrated a general trend towards less secretion of all types of collagen (particularly for TGFf3, EGF, and IL-2), while TFBs seemed less repressive.In TFBs the cytokine-induced responses for collagen types I and III tended to be discordant, and for types I and IV EGF inhibited, while TGFI3 stimulated the secretory process.These findings speak collectively for the presence of a functional heterogeneity among organ-based populations of syngeneic fibroblasts in normal tissues.Fibrosis in parenchymal organs is the pernicious end-product of progressive deposition of extracellular matrix remodelling normal somatic architecture [1-31.Such damage is typically preceded by inflammatory events which activate a variety of tissue resident cells including fibroblasts [4, 51.The fibrogenesis of organs, with its attendant distortion of normal structures, is also frequently the late consequence of autoimmune injury

Because the small leucine-rich proteoglycan decorin has been implicated in regulation of collagen fibrillogenesis leading to proper extracellular matrix assembly, we hypothesized it could play a key role in cardiac fibrosis following myocardial infarction. In this study we ligated the left anterior descending coronary artery in wildtype and decorin-null mice to produce large infarcts in the anterior wall of the left ventricle. At early stages post-coronary occlusion the myocardial infarction size did not appreciably differ between the two genotypes. However, we found a wider distribution of collagen fibril sizes with less organization and loose packing in mature scar from decorin-null mice. Thus, we tested the hypothesis that these abnormal collagen fibrils would adversely affect post-infarction mechanics and ventricular remodeling. Indeed, scar size, right ventricular remote hypertrophy, and left ventricular dilatation were greater in decorin-null animals compared with wildtype littermates 14 days after acute myocardial infarction. Echocardiography revealed depressed left ventricular systolic function between 4 and 8 weeks post-ischemia in the decorin-null animals. These changes indicate that decorin is required for the proper fibrotic evolution of myocardial infarctions, and that its absence leads to abnormal scar tissue formation. This might contribute to aneurysmal ventricular dilatation, remote hypertrophy, and depressed ventricular function.

The Wnt-5A gene codes for a secreted cysteine-rich growth factor that mediates cell to cell signaling via a paracrine mechanism during development and ontogeny. We have recently determine the genomic organization and chromosomal mapping of the human Wnt-5A, and observed distinct patterns of expression in developing human embryos. In this report, we have performed a detailed expression analysis of 100 adult human tissues and tumors and 10 human cell lines. Our data show a widespread expression of Wnt-5A in adult tissues and cells, and aberrant mRNA levels in lungs, breast, and prostate carcinomas and in melanomas. The up-regulation of Wnt-5A in human malignancy was not due to either gene rearrangement or amplification. These findings document an abnormal expression of this growth factor in malignancy and implicate Wnt-5A in the genesis of human cancer.

Decorin is a well-known, ubiquitous proteoglycan that is a normal component of the ECM. Upon transgenic expression of decorin, tumor cells with diverse histogenetic background overexpress p21WAF1, a potent inhibitor of cyclin-dependent kinase activity, become arrested in G1, and fail to generate tumors in immunocompromised animals. Because decorin is a secreted protein, it has been recently suggested that decorin could act as an autocrine and paracrine regulator of tumor growth. Here, we demonstrate that adenovirus (Ad)-mediated transfer and expression of human decorin cDNA induced in vivo apoptosis of xenograft tumor cells in nude mice. This oncolytic activity was observed when the Ad vector encoding the decorin cDNA was injected intratumorally (i.t.) or i.v. Importantly, i.t. injection of the decorin Ad vector led to growth inhibition of the injected tumor associated with similar growth inhibition of a distant contralateral tumor, demonstrating a distant decorin antitumoral effect. Immunochemistry against human decorin and decorin quantitation in tumors confirmed that decorin migrated to the tumor distant site. Furthermore, decorin effect was specific to tumor cells, because neither apoptosis nor growth inhibition were observed in nontumoral human cells such as hepatocytes, endothelial cells, and fibroblasts, despite p21 overexpression.

The metabolism of heparan sulfate proteoglycan, a major product of human colon carcinoma cells, was investigated in a series of pulse-chase experiments using a combination of quantitative biochemistry and electron microscope autoradiography. This was possible primarily because these cells incorporate [35S]sulfate exclusively into heparan sulfate proteoglycan, thus allowing the possibility of correlating the two sets of information. The results showed a progressive movement of the newly synthesized proteoglycan from the Golgi to the cell surface, where it became closely associated with the plasma membrane and was labeled ultrastructurally by both ruthenium red and radiosulfate. Subsequently, about 55% was released into the medium (t1/2 approximately 2.5 h) where it resided as intact macromolecule and was neither endocytosed nor degraded further. The remaining 45% was internalized and converted into smaller species through a series of degradative steps. Initially (Step 1) there was proteolytic cleavage of the protein core and partial endoglycosidic cleavage of the heparan sulfate chains (t1/2 approximately 6 h), with generation of larger glycosaminoglycan-peptide intermediates with chains of Mr approximately 10,000, about one-third their original size. These components were subsequently converted (Step 2) to yet smaller, limiting fragments of Mr approximately 5,000, which were finally depolymerized (Step 3) with quantitative release of free sulfate. The intracellular degradation of the proteoglycan, particularly Steps 2 and 3, was markedly inhibited by choloroquine, implicating the involvement of acidic compartments in the catabolism of these macromolecules. This was corroborated by the autoradiographic studies which showed the close association of 35S-labeled products with secondary lysosomes. However, the initial degradation of the proteoglycan might have occurred in a prelysosomal compartment since Step 1 was not totally blocked by chloroquine. The combined results indicate that the intracellular degradation of heparan sulfate follows structural as well as functional compartmentalization and provide a model that may be shared by other cell systems.

We recently established a critical role for the growth factor progranulin in bladder cancer insofar as progranulin promotes urothelial cancer cell motility and contributes, as an autocrine growth factor, to the transformed phenotype by modulating invasion and anchorage-independent growth.In addition, progranulin expression is upregulated in invasive bladder cancer tissues compared to normal controls.However, the molecular mechanisms of progranulin action in bladder cancer have not been fully elucidated.In this study, we searched for novel progranulininteracting proteins using pull-down assays with recombinant progranulin and proteomics.We discovered that drebrin, an F-actin binding protein, bound progranulin in urothelial cancer cells.We characterized drebrin function in urothelial cancer cell lines and showed that drebrin is critical for progranulin-dependent activation of the Akt and MAPK pathways and modulates motility, invasion and anchorage-independent growth.In addition, drebrin regulates tumor formation in vivo and its expression is upregulated in bladder cancer tissues compared to normal tissue controls.Our data are translationally relevant as indicate that drebrin exerts an essential functional role in the regulation of progranulin action and may constitute a novel target for therapeutic intervention in bladder tumors.In addition, drebrin may serve as novel biomarker for bladder cancer.

Macroautophagy is a fundamental and evolutionarily conserved catabolic process that eradicates damaged and aging macromolecules and organelles in eukaryotic cells. Decorin, an archetypical small leucine-rich proteoglycan, initiates a protracted autophagic program downstream of VEGF receptor 2 (VEGFR2) signaling that requires paternally expressed gene 3 (PEG3). We have discovered that PEG3 is an upstream transcriptional regulator of transcription factor EB (TFEB), a master transcription factor of lysosomal biogenesis, for decorin-evoked endothelial cell autophagy. We found a functional requirement of PEG3 for TFEB transcriptional induction and nuclear translocation in human umbilical vein endothelial and PAER2 cells. Mechanistically, inhibiting VEGFR2 or AMP-activated protein kinase (AMPK), a major decorin-activated energy sensor kinase, prevented decorin-evoked TFEB induction and nuclear localization. In conclusion, our findings indicate a non-canonical (nutrient- and energy-independent) mechanism underlying the pro-autophagic bioactivity of decorin via PEG3 and TFEB. Macroautophagy is a fundamental and evolutionarily conserved catabolic process that eradicates damaged and aging macromolecules and organelles in eukaryotic cells. Decorin, an archetypical small leucine-rich proteoglycan, initiates a protracted autophagic program downstream of VEGF receptor 2 (VEGFR2) signaling that requires paternally expressed gene 3 (PEG3). We have discovered that PEG3 is an upstream transcriptional regulator of transcription factor EB (TFEB), a master transcription factor of lysosomal biogenesis, for decorin-evoked endothelial cell autophagy. We found a functional requirement of PEG3 for TFEB transcriptional induction and nuclear translocation in human umbilical vein endothelial and PAER2 cells. Mechanistically, inhibiting VEGFR2 or AMP-activated protein kinase (AMPK), a major decorin-activated energy sensor kinase, prevented decorin-evoked TFEB induction and nuclear localization. In conclusion, our findings indicate a non-canonical (nutrient- and energy-independent) mechanism underlying the pro-autophagic bioactivity of decorin via PEG3 and TFEB.

Objective To elucidate the role of decorin, a small leucine‐rich proteoglycan, in the degradation of cartilage matrix during the progression of post‐traumatic osteoarthritis ( OA ). Methods Three‐month–old decorin‐null (Dcn −/− ) and inducible decorin‐knockout (Dcn i KO ) mice were subjected to surgical destabilization of the medial meniscus ( DMM ) to induce post‐traumatic OA . The OA phenotype that resulted was evaluated by assessing joint morphology and sulfated glycosaminoglycan ( sGAG ) staining via histological analysis (n = 6 mice per group), surface collagen fibril nanostructure via scanning electron microscopy (n = 4 mice per group), tissue modulus via atomic force microscopy–nanoindentation (n = 5 or more mice per group) and subchondral bone structure via micro–computed tomography (n = 5 mice per group). Femoral head cartilage explants from wild‐type and Dcn −/− mice were stimulated with the inflammatory cytokine interleukin‐1β ( IL ‐1β) in vitro (n = 6 mice per group). The resulting chondrocyte response to IL ‐1β and release of sGAG s were quantified. Results In both Dcn −/− and Dcn i KO mice, the absence of decorin resulted in accelerated sGAG loss and formation of highly aligned collagen fibrils on the cartilage surface relative to the control ( P &lt; 0.05). Also, Dcn −/− mice developed more salient osteophytes, illustrating more severe OA . In cartilage explants treated with IL ‐1β, loss of decorin did not alter the expression of either anabolic or catabolic genes. However, a greater proportion of sGAG s was released to the media from Dcn −/− mouse explants, in both live and devitalized conditions ( P &lt; 0.05). Conclusion In post‐traumatic OA , decorin delays the loss of fragmented aggrecan and fibrillation of cartilage surface, and thus, plays a protective role in ameliorating cartilage degeneration.

The need for more effective cancer therapies is omnipresent as the ever-complex, and highly adaptive, mechanisms of tumor biology allow this disease to elude even the most stringent treatment options. The expanding field of proteoglycan signaling is enticing as a reservoir of potential drug targets and prospects for novel therapeutic strategies. The newest trend in proteoglycan biology is the interplay between extracellular signaling and autophagy fueled by the close link between autophagy and angiogenesis. Here we summarize the most current evidence surrounding proteoglycan signaling in both of these biological processes featuring the well-known suspects, decorin and perlecan, as well as other up-and-coming neophytes in this evolving signaling web.

Abstract The extracellular matrix is a network of secreted macromolecules that provides a harmonious meshwork for the growth and homeostatic development of organisms. It conveys multiple signaling cascades affecting specific surface receptors that impact cell behavior. During cancer growth, this bioactive meshwork is remodeled and enriched in newly formed blood vessels, which provide nutrients and oxygen to the growing tumor cells. Remodeling of the tumor microenvironment leads to the formation of bioactive fragments that may have a distinct function from their parent molecules, and the balance among these factors directly influence cell viability and metastatic progression. Indeed, the matrix acts as a gatekeeper by regulating the access of cancer cells to nutrients. Here, we will critically evaluate the role of selected matrix constituents in regulating tumor angiogenesis and provide up-to-date information concerning their primary mechanisms of action.

Multimerin-2 is an extracellular matrix glycoprotein and member of the elastin microfibril interface-located (EMILIN) family of proteins. Multimerin-2 is deposited along blood vessels and we previously demonstrated that it regulates the VEGFA/VEGFR2 signaling axis and angiogenesis. However, its role in modulating vascular homeostasis remains largely unexplored. Here we identified Multimerin-2 as a key molecule required to maintain vascular stability. RNAi knockdown of Multimerin-2 in endothelial cells led to cell-cell junctional instability and increased permeability. Mechanistically cell-cell junction dismantlement occurred through the phosphorylation of VEGFR2 at Tyr951, activation of Src and phosphorylation of VE-cadherin. To provide an in vivo validation for these in vitro effects, we generated Multimerin-2−/− (Mmrn2−/−) mice. Although Mmrn2−/− mice developed normally and displayed no gross abnormalities, endothelial cells displayed cell junctional defects associated with increased levels of VEGFR2 phospho-Tyr949 (the murine counterpart of human Tyr951), impaired pericyte recruitment and increased vascular leakage. Of note, tumor associated vessels were defective in Mmrn2−/− mice, with increased number of small and often collapsed vessels, concurrent with a significant depletion of pericytic coverage. Consequently, the Mmrn2−/− vessels were less perfused and leakier, leading to increased tumor hypoxia. Chemotherapy efficacy was markedly impaired in Mmrn2−/− mice and this was associated with poor drug delivery to the tumor xenografts. Collectively, our findings demonstrate that Multimerin-2 is required for proper vessel homeostasis and stabilization, and unveil the possibility to utilize expression levels of this glycoprotein in predicting chemotherapy efficacy.

The cervix undergoes rapid and dramatic shifts in collagen and elastic fiber structure to achieve its disparate physiological roles of competence during pregnancy and compliance during birth. An understanding of the structure-function relationships of collagen and elastic fibers to maintain extracellular matrix (ECM) homeostasis requires an understanding of the mechanisms executed by non-structural ECM molecules. Small-leucine rich proteoglycans (SLRPs) play key functions in biology by affecting collagen fibrillogenesis and regulating enzyme and growth factor bioactivities. In the current study, we evaluated collagen and elastic fiber structure-function relationships in mouse cervices using mice with genetic ablation of decorin and/or biglycan genes as representative of Class I SLRPs, and lumican gene representative of Class II SLRP. We identified structural defects in collagen fibril and elastic fiber organization in nonpregnant mice lacking decorin, or biglycan or lumican with variable resolution of defects noted during pregnancy. The severity of collagen and elastic fiber defects was greater in nonpregnant mice lacking both decorin and biglycan and defects were maintained throughout pregnancy. Loss of biglycan alone reduced tissue extensibility in nonpregnant mice while loss of both decorin and biglycan manifested in decreased rupture stretch in late pregnancy. Collagen cross-link density was similar in the Class I SLRP null mice as compared to wild-type nonpregnant and pregnant controls. A broader range in collagen fibril diameter along with an increase in mean fibril spacing was observed in the mutant mice compared to wild-type controls. Collectively, these findings uncover functional redundancy and hierarchical roles of Class I and Class II SLRPs as key regulators of cervical ECM remodeling in pregnancy. These results expand our understating of the critical role SLRPs play to maintain ECM homeostasis in the cervix.

A characteristic rigid spatial arrangement of collagen fibrils in the stroma is critical for corneal transparency. This unique organization of collagen fibrils in corneal stroma can be impacted by the presence and interactions of proteoglycans and extracellular matrix (ECM) proteins in a corneal microenvironment. Earlier studies revealed that decorin, a leucine-rich proteoglycan in stroma, regulates keratocyte-collagen matrix assembly and wound healing in the cornea. This study investigated the role of decorin in the regulation of stromal fibrillogenesis and corneal transparency in vivo employing a loss-of-function genetic approach using decorin null (dcn−/−) and wild type (dcn+/+) mice and a standard alkali-injury model. A time-dependent ocular examinations with Slit lamp microscope in live animals assessed corneal clarity, haze, and neovascularization levels in normal and injured eyes. Morphometric changes in normal and injured dcn+/+ and dcn−/− corneas, post-euthanasia, were analyzed with Masson's Trichrome and Periodic Acid-Schiff (PAS) histology evaluations. The ultrastructure changes in all corneas were investigated with transmission electron microscopy (TEM). Injury to eye produced clinically relevant corneal haze and neovascularization in dcn−/− and dcn+/+ mice while corneas of uninjured eyes remained clear and avascular. A clinically significant haze and neovascularization appeared in injured dcn−/- corneas compared to the dcn+/+ corneas at day 21 post-injury and not at early tested times. Histological examinations revealed noticeably abnormal morphology and compromised collagen levels in injured dcn−/− corneas compared to the injured/normal dcn+/+ and uninjured dcn−/− corneas. TEM analysis exhibited remarkably uneven collagen fibrils size and distribution in the stroma with asymmetrical organization and loose packing in injured dcn−/− corneas than injured/normal dcn+/+ and uninjured dcn−/− corneas. The minimum and maximum inter-fibril distances were markedly irregular in injured dcn−/− corneas compared to all other corneas. Together, results of clinical, histological, and ultrastructural investigations in a genetic knockout model suggested that decorin influenced stromal fibrillogenesis and transparency in healing cornea.

The term heparan sulfate proteoglycan (HSPG) refers to a dichotomous structure, a protein coupled to a unique glycosaminoglycan chain characterized by a linear array of alternating disaccharide units. Both the protein core and the side chains undergo a series of modification reactions, which endow them with a very complex fabric (1). The final product is a sophisticated compound that can fluctuate in size, fine structure, and biology (2–5). The complexity of a usual heparan sulfate chain, for instance, can easily surpass that of common nucleic acids (6, 7). The field of proteoglycan research is nearly equally split into two factions: those who feel that heparan sulfate is the prima donna, with the protein core being just a carrier, and those who feel that there would be no role for heparan sulfate without the protein core. The truth, as usual, is in the middle. Both possess unique features that in some respects act synergistically to provide the maximal strength of signaling and biological activity. This Perspective series, motivated by the recent flurry of discoveries, has been coordinated to provide a balanced view of the most recent advances in the field of HSPG research. The development of novel experimental strategies for decoding the sequence of heparan sulfate (8–10), together with the availability of antibodies specific for heparan sulfate epitopes (11), holds promise for deciphering the functional specificity of heparan sulfate in tissues. The medical importance is underscored by the direct involvement of HSPGs in various human diseases involving the musculoskeletal system, such as the Schwartz-Jampel chondrodystrophic myotonia (12) and the dyssegmental dysplasia Silverman-Handmaker type (13), overgrowth syndrome (Simpson-Golabi-Behmel) (14), or hereditary bone disorders (hereditary multiple exostoses) (15).

Decorin is a leucine-rich, chondroitin/dermatan sulfate proteoglycan which binds collagen and growth factors. We have recently completed the genomic organization of human decorin and discovered two alternatively spliced leader exons, designated exon Ia and Ib, in the 5'-untranslated region. Initial analysis of the sequences upstream to these two exons showed that promoter Ia contained only two GC boxes while promoter Ib contained a CAAT and two TATA boxes in close proximity to the transcription start site. To determine if these 5'-flanking sequences exhibited promoter activity, chimeric chloramphenicol acetyltransferase expression plasmids containing the promoter region of either exon Ia or Ib were transfected into HeLa and MG-63 osteosarcoma cells. The results showed that only the region flanking exon Ib was functional. In vitro transcription assay generated two transcripts of 92 and 82 base pairs (bp) indicating that both TATA boxes could be used. Using stepwise 5' deletion analysis we found that the minimum promoter region at -140 bp from the transcription start site, which contained only the CAAT and the two TATA boxes, exhibited strong promoter activity. When a larger construct containing an additional 800 bp of upstream region was tested, a significant increase in transcriptional activity was observed. Interestingly, this promoter region contained several putative binding sites for ubiquitous factors (AP1, AP5, and NF-kappa B) and for transforming growth factor-beta and a 150-bp homopurine/homopyrimidine element with several mirror repeats. When contained in a supercoiled plasmid, this sequence exhibited sensitivity to endonuclease S1, an enzyme that preferentially digests single-stranded DNA. Precise S1 mapping, obtained by direct sequencing of nine distinct S1-generated clones, revealed that in all cases the borders of the sensitive sequence resided within the pur/pyr segment. We propose that this region of the promoter could adopt an intramolecular hairpin triplex structure in vivo and may play a role in the chromatin organization at the decorin gene locus. In addition, this region was able to up-regulate a minimal heterologous promoter in transient transfection assays. The results show that the structure of the decorin gene promoter is different from that of any other proteoglycan promoter characterized so far and indicate that the pur/pyr segment plays a role in the regulation of gene transcription.

Abstract The cellular and molecular mechanisms regulating the reversible accumulation of nonhelical, underhydroxylated procollagen in the rough endoplasmic reticulum (RER) remain obscure. To clarify these mechanisms, we isolated chondrocytes from chick vertebral cartilage and kept them in scorbutic monolayer cultures. By Day 9 of culture, the chondrocytes had accumulated a large amount of underhydroxylated Type II procollagen in their RER. Within 1 h of ascorbate treatment, the accumulated procollagen was hydroxylated; this was accompanied by a slight stimulation of procollagen secretion and was followed by a marked stimulation starting between 2 and 3 h of treatment. Secretion of the accumulated procollagen was completed by about 24 h of treatment. Strikingly, the marked stimulation of procollagen secretion at 2-3 h of treatment was associated with marked remodeling of the RER. This organelle came to consist of a few, unusually large cisternae (sacs) and many flat cisternae while the RER in untreated cells consisted of uniform, oval cisternae. The RER remodeling was accompanied by a comparable redistribution of the accumulated Type II procollagen stored in it. The RER sacs and flat cisternae invariably communicated directly and were still detectable by 8 h but not by 24 h of treatment. RER remodeling and procollagen redistribution also occurred in untreated chondrocytes that had been shifted to 23 degrees C for 2-3 h. Together, the data indicate that folding of the accumulated procollagen molecules into their normal helical configuration is followed by procollagen redistribution within, and remodeling of, the RER. These processes may have a role in stimulating procollagen export from the RER and secretion.

Despite the growing evidence implicating proteoglycans in the control of cell proliferation and differentiation, little is known about the factors that control their metabolism in neoplasia or the mechanisms through which these macromolecules may influence neoplastic growth.The primary objective of the present study was to test whether human colon carcinoma cells released soluble mediators capable of stimulating the synthesis of proteoglycans in normal colon fibroblasts in vitro.Serum-free medium conditioned by colon carcinoma cells (TCM) was capable of stimulating severalfold the synthesis and secretion of proteoglycans in normal colon fibroblasts without inducing a mitogenic response.This effect was a true stimulation of proteoglycan biosynthesis since the kinetics of turnover were identical in the presence or absence of TCM.Characterization of the proteoglycans synthesized in the absence of TCM revealed that colon fibroblasts synthesized at least three species of proteoglycans including a heparan sulfate proteoglycan which was associated primarily with the cell layer and two populations of proteoglycans which were predominantly released into the medium and contained chondroitin-dermatan sulfate side chains.When fibroblasts were exposed to TCM, they synthesized and released higher amounts of proteoglycans which had overall similar density, molecular weight, and polydispersity but differed from controls in that they contained significantly higher proportions of chondroitin sulfate side chains.Partial characterization of TCM strongly indicated that the stimulatory activity comprised a family of polypeptides, with molecular weight between 5.4 and 6.0 % lo', which were heat stable and acid/alkali labile.Neoplastic modulation of proteoglycan metabolism in normal mesenchymal cells may represent an additional mechanism through which tumor cells can alter their surrounding environment.It is well established that the development of a vascular supply and concomitant stromal support are vital for the growth and evolution of malignant neoplasms (1).The supporting structures enable the tumor to acquire a characteristic

Abnormal expression of proteoglycans has been implicated in cancer and metastasis primarily because these macromolecules are involved in the control of cell growth and matrix assembly. In this report, we have investigated the expression and immunolocalization of perlecan, a major heparan sulfate proteoglycan of basement membranes and pericellular matrices, in human metastatic melanomas. Twenty-six of the 27 tumor samples showed a significant increase (up to 15-fold) in the perlecan mRNA levels when compared with normal tissue. This change correlated with a vast deposition of perlecan protein core in the pericellular matrix of metastatic melanomas. Furthermore, we have established a relationship between perlecan expression in clonal melanoma cells (70W) stimulated with neurotrophins and their increased invasiveness. Interestingly, perlecan mRNA levels were up-regulated within 10 min of neurotrophin stimulation, indicating that perlecan is an early response gene. This upregulation also occurred prior to heparanase production, suggesting that perlecan expression and its regulation might play a pivotal role in the initial onset of invasion.

Abstract Endorepellin, a C-terminal fragment of the vascular basement membrane proteoglycan perlecan, inhibits angiogenesis via the α2β1-integrin receptor. Because this integrin is also implicated in platelet-collagen responses and because endorepellin or its fragments are generated in response to injury and inflammation, we hypothesized that endorepellin could also affect platelet biology. We discovered that endorepellin supported α2β1-dependent platelet adhesion, without appreciably activating or aggregating platelets. Notably, endorepellin enhanced collagen-evoked responses in platelets, in a src kinase-dependent fashion, and enhanced the collagen-inhibitory effect of an α2β1-integrin function-blocking antibody. Collectively, these results suggest that endorepellin/α2β1-integrin interaction and effects are specific and dependent on cell type, differ from those emanated by exposure to collagen, and may be due to cellular differences in α2β1-integrin activation/ligand affinity state. These studies also suggest a heretofore unrecognized role for angiostatic basement membrane fragments in platelet biology.

Achilles tendons are a common source of pain and injury, and their pathology may originate from aberrant structure function relationships. Small leucine rich proteoglycans (SLRPs) influence mechanical and structural properties in a tendon-specific manner. However, their roles in the Achilles tendon have not been defined. The objective of this study was to evaluate the mechanical and structural differences observed in mouse Achilles tendons lacking class I SLRPs; either decorin or biglycan. In addition, empirical modeling techniques based on mechanical and image-based measures were employed. Achilles tendons from decorin-null (Dcn(-/-)) and biglycan-null (Bgn(-/-)) C57BL/6 female mice (N=102) were used. Each tendon underwent a dynamic mechanical testing protocol including simultaneous polarized light image capture to evaluate both structural and mechanical properties of each Achilles tendon. An empirical damage model was adapted for application to genetic variation and for use with image based structural properties to predict tendon dynamic mechanical properties. We found that Achilles tendons lacking decorin and biglycan had inferior mechanical and structural properties that were age dependent; and that simple empirical models, based on previously described damage models, were predictive of Achilles tendon dynamic modulus in both decorin- and biglycan-null mice.

Hyaluronan plays a key role in regulating inflammation and tumor angiogenesis. Of the three transmembrane hyaluronan synthases, HAS2 is the main pro-angiogenic enzyme responsible for excessive hyaluronan production. We discovered that HAS2 was degraded in vascular endothelial cells via autophagy evoked by nutrient deprivation, mTOR inhibition, or pro-autophagic proteoglycan fragments endorepellin and endostatin. Using live-cell and super-resolution confocal microscopy, we found that protracted autophagy evoked a dynamic interaction between HAS2 and ATG9A, a key transmembrane autophagic protein. This regulatory axis of HAS2 degradation occurred in various cell types and species and in vivo upon nutrient deprivation. Inhibiting in vivo autophagic flux via chloroquine showed increased levels of HAS2 in the heart and aorta. Functionally, autophagic induction via endorepellin or mTOR inhibition markedly suppressed extracellular hyaluronan production in vascular endothelial cells and inhibited ex vivo angiogenic sprouting. Thus, we propose autophagy as a novel catabolic mechanism regulating hyaluronan production in endothelial cells and demonstrate a new link between autophagy and angiogenesis that could lead to potential therapeutic modalities for angiogenesis.

We present a simplified method for conducting aortic ring assays which yields robust sprouting and high reproducibility targeted towards matrix biologists studying angiogenesis and extracellular matrix signaling. Main adjustments from previously established protocols include embedding aortic rings between two layers of 3D type I collagen matrix and supplementing with vascular endothelial media. We also introduce a concise and effective staining protocol for obtaining high-resolution images of intracellular and extracellular matrix proteins along with a more accurate protocol to quantify angiogenesis. Importantly, we present a novel method to perform biochemical analyses of vessel sprouting without contamination from the aortic ring itself. Overall, our refined method enables detection of low abundance and phosphorylated proteins and provides a straightforward ex vivo angiogenic assay that can be easily reproduced by those in the matrix biology field.

Extracellular matrix-evoked angiostasis and autophagy within the tumor microenvironment represent two critical, but unconnected, functions of the small leucine-rich proteoglycan, decorin. Acting as a partial agonist of vascular endothelial growth factor 2 (VEGFR2), soluble decorin signals via the energy sensing protein, AMP-activated protein kinase (AMPK), in the autophagic degradation of intracellular vascular endothelial growth factor A (VEGFA). Here, we discovered that soluble decorin evokes intracellular catabolism of endothelial VEGFA that is mechanistically independent of mTOR, but requires an autophagic regulator, paternally expressed gene 3 (PEG3). We found that administration of autophagic inhibitors such as chloroquine or bafilomycin A1, or depletion of autophagy-related 5 (ATG5), results in accumulation of intracellular VEGFA, indicating that VEGFA is a basal autophagic substrate. Mechanistically, decorin increased the VEGFA clearance rate by augmenting autophagic flux, a process that required RAB24 member RAS oncogene family (RAB24), a small GTPase that facilitates the disposal of autophagic compartments. We validated these findings by demonstrating the physiological relevance of this process in vivo. Mice starved for 48 h exhibited a sharp decrease in overall cardiac and aortic VEGFA that could be blocked by systemic chloroquine treatment. Thus, our findings reveal a unified mechanism for the metabolic control of endothelial VEGFA for autophagic clearance in response to decorin and canonical pro-autophagic stimuli. We posit that the VEGFR2/AMPK/PEG3 axis integrates the anti-angiogenic and pro-autophagic bioactivities of decorin as the molecular basis for tumorigenic suppression. These results support future therapeutic use of decorin as a next-generation protein therapy to combat cancer. Extracellular matrix-evoked angiostasis and autophagy within the tumor microenvironment represent two critical, but unconnected, functions of the small leucine-rich proteoglycan, decorin. Acting as a partial agonist of vascular endothelial growth factor 2 (VEGFR2), soluble decorin signals via the energy sensing protein, AMP-activated protein kinase (AMPK), in the autophagic degradation of intracellular vascular endothelial growth factor A (VEGFA). Here, we discovered that soluble decorin evokes intracellular catabolism of endothelial VEGFA that is mechanistically independent of mTOR, but requires an autophagic regulator, paternally expressed gene 3 (PEG3). We found that administration of autophagic inhibitors such as chloroquine or bafilomycin A1, or depletion of autophagy-related 5 (ATG5), results in accumulation of intracellular VEGFA, indicating that VEGFA is a basal autophagic substrate. Mechanistically, decorin increased the VEGFA clearance rate by augmenting autophagic flux, a process that required RAB24 member RAS oncogene family (RAB24), a small GTPase that facilitates the disposal of autophagic compartments. We validated these findings by demonstrating the physiological relevance of this process in vivo. Mice starved for 48 h exhibited a sharp decrease in overall cardiac and aortic VEGFA that could be blocked by systemic chloroquine treatment. Thus, our findings reveal a unified mechanism for the metabolic control of endothelial VEGFA for autophagic clearance in response to decorin and canonical pro-autophagic stimuli. We posit that the VEGFR2/AMPK/PEG3 axis integrates the anti-angiogenic and pro-autophagic bioactivities of decorin as the molecular basis for tumorigenic suppression. These results support future therapeutic use of decorin as a next-generation protein therapy to combat cancer.

The growth factor progranulin plays a critical role in bladder cancer by modulating tumor cell motility and invasion. Progranulin regulates remodeling of the actin cytoskeleton by interacting with drebrin, an actin binding protein that regulates tumor growth. We previously discovered that progranulin depletion inhibits epithelial-to-mesenchymal transition and markedly reduces in vivo tumor growth. Moreover, progranulin depletion sensitizes urothelial cancer cells to cisplatin treatment, further substantiating a pro-survival function of progranulin. Until recently, the progranulin signaling receptor remained unidentified, precluding a full understanding of progranulin action in tumor cell biology. We recently identified EphA2, a member of a large family of receptor tyrosine-kinases, as the functional receptor for progranulin. However, it is not established whether EphA2 plays an oncogenic role in bladder cancer. Here we demonstrate that progranulin, and not ephrin-A1, the canonical ligand for EphA2, is the predominant EphA2 ligand in bladder cancer. Progranulin evoked Akt- and Erk1/2-mediated EphA2 phosphorylation at Ser897, which could drive bladder tumorigenesis. We discovered that EphA2 depletion severely blunted progranulin-dependent motility and anchorage-independent growth, and sensitized bladder cancer cells to cisplatin treatment. We further defined the mechanisms of progranulin/EphA2-dependent motility by identifying liprin-α1 as a novel progranulin-dependent EphA2 interacting protein and establishing its critical role in cell motility. The discovery of EphA2 as the functional signaling receptor for progranulin and the identification of novel downstream effectors offer a new avenue for understanding the underlying mechanism of progranulin action and may constitute novel clinical and therapeutic targets in bladder cancer.

ObjectiveTo delineate the activities of decorin and biglycan in the progression of post-traumatic osteoarthritis (PTOA).DesignThree-month-old inducible biglycan (BgniKO) and decorin/biglycan compound (Dcn/BgniKO) knockout mice were subjected to the destabilization of the medial meniscus (DMM) surgery to induce PTOA. The OA phenotype was evaluated by assessing joint structure and sulfated glycosaminoglycan (sGAG) staining via histology, surface collagen fibril nanostructure and calcium content via scanning electron microscopy, tissue modulus via atomic force microscopy-nanoindentation, as well as subchondral bone structure and meniscus ossification via micro-computed tomography. Outcomes were compared with previous findings in the inducible decorin (DcniKO) knockout mice.ResultsIn the DMM model, BgniKO mice developed similar degree of OA as the control (0.44 [-0.18 1.05] difference in modified Mankin score), different from the more severe OA phenotype observed in DcniKO mice (1.38 [0.91 1.85] difference). Dcn/BgniKO mice exhibited similar histological OA phenotype as DcniKO mice (1.51 [0.97 2.04] difference vs control), including aggravated loss of sGAGs, salient surface fibrillation and formation of osteophyte. Meanwhile, Dcn/BgniKO mice showed further cartilage thinning than DcniKO mice, resulting in the exposure of underlying calcified tissues and aberrantly high surface modulus. BgniKO and Dcn/BgniKO mice developed altered subchondral trabecular bone structure in both Sham and DMM groups, while DcniKO and control mice did not.ConclusionIn PTOA, decorin plays a more crucial role than biglycan in regulating cartilage degeneration, while biglycan is more important in regulating subchondral bone structure. The two have distinct activities and modest synergy in the pathogenesis of PTOA.

Breast cancer (BrCa) relies on specific microRNAs to drive disease progression. Oncogenic miR-21 is upregulated in many cancers, including BrCa, and is associated with poor survival and treatment resistance. We sought to determine the role of miR-21 in BrCa tumor initiation, progression and treatment response. In a triple-negative BrCa model, radiation exposure increased miR-21 in both primary tumor and metastases. In vitro, miR-21 knockdown decreased survival in all BrCa subtypes in the presence of radiation. The role of miR-21 in BrCa initiation was evaluated by implanting wild-type miR-21 BrCa cells into genetically engineered mouse models where miR-21 was intact, heterozygous or globally ablated. Tumors were unable to grow in the mammary fat pads of miR-21−/− mice, and grew in ~50% of miR-21+/− and 100% in miR-21+/+ mice. The contribution of miR-21 to progression and metastases was tested by crossing miR-21−/− mice with mice that spontaneously develop BrCa. The global ablation of miR-21 significantly decreased the tumorigenesis and metastases of BrCa, while sensitizing tumors to radio- and chemotherapeutic agents via Fas/FasL-dependent apoptosis. Therefore, targeting miR-21 alone or in combination with various radio or cytotoxic therapies may represent novel and efficacious therapeutic modalities for the future treatment of BrCa patients.

Proteoglycans and selected extracellular matrix constituents are emerging as intrinsic and critical regulators of evolutionarily conversed, intracellular catabolic pathways. Often, these secreted molecules evoke sustained autophagy in a variety of cell types, tissues, and model systems. The unique properties of proteoglycans have ushered in a paradigmatic shift to broaden our understanding of matrix-mediated signaling cascades. The dynamic cellular pathway controlling autophagy is now linked to an equally dynamic and fluid signaling network embedded in a complex meshwork of matrix molecules. A rapidly emerging field of research encompasses multiple matrix-derived candidates, representing a menagerie of soluble matrix constituents including decorin, biglycan, endorepellin, endostatin, collagen VI and plasminogen kringle 5. These matrix constituents are pro-autophagic and simultaneously anti-angiogenic. In contrast, perlecan, laminin α2 chain, and lumican have anti-autophagic functions. Mechanistically, each matrix constituent linked to intracellular catabolic events engages a specific cell surface receptor that often converges on a common core of the autophagic machinery including AMPK, Peg3 and Beclin 1. We consider this matrix-evoked autophagy as non-canonical given that it occurs in an allosteric manner and is independent of nutrient availability or prevailing bioenergetics control. We propose that matrix-regulated autophagy is an important outside-in signaling mechanism for proper tissue homeostasis that could be therapeutically leveraged to combat a variety of diseases.

There is growing evidence that the two small leucine-rich proteoglycans biglycan and decorin regulate the assembly of connective tissues and alter cell behavior during development and pathological processes. In this study, we have used an experimental animal model of unilateral ureteral ligation and mice deficient in either biglycan or decorin. We discovered that pressure-induced injury to the wild-type kidneys led to overexpression of decorin, biglycan, fibrillin-1, and fibrillin-2. In contrast, in biglycan-deficient kidneys the overexpression of fibrillin-1 was markedly attenuated and this was associated with cystic dilatation of Bowman's capsule and proximal tubules. Notably, we found that in ligated kidneys from decorin-null mice, fibrillin-1 expression was initially enhanced to the same extent as in wild-type animals. However, long-term obstruction resulted in down-regulation of fibrillin-1 and concurrent cystic dilatation of Bowman's capsule in 33% of kidneys at 5 months after obstruction. In all of the genotypes, no differences in fibrillin-2 expression were observed. These in vivo data correlated with a significant induction of fibrillin-1 expression in renal fibroblasts and mesangial cells by recombinant biglycan and decorin. Our results indicate a novel role for decorin and biglycan during pressure-induced renal injury by stimulating fibrillin-1 expression.

Two cases of large, multiple splenic hamartomas in children with pancytopenia, bone marrow hyperplasia, lymphadenopathy, hepatosplenomegaly, frequent infections, growth retardation, and fever are reported. These symptoms were relieved by splenectomy, and have not recurred during follow-up periods of one year and nine years. The sharply circumscribed lesions comprised large portions of the resected spleens and were composed of dilated vascular channels filled with mononuclear cells and iummunoblasts. The lesions lacked splenic cords or trabeculae, lymphoid follicles, Reed-Sternberg cells, and granulomas or other evidence of infection. Splenic hamartomas are usually single small lesions found incidentally at necropsy or laparotomy. Splenic hamartomas associated with symptoms and hypersplenism are large, and often confluent multiple tumors. Recognition of their benign nature is important in light of the current practice of laparotomy for staging and diagnosis of malignant conditions.

Decorin, an archetypal member of the small leucine-rich proteoglycan gene family, regulates collagen fibrillogenesis and cell growth. To further explore its biological function, we examined the role of Decorin during zebrafish development. Zebrafish Decorin is a chondroitin sulfate proteoglycan that exhibits a high degree of conservation with its mammalian counterpart and displays a unique spatiotemporal expression pattern. Morpholino-mediated knockdown of zebrafish decorin identified a developmental role during medial-lateral convergence and anterior-posterior extension of the body plan, as well as in craniofacial cartilage formation. decorin morphants displayed a pronounced shortening of the head-to-tail axis as well as compression, flattening, and extension of the jaw cartilages. The morphant phenotype was efficiently rescued by zebrafish decorin mRNA. Unexpectedly, microinjection of excess zebrafish decorin mRNA or proteoglycan/protein core into one-cell stage embryos caused cyclopia. The morphant and overexpression phenotype represent a convergent extension defect. Our results indicate a central function for Decorin during early embryogenesis.

Primary open-angle glaucoma, a neurodegenerative disorder characterized by degeneration of optic nerve axons, is a frequent cause of vision loss and blindness worldwide. Several randomized multicenter studies have identified intraocular pressure as the major risk factor for its development, caused by an increased outflow resistance to the aqueous humor within the trabecular meshwork. However, the molecular mechanism for increased outflow resistance in POAG has not been fully established. One of the proposed players is the pro-fibrotic transforming growth factor (TGF)-β2, which is found in higher amounts in the aqueous humor of patients with POAG. In this study we elucidated the role of decorin, a small leucine-rich proteoglycan and known antagonist of TGF-β, in the region of aqueous humor outflow tissue. Utilizing decorin deficient mice, we discovered that decorin modulated TGF-β signaling in the canonical outflow pathways and the lack of decorin in vivo caused an increase in intraocular pressure. Additionally, the Dcn-/- mice showed significant loss of optic nerve axons and morphological changes in the glial lamina, typical features of glaucoma. Moreover, using human trabecular meshwork cells we discovered that soluble decorin attenuated TGF-β2 mediated synthesis and expression of typical downstream target genes including CCN2/CTGF, FN and COL IV. Finally, we found a negative reciprocal regulation of decorin and TGF-β, with a dramatic downregulation of decorin in the canonical outflow pathways of patients with primary open-angle glaucoma. Collectively, our results indicate that decorin plays an important role in the pathogenesis of primary open-angle glaucoma and offers novel perspectives in the treatment of this serious disease.

We have established a continuous cell line from the Engelbreth-Holm-Swarm (EHS) tumor, a transplantable murine neoplasm that has been extensively utilized to investigate basement membrane constituents. The EHS-derived cells, designated BAM cells, have been subcultured for over 40 passages and have maintained phenotypic and biological properties of the parent EHS tumor cells. BAM cells have retained an epithelioid morphology and the ability to induce EHS-like tumors in mice. Biochemical and immunochemical studies demonstrated that BAM cells synthesize laminin A and B chains, collagen type IV, entactin and the basement membrane specific heparan sulfate proteoglycan. Interestingly, the proteoglycan synthesized by BAM cells was a hybrid molecule containing 2-3 heparan sulfate chains of 25-35 kDa and 1 chondroitin sulfate chain of approximately 17 kDa attached to a 400-kDa protein core. This cell line will be useful to investigations concerning biosynthesis of basement membrane constituents and will be a valuable source of extracellular matrix for testing cellular properties such as attachment, locomotion and differentiation.

We have previously reported that the connective tissue stroma of human colon carcinoma contains elevated amounts of decorin, a small proteoglycan involved in the regulation of matrix formation and cell proliferation. These biochemical changes were correlated with increased mRNA levels and general hypomethylation of the decorin gene in human colon cancer DNA. In this report we use a quantitative polymerase chain reaction method coupled with digestion of the DNA template by methylation-sensitive restriction endonucleases to investigate in detail the location of hypomethylated sites in decorin gene. We demonstrate that a specific site in the 3' region of the gene, encompassing codons 360-361, is specifically hypomethylated in both colon carcinoma and benign polyp. In contrast, three HpaII sites, clustered in the 5' untranslated region, show full methylation in normal and neoplastic DNA. The lack of such changes in ulcerative colitis DNA suggests that chronic inflammation alone is not sufficient to alter cytosine methylation in the decorin gene. These results suggest the possibility that the 3' region of the decorin-coding sequence may be involved in the control of decorin gene expression.

Pure sarcomas of the esophagus are exceedingly rare. We report a case of esophageal synovial sarcoma which occurred in an adolescent. The tumor was locally resected, sparing the patient esophagectomy. After postoperative radiation therapy, the patient remains alive and well without evidence of disease 28 mo after operation. The unique nature of polypoid sarcoma of the esophagus, and the potential for cure without esophagectomy, is discussed.

Glycosaminoglycans of a malignant pleural mesothelioma have been characterized histochemically and biochemically and compared with those of normal lung, pleural plaque, lung carcinoma, and other connective tissue neoplasms. Chondroitin sulfate constituted the major glycosaminoglycan (approximately 80% of total) present in the pleural mesothelioma while hyaluronic acid was present in only trace amounts (approximately 3% of total). In particular chondroitin 6-sulfate was the predominant isomer, constituting 80% of the total chondroitin sulfate. Control tissue exhibited different proportions of glycosaminoglycans and none of them contained as high an absolute concentration of chondroitin sulfate as the mesothelioma. These findings differ from previous reports demonstrating increased concentration of hyaluronic acid in mesothelioma and suggest the possible existence of a biochemically different form of this neoplasm.

Despite extensive clinical and experimental studies over the past decades, the pathogenesis and progression to the castration-resistant stage of prostate cancer remains largely unknown. Progranulin, a secreted growth factor, strongly binds the heparin-sulfate proteoglycan perlecan, and counteracts its biological activity. We established that progranulin acts as an autocrine growth factor and promotes prostate cancer cell motility, invasion, and anchorage-independent growth. Progranulin was overexpressed in prostate cancer tissues vis-à-vis non-neoplastic tissues supporting the hypothesis that progranulin may play a key role in prostate cancer progression. However, progranulin's mode of action is not well understood and proteins regulating progranulin signaling have not been identified. Sortilin, a single-pass type I transmembrane protein of the Vps10 family, binds progranulin in neurons and targets progranulin for lysosomal degradation. Significantly, in DU145 and PC3 cells, we detected very low levels of sortilin associated with high levels of progranulin production and enhanced motility. Restoring sortilin expression decreased progranulin levels, inhibited motility and anchorage-independent growth and destabilized Akt. These results demonstrated a critical role for sortilin in regulating progranulin and suggest that sortilin loss may contribute to prostate cancer progression. Here, we provide the novel observation that progranulin downregulated sortilin protein levels independent of transcription. Progranulin induced sortilin ubiquitination, internalization via clathrin-dependent endocytosis and sorting into early endosomes for lysosomal degradation. Collectively, these results constitute a regulatory feed-back mechanism whereby sortilin downregulation ensures sustained progranulin-mediated oncogenesis.

We have recently demonstrated a critical role for progranulin in bladder cancer. Progranulin contributes, as an autocrine growth factor, to the transformed phenotype by modulating Akt-and MAPK-driven motility, invasion and anchorage-independent growth. Progranulin also induces F-actin remodeling by interacting with the F-actin binding protein drebrin. In addition, progranulin is overexpressed in invasive bladder cancer compared to normal tissue controls, suggesting that progranulin might play a key role in driving the transition to the invasive phenotype of urothelial cancer. However, it is not established whether targeting progranulin could have therapeutic effects on bladder cancer. In this study, we stably depleted urothelial cancer cells of endogenous progranulin by shRNA approaches and determined that progranulin depletion severely inhibited the ability of tumorigenic urothelial cancer cells to migrate, invade and grow in anchorage-independency. We further demonstrate that progranulin expression is critical for tumor growth in vivo, in both xenograft and orthotopic tumor models. Notably, progranulin levels correlated with response to cisplatin treatment and were upregulated in bladder tumors. Our data indicate that progranulin may constitute a novel target for therapeutic intervention in bladder tumors. In addition, progranulin may serve as a novel biomarker for bladder cancer.

The extracellular matrix is engaged in an ever-evolving and elegant ballet of dynamic reciprocity that directly and bi-directionally regulates cell behavior. Homeostatic and pathophysiological changes in cell-matrix signaling cascades manifest as complex matrix phenotypes. Indeed, the extracellular matrix can be implicated in virtually every known human disease, thus, making it the most critical and dynamic “organ” in the human body. The overall goal of this Special Issue is to provide an accurate and inclusive functional definition that addresses the inherent complexity of matrix phenotypes. This goal is summarily achieved via a corpus of expertly written articles, reviews and original research, focused at answering this question empirically and fundamentally via state-of-the-art methods and research strategies.

Our earlier decorin (Dcn) gene overexpression studies found that the targeted Dcn gene transfer into the cornea inhibited corneal angiogenesis in vivo using a rabbit model. In this study, we tested the hypothesis that anti-angiogenic effects of decorin in the cornea are mediated by alterations in a normal physiologic balance of pro- and anti-angiogenic factors using decorin deficient (Dcn-/-) and wild type (Dcn+/+) mice. Corneal neovascularization (CNV) in Dcn-/- and Dcn+/+ mice was produced with a standard chemical injury technique. The clinical progression of CNV in mice was monitored with stereo- and slit-lamp microscopes, and histopathological hematoxylin and eosin (H&E) staining. Protein and mRNA expression of pro- and anti-angiogenic factors in the cornea were evaluated using immunofluorescence and quantitative real-time PCR, respectively. Slit-lamp clinical eye examinations revealed significantly more CNV in Dcn-/- mice than the Dcn+/+ mice post-injury (p < 0.05) and AAV5-Dcn gene therapy significantly reduced CNV in Dcn-/- mice compered to no AAV5-Dcn gene therapy controls (p < 0.001). H&E-stained corneal sections exhibited morphology with several neovessels in injured corneas of the Dcn-/- mice than the Dcn+/+ mice. Immunofluorescence of corneal sections displayed significantly higher expression of α-smooth muscle actin (α-SMA) and endoglin proteins in Dcn-/- mice than Dcn+/+ mice (p < 0.05). Quantitative real-time PCR found significantly increased mRNA levels of pro-angiogenic factors endoglin (2.53-fold; p < 0.05), Vegf (2.47-fold; p < 0.05), and Pecam (2.14-fold; p < 0.05) and anti-angiogenic factor Vegfr2 (1.56-fold; p < 0.05) in the normal cornea of the Dcn-/- mice than the Dcn+/+ mice. Furthermore, neovascularized Dcn-/- mice corneas showed greater increase in mRNA expression of pro-angiogenic factors endoglin (4.58-fold; p < 0.0001), Vegf (4.16-fold; p < 0.0001), and Pdgf (2.15-fold; p < 0.0001) and reduced expression of anti-angiogenic factors Ang2 (0.12-fold; p < 0.05), Timp1 (0.22-fold; p < 0.05), and Vegfr2 (0.67-fold; p > 0.05) compared to neovascularized Dcn+/+ mice corneas. These gene deficience studies carried with transgenic Dcn-/- mice revealed decorin's role in influencing a physiologic balance between pro-and anti-angiogenic factors in the normal and injured cornea. We infer that the functional deletion of Dcn promotes irregular corneal repair and aggravates CNV.

Perlecan is a major heparan sulfate proteoglycan of basement membranes and cell surfaces. Because of its strategic location and ability to store and protect growth factors, perlecan has been implicated in the control of tumor cell growth and metastatic behavior. To test the role of perlecan in malignancy, we generated several stably transfected clones of HT-1080, a human fibrosarcoma cell line, harboring a perlecan cDNA in the antisense orientation. Surprisingly, clones with a reduced synthesis of perlecan mRNA and protein core grew faster, formed larger colonies in semisolid agar, and induced faster formation of s.c. tumors in nude mice than the wild-type cells. Their growth properties in vitro were independent of exogenous basic fibroblast growth factor. Reduction of perlecan expression was associated with three distinct properties typical of tumor cells with a more aggressive phenotype: enhanced migration through 8-microm-pore filter, increased invasion in Matrigel-coated filters, and heightened adhesiveness to type IV collagen substrata. These results thus provide the first evidence that perlecan may inhibit the growth and invasiveness of fibrosarcoma cells in a basic fibroblast growth factor-independent pathway and raise the possibility that perlecan may prevent the infiltration of host tissues in mesenchymal neoplasms.

Abstract We provide direct evidence for the presence of unsulfated, but fully elongated heparan glycosaminoglycans covalently linked to the protein core of a heparan sulfate proteoglycan synthesized by human colon carcinoma cells. Chemical and enzymatic studies revealed that a significant proportion of these chains contained glucuronic acid and N-acetylated glucosamine moieties, consistent with N-acetylheparosan, an established precursor of heparin and heparan sulfate. The presence of unsulfated chains was not dependent upon the exogenous supply of sulfate since their synthesis, structure, or relative amount did not vary with low exogenous sulfate concentrations. Culture in sulfate-free medium also failed to generate undersulfated heparan sulfate-proteoglycan, but revealed an endogenous source of sulfate which was primarily derived from the catabolism of the sulfur-containing amino acids methionine and cysteine. Furthermore, the presence of unsulfated chains was not due to a defect in the sulfation process because pulse-chase experiments showed that they could be converted into the fully sulfated chains. However, their formation was inhibited by limiting the endogenous supply of hexosamine. The results also indicated the coexistence of the unsulfated and sulfated chains on the same protein core and further suggested that the sulfation of heparan sulfate may occur as an all or nothing phenomenon. Taken together, the results support the current biosynthetic model developed for the heparin proteoglycan in which unsulfated glycosaminoglycans are first elongated on the protein core, and subsequently modified and sulfated. These data provide the first evidence for the presence of such an unsulfated precursor in an intact cellular system.