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.

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.

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.

Filamentous Bacteriophage Produced by Pseudomonas aeruginosa Alters the Inflammatory Response and Promotes Noninvasive Infection In Vivo.

Pseudomonas aeruginosa is an important opportunistic human pathogen that lives in biofilm-like cell aggregates at sites of chronic infection, such as those that occur in the lungs of patients with cystic fibrosis and nonhealing ulcers. During growth in a biofilm, P. aeruginosa dramatically increases the production of filamentous Pf bacteriophage (Pf phage). Previous work indicated that when in vivo Pf phage production was inhibited, P. aeruginosa was less virulent. However, it is not clear how the production of abundant quantities of Pf phage similar to those produced by biofilms under in vitro conditions affects pathogenesis. Here, using a murine pneumonia model, we show that the production of biofilm-relevant amounts of Pf phage prevents the dissemination of P. aeruginosa from the lung. Furthermore, filamentous phage promoted bacterial adhesion to mucin and inhibited bacterial invasion of airway epithelial cultures, suggesting that Pf phage traps P. aeruginosa within the lung. The in vivo production of Pf phage was also associated with reduced lung injury, reduced neutrophil recruitment, and lower cytokine levels. Additionally, when producing Pf phage, P. aeruginosa was less prone to phagocytosis by macrophages than bacteria not producing Pf phage. Collectively, these data suggest that filamentous Pf phage alters the progression of the inflammatory response and promotes phenotypes typically associated with chronic infection.

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.

Filamentous Bacteriophage Promote Biofilm Assembly and Function.

Biofilms-communities of bacteria encased in a polymer-rich matrix-confer bacteria with the ability to persist in pathologic host contexts, such as the cystic fibrosis (CF) airways. How bacteria assemble polymers into biofilms is largely unknown. We find that the extracellular matrix produced by Pseudomonas aeruginosa self-assembles into a liquid crystal through entropic interactions between polymers and filamentous Pf bacteriophages, which are long, negatively charged filaments. This liquid crystalline structure enhances biofilm function by increasing adhesion and tolerance to desiccation and antibiotics. Pf bacteriophages are prevalent among P. aeruginosa clinical isolates and were detected in CF sputum. The addition of Pf bacteriophage to sputum polymers or serum was sufficient to drive their rapid assembly into viscous liquid crystals. Fd, a related bacteriophage of Escherichia coli, has similar biofilm-building capabilities. Targeting filamentous bacteriophage or the liquid crystalline organization of the biofilm matrix may represent antibacterial strategies.

Expression of V3 Versican by Rat Arterial Smooth Muscle Cells Promotes Differentiated and Anti-inflammatory Phenotypes.

Arterial smooth muscle cells (ASMCs) undergo phenotypic changes during development and pathological processes in vivo and during cell culture in vitro. Our previous studies demonstrated that retrovirally mediated expression of the versican V3 splice variant (V3) by ASMCs retards cell proliferation and migration in vitro and reduces neointimal thickening and macrophage and lipid accumulation in animal models of vascular injury and atherosclerosis. However, the molecular pathways induced by V3 expression that are responsible for these changes are not yet clear. In this study, we employed a microarray approach to examine how expression of V3 induced changes in gene expression and the molecular pathways in rat ASMCs. We found that forced expression of V3 by ASMCs affected expression of 521 genes by more than 1.5-fold. Gene ontology analysis showed that components of the extracellular matrix were the most significantly affected by V3 expression. In addition, genes regulating the formation of the cytoskeleton, which also serve as markers of contractile smooth muscle cells (SMCs), were significantly up-regulated. In contrast, components of the complement system, chemokines, chemokine receptors, and transcription factors crucial for regulating inflammatory processes were among the genes most down-regulated. Consistently, we found that the level of myocardin, a key transcription factor promoting contractile SMC phenotype, was greatly increased, and the proinflammatory transcription factors NFκB1 and CCAAT/enhancer-binding protein ß were significantly attenuated in V3-expressing SMCs. Overall, these findings demonstrate that V3 expression reprograms ASMCs promoting differentiated and anti-inflammatory phenotypes.

Expression of versican V3 by arterial smooth muscle cells alters tumor growth factor ß (TGFß)-, epidermal growth factor (EGF)-, and nuclear factor κB (NFκB)-dependent signaling pathways, creating a microenvironment that resists monocyte adhesion.

Monocyte/macrophage accumulation plays a critical role during progression of cardiovascular diseases, such as atherosclerosis. Our previous studies demonstrated that retrovirally mediated expression of the versican V3 splice variant (V3) by arterial smooth muscle cells (ASMCs) decreases monocyte adhesion in vitro and macrophage accumulation in a model of lipid-induced neointimal formation in vivo. We now demonstrate that V3-expressing ASMCs resist monocyte adhesion by altering the composition of the microenvironment surrounding the cells by affecting multiple signaling pathways. Reduction of monocyte adhesion to V3-expressing ASMCs is due to the generation of an extracellular matrix enriched in elastic fibers and depleted in hyaluronan, and reduction of the proinflammatory cell surface vascular cell adhesion molecule 1 (VCAM1). Blocking these changes reverses the protective effect of V3 on monocyte adhesion. The enhanced elastogenesis induced by V3 expression is mediated by TGFß signaling, whereas the reduction in hyaluronan cable formation induced by V3 expression is mediated by the blockade of epidermal growth factor receptor and NFκB activation pathways. In addition, expression of V3 by ASMCs induced a marked decrease in NFκB-responsive proinflammatory cell surface molecules that mediate monocyte adhesion, such as VCAM1. Overall, these results indicate that V3 expression by ASMCs creates a microenvironment resistant to monocyte adhesion via differentially regulating multiple signaling pathways.

ADAMTS-4 and biglycan are expressed at high levels and co-localize to podosomes during endothelial cell tubulogenesis in vitro.

Proteolysis of the extracellular matrix influences vascular growth. We examined the expression of ADAMTS-1, -4, and -5 metalloproteinases and their proteoglycan substrates versican, decorin, and biglycan as human umbilical vein endothelial cells (HUVECs) formed tubes within type I collagen gels in vitro. Tubulogenic and control HUVEC cultures expressed low levels of ADAMTS-1 and -5 mRNAs, but ADAMTS-4 mRNA was relatively abundant and was significantly elevated (as was ADAMTS-4 protein) in tubulogenic cultures versus controls. Immunocytochemistry revealed ADAMTS-4 in f-actin- and cortactin-positive podosome-like puncta in single cells and mature tubes. Tubulogenic and control cultures expressed low levels of versican and decorin mRNAs; however, peak levels of biglycan mRNA were 400- and 16,000-fold that of versican and decorin, respectively. Biglycan mRNA was highest at 3 hr, declined steadily through day 7 and, at 12 hr and beyond, was significantly lower in tubulogenic cultures than in controls. Western blots of extracellular matrix from tubulogenic cultures contained bands corresponding to biglycan and its cleavage products. By immunocytochemistry, biglycan was found in the pericellular matrix surrounding endothelial tubes and in cell-associated puncta that co-localized with ADAMTS-4 and cortactin. Collectively, our results suggest that ADAMTS-4 and its substrate biglycan are involved in tubulogenesis by endothelial cells.

Endothelial deletion of ADAM17 in mice results in defective remodeling of the semilunar valves and cardiac dysfunction in adults.

Global inactivation of the metalloproteinase ADAM17 during mouse development results in perinatal lethality and abnormalities of the heart, including late embryonic cardiomegaly and thickened semilunar and atrioventricular valves. These defects have been attributed in part to a lack of ADAM17-mediated processing of HB-EGF, as absence of soluble HB-EGF results in similar phenotypes. Because valvular mesenchymal cells are largely derived from cardiac endothelial cells, we generated mice with a floxed Adam17 allele and crossed these animals with Tie2-Cre transgenics to focus on the role of endothelial ADAM17 in valvulogenesis. We find that although hearts from late-stage embryos with ablation of endothelial ADAM17 appear normal, an increase in valve size and cell number is evident, but only in the semilunar cusps. Unlike Hbegf(-/-) valves, ADAM17-null semilunar valves do not differ from controls in acute cell proliferation at embryonic day 14.5 (E14.5), suggesting compensatory processing of HB-EGF. However, levels of the proteoglycan versican are significantly reduced in mutant hearts early in valve remodeling (E12.5). After birth, aortic valve cusps from mutants are not only hyperplastic but also show expansion of the glycosaminoglycan-rich component, with the majority of adults exhibiting aberrant compartmentalization of versican and increased deposition of collagen. The inability of mutant outflow valve precursors to transition into fully mature cusps is associated with decreased postnatal viability, progressive cardiomegaly, and systolic dysfunction. Together, our data indicate that ADAM17 is required in valvular endothelial cells for regulating cell content as well as extracellular matrix composition and organization in semilunar valve remodeling and homeostasis.

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 and hyaluronan binding proteins are normal components of mouse pancreatic islets and are differentially expressed by islet endocrine cell types.

The pancreatic islet comprises endocrine, vascular, and neuronal cells. Signaling among these cell types is critical for islet function. The extracellular matrix (ECM) is a key regulator of cell-cell signals, and while some islet ECM components have been identified, much remains unknown regarding its composition. We investigated whether hyaluronan, a common ECM component that may mediate inflammatory events, and molecules that bind hyaluronan such as versican, tumor necrosis factor-stimulated gene 6 (TSG-6), and components of inter-α-trypsin inhibitor (IαI), heavy chains 1 and 2 (ITIH1/ITIH2), and bikunin, are normally produced in the pancreatic islet. Mouse pancreata and isolated islets were obtained for microscopy (with both paraformaldehyde and Carnoy's fixation) and mRNA. Hyaluronan was present predominantly in the peri-islet ECM, and hyaluronan synthase isoforms 1 and 3 were also expressed in islets. Versican was produced in α cells; TSG-6 in α and ß cells; bikunin in α, ß, and δ cells; and ITIH1/ITIH2 predominantly in ß cells. Our findings demonstrate that hyaluronan, versican, TSG-6, and IαI are normal islet components and that different islet endocrine cell types contribute these ECM components. Thus, dysfunction of either α or ß cells likely alters islet ECM composition and could thereby further disrupt islet function.

Diabetes promotes an inflammatory macrophage phenotype and atherosclerosis through acyl-CoA synthetase 1.

The mechanisms that promote an inflammatory environment and accelerated atherosclerosis in diabetes are poorly understood. We show that macrophages isolated from two different mouse models of type 1 diabetes exhibit an inflammatory phenotype. This inflammatory phenotype associates with increased expression of long-chain acyl-CoA synthetase 1 (ACSL1), an enzyme that catalyzes the thioesterification of fatty acids. Monocytes from humans and mice with type 1 diabetes also exhibit increased ACSL1. Furthermore, myeloid-selective deletion of ACSL1 protects monocytes and macrophages from the inflammatory effects of diabetes. Strikingly, myeloid-selective deletion of ACSL1 also prevents accelerated atherosclerosis in diabetic mice without affecting lesions in nondiabetic mice. Our observations indicate that ACSL1 plays a critical role by promoting the inflammatory phenotype of macrophages associated with type 1 diabetes; they also raise the possibilities that diabetic atherosclerosis has an etiology that is, at least in part, distinct from the etiology of nondiabetic vascular disease and that this difference is because of increased monocyte and macrophage ACSL1 expression.

Monocyte-to-macrophage differentiation: synthesis and secretion of a complex extracellular matrix.

Although monocyte- and macrophage-derived molecules are known to promote extracellular matrix (ECM) disruption and destabilization, it is less appreciated that they also synthesize molecules contributing to ECM formation, stabilization, and function. We have identified and characterized the synthesis of proteoglycans and related proteins, some not previously known to be associated with macrophages. Proteoglycan extracts of [(35)S]sulfate- and (35)S-trans amino acid-radiolabeled culture media from THP-1 monocytes induced to differentiate by treatment with phorbol myristate acetate revealed three major proteins of ~25, 90, and 100 kDa following chondroitin ABC lyase digestion. The 25-kDa protein was predominant for monocytes, whereas the 90- and 100-kDa proteins were predominant for macrophages. Tandem mass spectrometry identified (i) the 25-kDa core protein as serglycin, (ii) the 90-kDa core protein as inter-α-inhibitor heavy chain 2 (IαIHC2), and (iii) the 100-kDa core as amyloid precursor-like protein 2 (APLP2). Differentiation was also associated with (i) a >500-fold increase in mRNA for TNF-stimulated gene-6, an essential cofactor for heavy chain-mediated matrix stabilization; (ii) a >800-fold increase in mRNA for HAS2, which is responsible for hyaluronan synthesis; and (iii) a 3-fold increase in mRNA for versican, which interacts with hyaluronan. Biochemical evidence is also presented for an IαIHC2-APLP2 complex, and immunohistochemical staining of human atherosclerotic lesions demonstrates similar staining patterns for APLP2 and IαIHC2 with macrophages, whereas serglycin localizes to the underlying glycosaminoglycan-rich region. These findings indicate that macrophages synthesize many of the molecules participating in ECM formation and function, suggesting a novel role for these molecules in the differentiation of macrophages in the development of atherosclerosis.

ECM components guide IL-10 producing regulatory T-cell (TR1) induction from effector memory T-cell precursors.

We describe a role for ECM as a biosensor for inflammatory microenvironments that plays a critical role in peripheral immune tolerance. We show that hyaluronan (HA) promotes induction of Foxp3- IL-10-producing regulatory T cells (TR1) from conventional T-cell precursors in both murine and human systems. This is, to our knowledge, the first description of an ECM component inducing regulatory T cells. Intact HA, characteristic of healing tissues, promotes induction of TR1 capable of abrogating disease in an IL-10-dependent mouse colitis model whereas fragmentary HA, typical of inflamed tissues, does not, indicating a decisive role for tissue integrity in this system. The TR1 precursor cells in this system are CD4(+)CD62L(-)FoxP3(-), suggesting that effector memory cells assume a regulatory phenotype when they encounter their cognate antigen in the context of intact HA. Matrix integrity cues might thereby play a central role in maintaining peripheral tolerance. This TR1 induction is mediated by CD44 cross-linking and signaling through p38 and ERK1/2. This induction is suppressed, also in a CD44-dependent manner, by osteopontin, a component of chronically inflamed ECM, indicating that CD44 signaling serves as a nexus for fate decisions regarding TR1 induction. Finally, we demonstrate that TR1 induction signals can be recapitulated using synthetic matrices. These results reveal important roles for the matrix microenvironment in immune regulation and suggest unique strategies for immunomodulation.

Neointima formed by arterial smooth muscle cells expressing versican variant V3 is resistant to lipid and macrophage accumulation.

OBJECTIVE: Extracellular matrix (ECM) of neointima formed following angioplasty contains elevated levels of versican, loosely arranged collagen, and fragmented deposits of elastin, features associated with lipid and macrophage accumulation. ECM with a low versican content, compact structure, and increased elastic fiber content can be achieved by expression of versican variant V3, which lacks chondroitin sulfate glycosaminoglycans. We hypothesized that V3-expressing arterial smooth muscle cells (ASMC) can be used to form a neointima resistant to lipid and macrophage accumulation associated with hypercholesterolemia. METHODS AND RESULTS: ASMC transduced with V3 cDNA were seeded into ballooned rabbit carotid arteries, and animals were fed a chow diet for 4 weeks, followed by a cholesterol-enriched diet for 4 weeks, achieving plasma cholesterol levels of 20 to 25 mmol/L. V3 neointimae at 8 weeks were compact, multilayered, and elastin enriched. They were significantly thinner (57%) than control neointimae; contained significantly more elastin (118%), less collagen (22%), and less lipid (76%); and showed significantly reduced macrophage infiltration (85%). Mechanistic studies demonstrated that oxidized low-density lipoprotein stimulated the formation of a monocyte-binding ECM, which was inhibited in the presence of V3 expressing ASMC. CONCLUSION: These results demonstrate that expression of V3 in vessel wall creates an elastin-rich neointimal matrix that in the presence of hyperlipidemia is resistant to lipid deposition and macrophage accumulation.

Differentiation of cardiomyocytes from human embryonic stem cells is accompanied by changes in the extracellular matrix production of versican and hyaluronan.

Proteoglycans and hyaluronan play critical roles in heart development. In this study, human embryonic stem cells (hESC) were used as a model to quantify the synthesis of proteoglycans and hyaluronan in hESC in the early stages of differentiation, and after directed differentiation into cardiomyocytes. We demonstrated that both hESC and cardiomyocyte cultures synthesize an extracellular matrix (ECM) enriched in proteoglycans and hyaluronan. During cardiomyocyte differentiation, total proteoglycan and hyaluronan decreased and the proportion of proteoglycans bearing heparan sulfate chains was reduced. Versican, a chondroitin sulfate proteoglycan, accumulated in hESC and cardiomyocyte cultures. Furthermore, versican synthesized by hESC contained more N- and O-linked oligosaccharide than versican from cardiomyocytes. Transcripts for the versican variants, V0, V1, V2, and V3, increased in cardiomyocytes compared to hESC, with V1 most abundant. Hyaluronan in hESC had lower molecular weight than hyaluronan from cardiomyocyte cultures. These changes were accompanied by an increase in HAS-1 and HAS-2 mRNA in cardiomyocyte cultures, with HAS-2 most abundant. Interestingly, HAS-3 was absent from the cardiomyocyte cultures, but expressed by hESC. These results indicate that human cardiomyocyte differentiation is accompanied by specific changes in the expression and accumulation of ECM components and suggest a role for versican and hyaluronan in this process.

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.

Synthesis and organization of hyaluronan and versican by embryonic stem cells undergoing embryoid body differentiation.

Embryonic stem cells (ESCs) provide a convenient model to probe the molecular and cellular dynamics of developmental cell morphogenesis. ESC differentiation in vitro via embryoid bodies (EBs) recapitulates many aspects of early stages of development, including the epithelial-mesenchymal transition (EMT) of pluripotent cells into more differentiated progeny. Hyaluronan and versican are important extracellular mediators of EMT processes, yet the temporal expression and spatial distribution of these extracellular matrix (ECM) molecules during EB differentiation remains undefined. Thus, the objective of this study was to evaluate the synthesis and organization of hyaluronan and versican by using murine ESCs during EB differentiation. Hyaluronan and versican (V0 and V1 isoforms), visualized by immunohistochemistry and evaluated biochemically, accumulated within EBs during the course of differentiation. Interestingly, increasing amounts of a 70-kDa proteolytic fragment of versican were also detected over time, along with ADAMTS-1 and -5 protein expression. ESCs expressed each of the hyaluronan synthases (HAS) -1, -2, and -3 and versican splice variants (V0, V1, V2, and V3) throughout EB differentiation, but HAS-2, V0, and V1 were expressed at significantly increased levels at each time point examined. Hyaluronan and versican exhibited overlapping expression patterns within EBs in regions of low cell density, and versican expression was excluded from clusters of epithelial (cytokeratin-positive) cells but was enriched within the vicinity of mesenchymal (N-cadherin-positive) cells. These results indicate that hyaluronan and versican synthesized by ESCs within EB microenvironments are associated with EMT processes and furthermore suggest that endogenously produced ECM molecules play a role in ESC differentiation. This manuscript contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials.

Polyinosine-polycytidylic acid stimulates versican accumulation in the extracellular matrix promoting monocyte adhesion.

Viral infections are known to exacerbate asthma and other lung diseases in which chronic inflammatory processes are implicated, but the mechanism is not well understood. The viral mimetic, polyinosine-polycytidylic acid, causes accumulation of a versican- and hyaluronan-enriched extracellular matrix (ECM) by human lung fibroblasts with increased capacity for monocyte adhesion. The fivefold increase in versican retention in this ECM is due to altered compartmentalization, with decreased degradation of cell layer-associated versican, rather than an increase in total accumulation in the culture. This is consistent with decreased mRNA levels for all of the versican splice variants. Reduced versican degradation is further supported by low levels of the epitope, DPEAAE, a product of versican digestion by a disintegrin-like and metallopeptidase with thrombospondin type 1 motif enzymes, in the ECM. The distribution of hyaluronan is similarly altered with a 3.5-fold increase in the cell layer. Pulse-chase studies of radiolabeled hyaluronan show a 50% reduction in the rate of loss from the cell layer over 24 hours. Formation of monocyte-retaining, hyaluronidase-sensitive ECMs can be blocked by the presence of anti-versican antibodies. In comparison, human lung fibroblasts treated with the cytokines, IL-1beta plus TNF-alpha, synthesize increased amounts of hyaluronan, but do not retain it or versican in the ECM, which, in turn, does not retain monocytes. These results highlight an important role for versican in the hyaluronan-dependent binding of monocytes to the ECM of lung fibroblasts stimulated with polyinosine-polycytidylic acid.