Major chondroitin sulfate proteoglycans identified in L6J1 myoblast culture

The major proteoglycans from L6J1 rat myoblast culture were identified. The proteoglycans were isolated from different constituents of cell culture: culture medium, extracellular matrix (ECM), and myoblasts. To identify their core proteins, the proteoglycans were treated with enzymes specifically digesting chondroitin/dermatan sulfates or chondroitin sulfates. Subsequent electrophoresis and mass spectrometry revealed versican, collagen XII, and inter-α-trypsin inhibitor classified as chondroitin sulfate proteoglycans and biglycan known to be chondroitin/dermatan sulfate proteoglycan. Versican was identified in ECM and the other proteoglycans in the culture medium. Such difference in localization is likely to be a consequence of different biological functions. Versican, collagen XII, and biglycan are synthesized by myoblasts and inter-α-trypsin inhibitor originates from fetal bovine serum (a culture medium component).     

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.     

Expression of versican and ADAMTS1, 4, and 5 during bone development in the rat mandible and hind limb.

Extracellular matrix (ECM) remodeling is achieved by both production and degradation of ECM molecules during bone development. ADAMTS (a disintegrin and metalloprotease with thrombospondin type 1 motifs) constitutes a family of extracellular proteases which are implicated in cleaving the protein versican. The present study was designed to investigate the expression of versican and ADAMTS1, 4, and 5 mRNA during bone development in rat mandibles and hind limbs by RT-PCR and in situ hybridization. Versican was localized by immunohistochemistry. The process of bone development from day 14 postcoitum through week 6 postnatum was divided into the beginning of osteogenesis, woven bone, and lamellar bone stages. Versican protein was abundant in the woven bone matrix, but decreased in the lamellar bone matrix. Versican mRNA was prominent in some osteoblasts with corresponding localization of the cognate protein. The temporal and spatial mRNA expression pattern of ADAMTS1, 4, and 5 was comparable to that of versican. These results suggest that woven bone rich in versican alters into lamellar bone containing little versican during bone development in both mandibles and hind limbs, where some osteoblasts may be involved in production as well as degradation of versican by secreting ADAMTS1, 4, and 5.     

The roles of versican V1 and V2 isoforms in cell proliferation and apoptosis

Versican is a large chondroitin sulfate proteoglycan belonging to the lectican family. Alternative splicing of versican generates at least four isoforms named V0, V1, V2, and V3. We have shown that the versican V1 isoform not only enhanced cell proliferation, but also modulated cell cycle progression and protected the cells from apoptosis. Futhermore, the V1 isoform was able to not only activate proto-oncogene EGFR expression and modulate its downstream signaling pathway, but also induce p27 degradation and enhance CDK2 kinase activity. As well, the V1 isoform down-regulated the expression of the proapoptotic protein Bad. By contrast, the V2 isoform exhibited opposite biological activities by inhibiting cell proliferation and down-regulated the expression of EGFR and cyclin A. Furthermore, V2 did not contribute apoptotic resistance to the cells. In light of these results, we are reporting opposite functions for the two versican isoforms whose expression is differentially regulated. Our studies suggest that the roles of these two isoforms are associated with the subdomains CSbeta and CSalpha, respectively. These results were confirmed by silencing the expression of versican V1 with small interfering RNA (siRNA), which abolished V1-enhanced cell proliferation and V1-induced reduction of apoptosis.     

Calcium-dependent self-association of the C-type lectin domain of versican

Versican is a large (1-2 x 10(6) Da) chondroitin-sulfate proteoglycan that can form large aggregates by means of interaction with hyaluronan and also binds to a series of other extracellular matrix proteins, chemokines and cell-surface molecules. Versican is a multifunctional molecule with roles in cell adhesion, matrix assembly, cell migration and proliferation. Characterization of the binding interactions mediated by the various domains of versican is a first step towards understanding the functions of versican and interacting molecules in the extracellular matrix. In this study we investigated a recombinant construct corresponding to the C-type lectin domain of versican and demonstrated a calcium-dependent self-association of this region by blot overlay and plasmon surface resonance assays. Electron microscopy provided further evidence of the relevance of the binding reaction by demonstrating a mixture of monomers, dimers and complex aggregates of recombinant versican C-type lectin domain. This binding reaction could contribute to the ability of versican to organize formation of the proteoglycan extracellular matrix by inducing binding of individual versican molecules or by modulating binding reactions to other matrix components.     

Pancreatic tumor cells influence the composition of the extracellular matrix

The malignant behavior of cancers depends on the microenvironmental context. We investigated compositional alterations of the extracellular matrix (ECM) in pancreatic cancer, with special emphasis on the proteoglycans decorin, lumican, and versican. Compared with normal controls (n=18), marked overexpression of these proteoglycans was observed in pancreatic cancer tissues (n=30) by quantitative RT-PCR (p<0.0001). Immunohistochemistry revealed abundance of proteoglycans in the ECM of pancreatic cancer specimens, whereas tumor cells themselves were devoid of either decorin, lumican or versican. RT-PCR confirmed pancreatic stellate cells (PSCs) as the major source of these proteins. Interestingly, TGFbeta1 and conditioned medium derived from pancreatic cancer cell lines synergistically suppressed the expression of known anti-tumor factors decorin and lumican, but stimulated the expression of pro-metastatic factor versican in cultured PSCs. These findings indicate that malignant cells can actively influence the composition of the ECM through TGFbeta1 and other soluble factors, altering their microenvironment in a tumor-favorable way.     

Expression of Versican and ADAMTS During Rat Tooth Eruption

Summary A disintegrin and metalloprotease with thrombospondin type 1 motifs (ADAMTS) is a family of extracellular proteases and implicated in cleaving proteoglycans, such as aggrecan, versican and brevican. No information is available about expression or localization of these ADAMTSs in teeth. Versican is a large chondroitin sulfate proteoglycan that is present in a variety of connective tissue including dental pulp, dentin, cementum and periodontal ligaments. The present study was designed to investigate expression of ADAMTSs and versican during rat tooth eruption. Rat maxillary first molars in weeks 1, 2, 3, 4 and 6 were examined. The mRNA expression of ADAMTS1, ADAMTS4, ADAMTS5 and versican was localized using in situ hybridization. ADAMTS1, ADAMTS4, ADAMTS5 and versican were expressed in dental pulp cells, odontoblasts, cementoblasts, cementocytes, periodontal ligament cells, osteoblasts and osteocytes. The temporal and spatial expression pattern in these cellular phenotypes was comparable among ADAMTSs and versican. The present study suggests that dental pulp cells, odontoblasts, cementoblasts, cementocytes, periodontal ligament cells, osteoblasts and osteocytes may be involved in both production and degradation of versican with secreting ADAMTS1, ADAMTS4 and ADAMTS5.     

Interleukin-11 promotes the progress of gastric carcinoma via abnormally expressed versican

Versican, a ubiquitous component of the extracellular matrix (ECM), accumulates both in tumor stroma and cancer cells and is highly regulated by various cytokines. The aberrant expression of versican and its isoforms is known to modulate cell proliferation, differentiation, and migration, all of which are features of the invasion and metastasis of cancer; versican is also known to favour the homeostasis of the ECM. Interleukin-11 (IL-11) is an important cytokine that exhibits a wide variety of biological effects in gastric cancer development. Here, we analysed the expression of versican isoforms and found that the major isoforms expressed by both gastric carcinoma tissue and gastric cell lines were V0 and V1, and V1 was significantly higher in gastric carcinoma tissue. The treatment of the gastric cell lines AGS and MKN45 with rhIL-11 resulted in a significant increase in the expression of V0 and V1. Exogenous IL-11 increased migration in AGS and MKN45 cells, whereas these effects were reversed when the expression of V0 and V1 were abolished by siRNA targeting versican V0/V1. Collectively, these findings suggest that the abnormally expressed versican and its isoforms participate, at least in part, in the progress of gastric carcinoma triggered by IL-11.     

Versican modulates gap junction intercellular communication

Versican is a large chondroitin sulfate proteoglycan and belongs to the family of lecticans. Versican possesses two globular domains, G1 and G3 domain, separated by a CS-attachment region. The CS-attachment region present in the middle region is divided into two spliced domains named CSalpha and beta. Alternative splicing of versican generates at least four versican isoforms named V0, V1, V2, and V3. We have successfully cloned the full-length cDNA of chick versican isoforms V1 and V2 and found that versican isoform V1 induced mesenchymal-epithelial transition in NIH3T3 cells. Mesenchymal-epithelial transition induced by V1 in NIH3T3 cells is characterized by expression of E-cadherin and occludin, two epithelial markers, and reduced expression of fibroblastic marker vimentin (Sheng et al., 2006, Mol Biol Cell. 17, 2009-2020). In the present studies, we found that versican V1 isoform not only induced cell transition, but also increased intercellular communication via gap junction channels composed of connexin proteins. Our results showed that V1 induces plasma membrane localization of connexin 43, resulting in increased cell communication. This was further confirmed by blocking assays. Gap junctions mediated the transfer of small cytoplasmic molecules and the diffusion of second messenger molecules between adjacent cells. The ability of versican in regulating gap junction implied a potential role of versican in coordinating functions.     

The increased accumulation of structurally modified versican and decorin is related with the progression of laryngeal cancer

Versican and decorin, two proteoglycans (PGs) with contradictory roles in the pathophysiology of cancer, comprise important stromal components in many tumor types and play a crucial role in the progression of cancer. In this study, we provide direct evidence for a significant and stage-related accumulation of versican and decorin in the tumor-associated stroma of laryngeal squamous cell carcinoma (LSCC) in comparison to normal larynx. Both PGs were found to be co-localized within the peritumorous stroma. In addition, the accumulated versican and decorin were markedly modified on both protein core and glycosaminoglycan (GAG) levels. Decorin, which was present under both glycanated and non-glycanated forms, perceptibly increased with the progression of LSCC, compared to the normal larynx. Tumor-associated glycanated decorin was found to contain significant amounts of dermatan sulfate (DS) sequences. Versican was also found to undergo stage-related structural modifications since a marked heterogeneity of protein cores was observed, being intense in late stage of laryngeal cancer. The increased accumulation of both versican and decorin was associated with a significant stage-related increase of the molar ratio of Delta di-mono4S to Delta di-mono6S up to approximately threefold in LSCC compared to the normal ones. The modified chemical structure of both PGs could be associated with the degree of aggressiveness of laryngeal squamous cell carcinomas.     

Versican is induced in infiltrating monocytes in myocardial infarction

Versican, a large chondroitin sulfate proteoglycan, plays a role in conditions such as wound healing and tissue remodelling. To test the hypothesis that versican expression is transiently upregulated and plays a role in the infarcted heart, we examined its expression in a rat model of myocardial infarction. Northern blot analysis demonstrated increased expression of versican mRNA. Quantitative real-time RT-PCR analysis revealed that versican mRNA began to increase as early as 6 h and reached its maximal level 2 days after coronary artery ligation. Versican mRNA then gradually decreased, while the mRNA of decorin, another small proteoglycan, increased thereafter. Versican mRNA was localized in monocytes, as indicated by CD68-positive staining, around the infarct tissue. The induction of versican mRNA was accelerated by ischemia/reperfusion (I/R), which was characterized by massive cell infiltration and enhanced inflammatory response. To examine the alteration of versican expression in monocytes/macrophages, we isolated human peripheral blood mononuclear cells and stimulated them with granulocyte/macrophage colony-stimulating factor (GM-CSF). Stimulation of mononuclear cells with GM-CSF increased the expression of versican mRNA as well as cytokine induction. The production of versican by monocytes in the infarct area represents a novel finding of the expression of an extracellular matrix gene by monocytes in the infarcted heart. We suggest that upregulation of versican in the infarcted myocardium may have a role in the inflammatory reaction, which mediates subsequent chemotaxis in the infarcted heart. (Mol Cell Biochem xxx: 47–56, 2005)     

Hyaluronan and versican in the control of human T-lymphocyte adhesion and migration

The ability of lymphocytes to migrate freely through connective tissues is vital to efficient immune function. How the extracellular matrix (ECM) may affect T-cell adhesion and migration is not well understood. We have examined the adhesion and migration of activated human T-lymphocytes on ECM made by fibroblast-like synoviocytes and lung fibroblasts. These cells were minimally interactive until treated with a viral mimetic, Poly I:C. This treatment promoted myofibroblast formation and engendered a higher-order structured ECM, rich in versican and hyaluronan, to which T-cells avidly adhered in a hyaluronidase-sensitive manner. This Poly I:C-induced matrix impeded T-cell spreading and migration on and through synoviocyte monolayers, while hyaluronidase treatment or adding versican antibody during matrix formation reversed the effect on T-cell migration. Hyaluronidase also reversed the spread myofibroblast morphology. These data suggest that the viscous hyaluronan- and versican-rich matrix binds and constrains T-lymphocytes. Using purified matrix components and solid state matrices of defined composition, we uncovered a role for versican in modulating hyaluronan-T-cell interactions. Versican prevented T-cell binding to soluble hyaluronan, as well as the amoeboid shape change on hyaluronan-coated dishes and T-cell penetration of collagen gels. Together, these data suggest that hyaluronan and versican play a role in T-cell trafficking and function in inflamed tissues.     

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.     

Versican mediates mesenchymal-epithelial transition

Versican is a large extracellular chondroitin sulfate proteoglycan that belongs to the family of lecticans. Alternative splicing of versican generates at least four isoforms named V0, V1, V2, and V3. We show here that ectopic expression of versican V1 isoform induced mesenchymal-epithelial transition (MET) in NIH3T3 fibroblasts, and inhibition of endogenous versican expression abolished the MET in metanephric mesenchyme. MET in NIH3T3 cells was demonstrated by morphological changes and dramatic alterations in both membrane and cytoskeleton architecture. Molecular analysis showed that V1 promoted a "switch" in cadherin expression from N- to E-cadherin, resulting in epithelial specific adhesion junctions. V1 expression reduced vimentin levels and induced expression of occludin, an epithelial-specific marker, resulting in polarization of V1-transfected cells. Furthermore, an MSP (methylation-specific PCR) assay showed that N-cadherin expression was suppressed through methylation of its DNA promoter. Exogenous expression of N-cadherin in V1-transfected cells reversed V1's effect on cell aggregation. Reduction of E-cadherin expression by Snail transfection and siRNA targeting E-cadherin abolished V1-induced morphological alteration. Transfection of an siRNA construct targeting versican also reversed the changed morphology induced by V1 expression. Silencing of endogenous versican prevented MET of metanephric mesenchyme. Taken together, our results demonstrate the involvement of versican in MET: expression of versican is sufficient to induce MET in NIH3T3 fibroblasts and reduction of versican expression decreased MET in metanephric mesenchyme.     

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.     

Isolation of a large aggregating proteoglycan from human brain.

A large proteoglycan (365 kDa), identified with monoclonal antibodies raised against chondroitin sulfate, was isolated from human brain. The isolation required anion-exchange chromatography followed by gel filtration through a Sephacryl S-500 column. The proteoglycan bound specifically to [3H]hyaluronate (HA). The binding was not reduced by high salt concentrations (up to 4 M) and was inhibited at low pH (< 4.0). The binding was inhibited by the octamer and decamer (but not the hexamer) oligosaccharides of HA. Limited proteolysis of the proteoglycan gave rise to a relatively stable polypeptide (80 kDa). The amino-terminal sequence of the 80-kDa polypeptide was identical to the cDNA-derived amino-terminal sequence of versican, a large human fibroblast proteoglycan. A monoclonal antibody raised against bovine proteoglycans and recognizing the versican core protein reacted by immunoblotting with the proteoglycan isolated from human brain. The antibody was used to localize the proteoglycan in acetone-fixed cryostat sections of bovine spinal cord. The localization of the proteoglycan in the central nervous system was identical to that previously reported for glial hyaluronate-binding protein (GHAP), a 60-kDa glycoprotein of the brain extracellular matrix (ECM). However, a major difference was observed with respect to the sensitivity of the two antigens to hyaluronidase. As previously reported, GHAP was released from the tissue by hyaluronidase digestion, whereas the proteoglycan persisted under these conditions. We conclude that the protein-hyaluronate aggregates in brain ECM contain both GHAP and versican, that GHAP is only retained in the ECM by its interaction with hyaluronate, and that the proteoglycan is anchored in some other manner and probably connects cell surfaces with the ECM since it was not released by hyaluronidase digestion.