V3: an enigmatic isoform of the proteoglycan versican.

V3 is an isoform of the extracellular matrix (ECM) proteoglycan (PG) versican generated through alternative splicing of the versican gene such that the two major exons coding for sequences in the protein core that support chondroitin sulfate (CS) glycosaminoglycan (GAG) chain attachment are excluded. Thus, versican V3 isoform carries no GAGs. A survey of PubMed reveals only 50 publications specifically on V3 versican, so it is a very understudied member of the versican family, partly because to date there are no antibodies that can distinguish V3 from the CS-carrying isoforms of versican, that is, to facilitate functional and mechanistic studies. However, a number of in vitro and in vivo studies have identified the expression of the V3 transcript during different phases of development and in disease, and selective overexpression of V3 has shown dramatic phenotypic effects in "gain and loss of function" studies in experimental models. Thus, we thought it would be useful and instructive to discuss the discovery, characterization, and the putative biological importance of the enigmatic V3 isoform of versican.

Versican Deficiency Significantly Reduces Lung Inflammatory Response Induced by Polyinosine-Polycytidylic Acid Stimulation.

Viral infection is an exacerbating factor contributing to chronic airway diseases, such as asthma, via mechanisms that are still unclear. Polyinosine-polycytidylic acid (poly(I:C)), a Toll-like receptor 3 (TLR3) agonist used as a mimetic to study viral infection, has been shown to elicit inflammatory responses in lungs and to exacerbate pulmonary allergic reactions in animal models. Previously, we have shown that poly(I:C) stimulates lung fibroblasts to accumulate an extracellular matrix (ECM), enriched in hyaluronan (HA) and its binding partner versican, which promotes monocyte adhesion. In the current study, we aimed to determine the in vivo role of versican in mediating inflammatory responses in poly(I:C)-induced lung inflammation using a tamoxifen-inducible versican-deficient mouse model (Vcan-/- mice). In C57Bl/6 mice, poly(I:C) instillation significantly increased accumulation of versican and HA, especially in the perivascular and peribronchial regions, which were enriched in infiltrating leukocytes. In contrast, versican-deficient (Vcan-/-) lungs did not exhibit increases in versican or HA in these regions and had strikingly reduced numbers of leukocytes in the bronchoalveolar lavage fluid and lower expression of inflammatory chemokines and cytokines. Poly(I:C) stimulation of lung fibroblasts isolated from control mice generated HA-enriched cable structures in the ECM, providing a substrate for monocytic cells in vitro, whereas lung fibroblasts from Vcan-/- mice did not. Moreover, increases in proinflammatory cytokine expression were also greatly attenuated in the Vcan-/- lung fibroblasts. These findings provide strong evidence that versican is a critical inflammatory mediator during poly(I:C)-induced acute lung injury and, in association with HA, generates an ECM that promotes leukocyte infiltration and adhesion.

Versican is produced by Trif- and type I interferon-dependent signaling in macrophages and contributes to fine control of innate immunity in lungs.

Growing evidence suggests that versican is important in the innate immune response to lung infection. Our goal was to understand the regulation of macrophage-derived versican and the role it plays in innate immunity. We first defined the signaling events that regulate versican expression, using bone marrow-derived macrophages (BMDMs) from mice lacking specific Toll-like receptors (TLRs), TLR adaptor molecules, or the type I interferon receptor (IFNAR1). We show that LPS and polyinosinic-polycytidylic acid [poly(I:C)] trigger a signaling cascade involving TLR3 or TLR4, the Trif adaptor, type I interferons, and IFNAR1, leading to increased expression of versican by macrophages and implicating versican as an interferon-stimulated gene. The signaling events regulating versican are distinct from those for hyaluronan synthase 1 (HAS1) and syndecan-4 in macrophages. HAS1 expression requires TLR2 and MyD88. Syndecan-4 requires TLR2, TLR3, or TLR4 and both MyD88 and Trif. Neither HAS1 nor syndecan-4 is dependent on type I interferons. The importance of macrophage-derived versican in lungs was determined with LysM/Vcan-/- mice. These studies show increased recovery of inflammatory cells in the bronchoalveolar lavage fluid of poly(I:C)-treated LysM/Vcan-/- mice compared with control mice. IFN-ß and IL-10, two important anti-inflammatory molecules, are significantly decreased in both poly(I:C)-treated BMDMs from LysM/Vcan-/- mice and bronchoalveolar lavage fluid from poly(I:C)-treated LysM/Vcan-/- mice compared with control mice. In short, type I interferon signaling regulates versican expression, and versican is necessary for type I interferon production. These findings suggest that macrophage-derived versican is an immunomodulatory molecule with anti-inflammatory properties in acute pulmonary inflammation.

Versican and the regulation of cell phenotype in disease.

BACKGROUND: Versican is an extracellular matrix (ECM) proteoglycan that is present in the pericellular environment of most tissues and increases in many different diseases. Versican interacts with cells to influence the ability of cells to proliferate, migrate, adhere and assemble an ECM. SCOPE OF REVIEW: The structure of the versican molecule is briefly reviewed and studies highlighting those factors that promote versican synthesis and degradation and their impact on cell phenotype in disease are discussed. Particular attention is given to vascular disease, but other diseases where versican is important are covered as well, most notably different forms of cancers. Attention is given to mechanisms(s) by which versican influences cell behaviors through either direct or indirect processes. Versican produced by either stromal cells or myeloid cells can have a major impact influencing immunity and inflammation. Finally, studies controlling versican accumulation that either delay or inhibit the progression of disease will be highlighted. MAJOR CONCLUSIONS: Versican is one component of the ECM that can influence the ability of cells to proliferate, migrate, adhere, and remodel the ECM. Targeting versican as a way to control cell phenotype offers a novel approach in the treatment of disease. SIGNIFICANCE: ECM molecules such as versican contribute to the structural integrity of tissues and interact with cells through direct and indirect means to regulate, in part, cellular events that form the basis of disease. This article is part of a Special Issue entitled Matrix-mediated cell behaviour and properties.

Perlecan Heparan Sulfate Is Required for the Inhibition of Smooth Muscle Cell Proliferation by All-trans-Retinoic Acid.

Smooth muscle cell (SMC) proliferation is a key process in stabilization of atherosclerotic plaques, and during restenosis after interventions. A clearer understanding of SMC growth regulation is therefore needed to design specific anti-proliferative therapies. Retinoic acid has been shown to inhibit proliferation of SMCs both in vitro and in vivo and to affect the expression of extracellular matrix molecules. To explore the mechanisms behind the growth inhibitory activity of retinoic acid, we hypothesized that retinoids may induce the expression of perlecan, a large heparan sulfate proteoglycan with anti-proliferative properties. Perlecan expression and accumulation was induced in murine SMC cultures by all-trans-retinoic acid (AtRA). Moreover, the growth inhibitory effect of AtRA on wild-type cells was greatly diminished in SMCs from transgenic mice expressing heparan sulfate-deficient perlecan, indicating that the inhibition is perlecan heparan sulfate-dependent. In addition, AtRA influenced activation and phosphorylation of PTEN and Akt differently in wild-type and mutant SMCs, consistent with previous studies of perlecan-dependent SMC growth inhibition. We demonstrate that AtRA regulates perlecan expression in SMCs and that the inhibition of SMC proliferation by AtRA is, at least in part, secondary to an increased expression of perlecan and dependent upon its heparan sulfate-chains.

Overall sulfation of heparan sulfate from pancreatic islet ß-TC3 cells increases maximal fibril formation but does not determine binding to the amyloidogenic peptide islet amyloid polypeptide.

Islet amyloid, a pathologic feature of type 2 diabetes, contains the islet ß-cell peptide islet amyloid polypeptide (IAPP) as its unique amyloidogenic component. Islet amyloid also contains heparan sulfate proteoglycans (HSPGs) that may contribute to amyloid formation by binding IAPP via their heparan sulfate (HS) chains. We hypothesized that ß-cells produce HS that bind IAPP via regions of highly sulfated disaccharides. Unexpectedly, HS from the ß-cell line ß-TC3 contained fewer regions of highly sulfated disaccharides compared with control normal murine mammary gland (NMuMG) cells. The proportion of HS that bound IAPP was similar in both cell lines (∼65%). The sulfation pattern of IAPP-bound versus non-bound HS from ß-TC3 cells was similar. In contrast, IAPP-bound HS from NMuMG cells contained frequent highly sulfated regions, whereas the non-bound material demonstrated fewer sulfated regions. Fibril formation from IAPP was stimulated equally by IAPP-bound ß-TC3 HS, non-bound ß-TC3 HS, and non-bound NMuMG HS but was stimulated to a greater extent by the highly sulfated IAPP-bound NMuMG HS. Desulfation of HS decreased the ability of both ß-TC3 and NMuMG HS to stimulate IAPP maximal fibril formation, but desulfated HS from both cell types still accelerated fibril formation relative to IAPP alone. In summary, neither binding to nor acceleration of fibril formation from the amyloidogenic peptide IAPP is dependent on overall sulfation in HS synthesized by ß-TC3 cells. This information will be important in determining approaches to reduce HS-IAPP interactions and ultimately prevent islet amyloid formation and its toxic effects in type 2 diabetes.

Platelet-derived growth factor differentially regulates the expression and post-translational modification of versican by arterial smooth muscle cells through distinct protein kinase C and extracellular signal-regulated kinase pathways.

The synthesis of proteoglycans involves steps that regulate both protein and glycosaminoglycan (GAG) synthesis, but it is unclear whether these two pathways are regulated by the same or different signaling pathways. We therefore investigated signaling pathways involved in platelet-derived growth factor (PDGF)-mediated increases in versican core protein and GAG chain synthesis in arterial smooth muscle cells (ASMCs). PDGF treatment of ASMCs resulted in increased versican core protein synthesis and elongation of GAG chains attached to the versican core protein. The effects of PDGF on versican mRNA were blocked by inhibiting either protein kinase C (PKC) or the ERK pathways, whereas the GAG elongation effect of PDGF was blocked by PKC inhibition but not by ERK inhibition. Interestingly, blocking protein synthesis in the presence of cycloheximide abolished the PDGF effect, but not in the presence of xyloside, indicating that GAG synthesis that results from PKC activation is independent from de novo protein synthesis. PDGF also stimulated an increase in the chondroitin-6-sulfate to chondroitin-4-sulfate ratio of GAG chains on versican, and this effect was blocked by PKC inhibitors. These data show that PKC activation is sufficient to cause GAG chain elongation, but both PKC and ERK activation are required for versican mRNA core protein expression. These results indicate that different signaling pathways control different aspects of PDGF-stimulated versican biosynthesis by ASMCs. These data will be useful in designing strategies to interfere with the synthesis of this proteoglycan in various disease states.

Heparan sulfate in perlecan promotes mouse atherosclerosis: roles in lipid permeability, lipid retention, and smooth muscle cell proliferation.

Heparan sulfate (HS) has been proposed to be antiatherogenic through inhibition of lipoprotein retention, inflammation, and smooth muscle cell proliferation. Perlecan is the predominant HS proteoglycan in the artery wall. Here, we investigated the role of perlecan HS chains using apoE null (ApoE0) mice that were cross-bred with mice expressing HS-deficient perlecan (Hspg2(Delta3/Delta3)). Morphometry of cross-sections from aortic roots and en face preparations of whole aortas revealed a significant decrease in lesion formation in ApoE0/Hspg2(Delta3/Delta3) mice at both 15 and 33 weeks. In vitro, binding of labeled mouse triglyceride-rich lipoproteins and human LDL to total extracellular matrix, as well as to purified proteoglycans, prepared from ApoE0/Hspg2(Delta3/Delta3) smooth muscle cells was reduced. In vivo, at 20 minutes influx of human (125)I-LDL or mouse triglyceride-rich lipoproteins into the aortic wall was increased in ApoE0/Hspg2(Delta3/Delta3) mice compared to ApoE0 mice. However, at 72 hours accumulation of (125)I-LDL was similar in ApoE0/Hspg2(Delta3/Delta3) and ApoE0 mice. Immunohistochemistry of lesions from ApoE0/Hspg2(Delta3/Delta3) mice showed decreased staining for apoB and increased smooth muscle alpha-actin content, whereas accumulation of CD68-positive inflammatory cells was unchanged. We conclude that the perlecan HS chains are proatherogenic in mice, possibly through increased lipoprotein retention, altered vascular permeability, or other mechanisms. The ability of HS to inhibit smooth muscle cell growth may also influence development as well as instability of lesions.

The synthesis and secretion of versican isoform V3 by mammalian cells: A role for N-linked glycosylation.

Versican is a large extracellular matrix (ECM) chondroitin sulfate (CS) proteoglycan found in most soft tissues, which is encoded by the VCAN gene. At least four major isoforms (V0, V1, V2, and V3) are generated via alternative splicing. The isoforms of versican are expressed and accumulate in various tissues during development and disease, where they contribute to ECM structure, cell growth and migration, and immune regulation, among their many functions. While several studies have identified the mRNA transcript for the V3 isoform in a number of tissues, little is known about the synthesis, secretion, and targeting of the V3 protein. In this study, we used lentiviral generation of doxycycline-inducible rat V3 with a C-terminal tag in stable NIH 3T3 cell lines and demonstrated that V3 is processed through the classical secretory pathway. We further show that N-linked glycosylation is required for efficient secretion and solubility of the protein. By site-directed mutagenesis, we identified amino acids 57 and 330 as the active N-linked glycosylation sites on V3 when expressed in this cell type. Furthermore, exon deletion constructs of V3 revealed that exons 11-13, which code for portions of the carboxy region of the protein (G3 domain), are essential for V3 processing and secretion. Once secreted, the V3 protein associates with hyaluronan along the cell surface and within the surrounding ECM. These results establish critical parameters for the processing, solubility, and targeting of the V3 isoform by mammalian cells and establishes a role for V3 in the organization of hyaluronan.

Retrovirally mediated overexpression of versican v3 by arterial smooth muscle cells induces tropoelastin synthesis and elastic fiber formation in vitro and in neointima after vascular injury.

Versican is an extracellular matrix (ECM) proteoglycan that is synthesized as multiple splice variants. In a recent study, we demonstrated that retroviral-mediated overexpression of the variant V3, which lacks chondroitin sulfate (CS) chains, altered arterial smooth muscle cell (ASMC) phenotype in short-term cell culture. We now report that V3-overexpressing ASMCs exhibit significantly increased expression of tropoelastin and increased formation of elastic fibers in long-term cell cultures. In addition, V3-overexpressing ASMCs seeded into ballooned rat carotid arteries continued to overexpress V3 and, at 4 weeks after seeding, produced a highly structured neointima significantly enriched in elastic fiber lamellae. In contrast to the hydrated, myxoid neointima produced by rounded or stellate vector-alone--transduced cells, V3-expressing cells produced a compact and highly ordered neointima, which contained elongated ASMCs that were arranged in parallel arrays and separated by densely packed collagen bundles and elastic fibers. These results indicate that a variant of versican is involved in elastic fiber assembly and may represent a novel therapeutic approach to facilitate the formation of elastic fibers.

Changes in perlecan expression during vascular injury: role in the inhibition of smooth muscle cell proliferation in the late lesion.

OBJECTIVE: Vascular smooth muscle cells (SMCs), activated by growth factors after arterial injury, migrate and proliferate to expand the intima of the blood vessel. During intimal expansion, proliferation is suppressed and an increasingly large proportion of the neointimal mass is composed of newly synthesized extracellular matrix (ECM). We sough to determine whether the ECM heparan sulfate proteoglycan (HSPG) perlecan, which inhibits SMC proliferation in vitro, also accumulates and limits SMC proliferation during neointimal expansion. METHODS AND RESULTS: Perlecan expression and accumulation were analyzed by immunohistochemistry and in situ hybridization during neointima formation after balloon catheter injury to the rat carotid artery. Perlecan expression was low in uninjured vessels and up to 7 days after injury, during maximal SMC proliferation. By 14 days after injury, perlecan was dramatically increased, and immunostaining remained heavy throughout the advanced lesion, 35 to 42 days after injury. Finally, explants of intimal tissue from 35- to 42-day neointimal lesions were digested with glycosaminoglycanases to determine whether endogenous HSPGs inhibit intimal SMC proliferation. SMCs within HS-depleted, but not chondroitinase ABC-treated or mock-incubated, explants were found to proliferate in response to platelet-derived growth factor BB. CONCLUSIONS: HSPGs, such as perlecan, may inhibit the proliferative response of SMCs after vascular injury.

Overexpression of decorin by rat arterial smooth muscle cells enhances contraction of type I collagen in vitro.

OBJECTIVE: Overexpression of decorin reduces neointimal thickening in balloon-injured carotid arteries of rats by decreasing the volume of neointimal extracellular matrix (ECM). We examined the hypothesis that decorin regulates ECM volume by stimulating cell-mediated contraction of collagen-rich ECMs. METHODS AND RESULTS: Rat arterial smooth muscle cells (ASMCs) transduced with bovine decorin cDNA by retroviral transfection (LDSN) exhibited enhanced contraction of collagen gels in vitro when compared with vector-only transduced (LXSN) cells. Addition of recombinant decorin to LXSN or LDSN cells did not stimulate contraction of collagen gels. Enhanced contraction of collagen by LDSN cells was unaffected by the metalloproteinase inhibitor GM6001. LDSN cells exhibited increased expression of type I collagen mRNA when compared with that of LXSN cells. Correspondingly, collagen gel contraction by LDSN cells was reduced by inhibition of collagen synthesis by 3,4-l-dehydroproline (L-DHP). Antibodies to alpha1beta1-integrin, but not to alpha2beta1-integrin, blocked collagen contraction by both LXSN and LDSN cells. However, LXSN and LDSN cells expressed similar levels of alpha1- and beta1-integrin mRNAs. CONCLUSIONS: Decorin synthesized de novo by ASMCs increases type I collagen synthesis and enhances contraction of collagen gels. Regulated synthesis of decorin may be a useful therapeutic approach to reduce ECM volume in vascular disease.

The regulated synthesis of versican, decorin, and biglycan: extracellular matrix proteoglycans that influence cellular phenotype.

The principal extracellular matrix (ECM) chondroitin/dermatan sulfate proteoglycans include members of two gene families--the large aggregating chondroitin sulfate proteoglycans (lecticans) and the small leucine-rich proteoglycans (SLRPs). These families of proteoglycans are widely distributed within the interstitial matrix, where they are known to bind a variety of both soluble and insoluble ligands. Extensive structural studies and data concerning the synthesis of these proteoglycans have been published over the last few years. This review focuses on the regulation of the expression of the lectican, versican, and the SLRPs--decorin and biglycan, as well--studied and widely distributed examples of these families of ECM proteoglycans. In addition, the effects of these proteoglycans on the formation of the ECM and the response of cells to growth factors and cytokines are examined as mechanisms by which versican, decorin and biglycan, both directly and indirectly influence cellular proliferation, migration, and phenotype.

Removal of heparan sulfate by heparinase treatment inhibits FGF-2-dependent smooth muscle cell proliferation in injured rat carotid arteries.

Smooth muscle cells (SMC) of the rat carotid arterial media proliferate and migrate in response to injury during the formation of a neointima. The interaction of fibroblast growth factor (FGF-2), which is released at the site of injury, with heparan sulfate proteoglycans (HSPGs) is necessary to induce signaling, which elicits an FGF-dependent mitogenic response by arterial smooth muscle cells, and also serves as a mechanism for storage of the growth factor within the extracellular matrix. However, whether these interactions are critical during neointimal formation has not been directly tested. In this study, a model of FGF-2-dependent medial SMC mitogenic response in balloon-injured rat carotid artery was used to test the effect of degradation of vessel wall heparan sulfate on subsequent SMC proliferation. Treatment of balloon-catheterized rat carotid arteries with chondroitin ABC lyase and/or heparin lyases eliminated heparan sulfates in the vessel wall, as determined by immunoperoxidase staining. In contrast, the distribution in the carotid vessel wall of the large core protein of perlecan, a major vessel wall HSPG that binds FGF-2, is not decreased. The effect of glycosaminoglycan digestion in situ on medial SMC proliferation in response to a bolus injection of FGF-2 after injury was determined by measuring the percentage of SMC nuclei that incorporated 5-bromo-2'-deoxyuridine (BrdU) 48 h after injury. Enzymatic removal of heparan sulfate reduced BrdU incorporation into medial SMC by 60-70% (P < 0.001) at 48 h after injury. Moreover, pre-incubation of FGF-2 with heparin prior to injection restored SMC replication to the levels present in injured vessels treated with buffer alone (P < 0.01). These experiments indicate that endogenous HSPGs are essential to promote FGF-2-driven medial SMC proliferation following injury, and that heparinase treatment can abrogate FGF-2-dependent responses in vivo.

Smooth muscle cell biglycan overexpression results in increased lipoprotein retention on extracellular matrix: implications for the retention of lipoproteins in atherosclerosis.

Lipoprotein retention on extracellular matrix (ECM) may play a central role in atherogenesis, and a specific extracellular matrix proteoglycan, biglycan, has been implicated in lipoprotein retention in human atherosclerosis. To test whether increased cellular biglycan expression results in increased retention of lipoproteins on ECM, rat aortic smooth muscle cells (SMCs) were transduced with a human biglycan cDNA-containing retroviral vector (LBSN) or with an empty retroviral vector (LXSN). To assess the importance of biglycan's glycosaminoglycan side chains in lipoprotein retention, ECM binding studies were also performed using RASMCs transduced with a retroviral vector encoding for a mutant, glycosaminoglycan-deficient biglycan (LBmutSN). Human biglycan mRNA and protein were confirmed in LBSN and LBmutSN RASMCs by Northern and Western blot analyses. HDL3+E binding to SMC ECM was increased significantly (as determined by 95% confidence intervals for binding curves) for LBSN as compared to either LXSN or LBmutSN cells; the increases for LBSN cell ECM were due primarily to an approximately 50% increase in binding sites (increased Bmax) versus LXSN cell ECM and of approximately 25% versus LBmutSN cell ECM. These results are consistent with the hypothesis that biglycan, through its glycosaminoglycan side chains, may mediate lipoprotein retention on atherosclerotic plaque ECM.

Reprint of: A rapid increase in macrophage-derived versican and hyaluronan in infectious lung disease.

The goals of this study were to characterize the changes in chondroitin sulfate proteoglycans and hyaluronan in lungs in acute response to gram-negative bacterial infection and to identify cellular components responsible for these changes. Mice were treated with intratracheal (IT) live Escherichia coli, E. coli lipopolysaccharide (LPS), or PBS. Both E. coli and LPS caused rapid selective increases in mRNA expression of versican and hyaluronan synthase (Has) isoforms 1 and 2 associated with increased immunohistochemical and histochemical staining for versican and hyaluronan in the lungs. Versican was associated with a subset of alveolar macrophages. To examine whether macrophages contribute to versican and hyaluronan accumulation, in vitro studies with primary cultures of bone marrow-derived and alveolar macrophages were performed. Unstimulated macrophages expressed very low levels of versican and hyaluronan synthase mRNA, with no detectible versican protein or hyaluronan product. Stimulation with LPS caused rapid increases in versican mRNA and protein, a rapid increase in Has1 mRNA, and concomitant inhibition of hyaluronidases 1 and 2, the major hyaluronan degrading enzymes. Hyaluronan could be detected following chloroquine pre-treatment, indicating rapid turnover and degradation of hyaluronan by macrophages. In addition, the effects of LPS, the M1 macrophage classical activation agonist, were compared to those of IL-4/IL-13 or IL-10, the M2a and M2c alternative activation agonists, respectively. Versican and Has1 increased only in response to M1 activation. Finally, the up-regulation of versican and Has1 in the whole lungs of wild-type mice following IT LPS was completely abrogated in TLR-4(-/-) mice. These findings suggest that versican and hyaluronan synthesis may play an important role in the innate immune response to gram-negative lung infection.

A rapid increase in macrophage-derived versican and hyaluronan in infectious lung disease.

The goals of this study were to characterize the changes in chondroitin sulfate proteoglycans and hyaluronan in lungs in acute response to gram-negative bacterial infection and to identify cellular components responsible for these changes. Mice were treated with intratracheal (IT) live Escherichia coli, E. coli lipopolysaccharide (LPS), or PBS. Both E. coli and LPS caused rapid selective increases in mRNA expression of versican and hyaluronan synthase (Has) isoforms 1 and 2 associated with increased immunohistochemical and histochemical staining for versican and hyaluronan in the lungs. Versican was associated with a subset of alveolar macrophages. To examine whether macrophages contribute to versican and hyaluronan accumulation, in vitro studies with primary cultures of bone marrow-derived and alveolar macrophages were performed. Unstimulated macrophages expressed very low levels of versican and hyaluronan synthase mRNA, with no detectible versican protein or hyaluronan product. Stimulation with LPS caused rapid increases in versican mRNA and protein, a rapid increase in Has1 mRNA, and concomitant inhibition of hyaluronidases 1 and 2, the major hyaluronan degrading enzymes. Hyaluronan could be detected following chloroquine pre-treatment, indicating rapid turnover and degradation of hyaluronan by macrophages. In addition, the effects of LPS, the M1 macrophage classical activation agonist, were compared to those of IL-4/IL-13 or IL-10, the M2a and M2c alternative activation agonists, respectively. Versican and Has1 increased only in response to M1 activation. Finally, the up-regulation of versican and Has1 in the whole lungs of wild-type mice following IT LPS was completely abrogated in TLR-4(-/-) mice. These findings suggest that versican and hyaluronan synthesis may play an important role in the innate immune response to gram-negative lung infection.

Inhibition of PDGF-B induction and cell growth by syndecan-1 involves the ubiquitin and SUMO-1 ligase, Topors.

Syndecans are receptors for soluble ligands, including heparin-binding growth factors, and matrix proteins. However, intracellular targets of syndecan-1 (Sdc-1)-mediated signaling are not fully understood. A yeast two-hybrid protein interaction screening of a mouse embryo library identified the ubiquitin and SUMO-1 E3 ligase, Topors, as a novel ligand of the Sdc-1 cytoplasmic domain (S1CD), a finding confirmed by ligand blotting and co-precipitation with Sdc-1 from cell lysates. Deletion mutagenesis identified an 18-amino acid sequence of Topors required for the interaction with the S1CD. By immunohistochemistry, Topors and Sdc-1 co-localized near the cell periphery in normal murine mammary gland (NMuMG) cells in vitro and in mouse embryonic epithelia in vivo. Finally, siRNA-mediated knockdown of Topors demonstrated that Topors is a growth promoter for murine arterial smooth muscle cells and is required for the inhibitory effect of Sdc-1 on cell growth and platelet-derived growth factor-B induction. These data suggest a novel mechanism for the inhibitory effects of Sdc-1 on cell growth that involves the interaction between the cytoplasmic domain of Sdc-1 and the SUMO-1 E3 ligase, Topors.

Hyaluronan accumulation is elevated in cultures of low density lipoprotein receptor-deficient cells and is altered by manipulation of cell cholesterol content.

The extracellular matrix molecule hyaluronan (HA) accumulates in human atherosclerotic lesions. Yet the reasons for this accumulation have not been adequately addressed. Because abnormalities in lipid metabolism promote atherosclerosis, we have asked whether disrupted cholesterol homeostasis alters HA accumulation in low density lipoprotein receptor-deficient cell cultures. Cultured aortic smooth muscle cells (ASMC) from Watanabe heritable hyperlipidemic (WHHL) rabbits and skin fibroblasts from homozygous patients with familial hypercholesterolemia accumulated 2-4-fold more HA than corresponding cells from age- and sex-matched normolipidemic rabbits and individuals. This occurred in both cell-associated and secreted HA fractions and was independent of cell density or medium serum concentration. WHHL ASMC cultures synthesized twice the proportion of high molecular mass HA (>2x10(6) Da) as normal rabbit ASMC but showed a lower capacity to degrade exogenous [3H]HA. Most importantly, cholesterol depletion or blocking cholesterol synthesis markedly reduced HA accumulation in WHHL ASMC cultures, whereas cholesterol replenishment or stimulation of cholesterol synthesis restored elevated HA levels. We conclude the following: 1) maintaining normal HA levels in cell cultures requires normal cell cholesterol homeostasis; 2) HA degradation may contribute to but is not the predominant mechanism to increase high molecular mass HA accumulation in low density lipoprotein receptor-deficient WHHL ASMC cultures; and 3) elevated accumulation of HA depends on cellular or membrane cholesterol content and, potentially, intact cholesterol-rich microdomains.

Characterization of chondroitin/dermatan sulfate proteoglycans synthesized by bovine retinal pericytes in culture.

Pericytes associate with the outside of endothelial cells in microvessels. Previous studies have shown that these cells synthesize glycosaminoglycans (GAGs) but the nature of the core proteins to which these GAGs are attached is unknown. In the present study, cultured bovine retinal pericytes were metabolically labeled with [(3)H]glucosamine, [(35)S]sodium sulfate or (35)S-labeled amino acids and the proteoglycans synthesized by these cells were purified by DEAE-Sephacel ion exchange and molecular sieve Sepharose CL-4B chromatography. Separated proteoglycans were digested with papain, heparitinase or chondroitin ABC lyase and the GAGs characterized by Sepharose CL-6B chromatography. Proteoglycans were also assessed by sodium dodecyl sulfate polyacrylamide gel electrophoresis before and after digestion with chondroitin ABC lyase. Pericytes predominantly synthesize and secrete chondroitin or dermatan sulfate proteoglycans (CS/DS PGs) rather than heparan sulfate proteoglycans (HSPGs). Two subclasses of CS/DS PGs are synthesized by pericytes; one is a high M(r) subclass with high charge density. This subclass eluted at the void volume of a Sepharose CL-4B molecular sieve column, was susceptible to chondroitin ABC lyase, and contained core proteins of ca. 550 and 450 kD which were recognized by antibody to versican. The other major subclass eluted at a K(av) ca. 0.45 on a Sepharose CL-4B molecular sieve column, was susceptible to chondroitin ABC lyase, and contained core proteins recognized by antibodies to either biglycan or decorin that separated as a broad band of ca. 50 kDa in SDS-PAGE. A small amount of HSPG was also synthesized by these cells and could be separated from the CS/DS PGs by DEAE-Sephacel chromatography using a linear gradient of 0.1-0.7 M NaCl. Release of GAG chains by protease digestion indicated that the length of GAG chains was approximately M(r) 45000 in biglycan and decorin, approximately M(r) 48000 in the small amount of HSPGs and approximately M(r) 66000 in versican. These proteoglycans resemble those synthesized by vascular smooth muscle cells but differ markedly from those synthesized by vascular endothelial cells.