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PubMed2006

V3 versican isoform expression has a dual role in human melanoma tumor growth and metastasis.

Versican is a large chondroitin sulfate proteoglycan produced by several tumor cell types, including malignant melanoma, which exists as four different splice variants. The presence of versican in the extracellular matrix plays a role in tumor cell growth, adhesion and migration, which could be altered by altering the ratio between versican isoforms. We have previously shown that overexpression of the V3 isoform of versican in human melanoma cell lines markedly reduces cell growth in vitro and in vivo, since V3-overexpressing (LV3SN) cultured cells as well as primary tumors arising from these cells grow slower than their vector-only counterparts (LXSN). In the present work, we have extended these observations to demonstrate that the delayed cell growth is due to multiple events since differences in proliferative index as well as in apoptosis are observed in LV3SN cells and tumors compared to LXSN. For example, LV3SN melanoma cells exhibit delayed activation of MAPK in response to EGF, we have also characterized further the primary tumors originated in nude mice from V3-transduced melanoma cells to determine if other events affect the V3 tumor phenotype. For example, hyaluronan content of LV3SN tumors was higher than in LXSN tumors, whereas other related matrix components and vascularization were unaffected. Furthermore, lung metastasis in nude mice occurred only in animals carrying LV3SN tumors, indicating a dual role for this molecule, both as an inhibitor of tumor growth and a metastasis inductor.

Apoptosis↗
PubMed2004

ADAMTS4 (aggrecanase-1) cleaves human brain versican V2 at Glu405-Gln406 to generate glial hyaluronate binding protein.

Human brain tissue from cerebellum and hippocampus was obtained between 2 h and 24 h post mortem and, after extraction in the presence of proteinase inhibitors, proteoglycans were purified by anion-exchange chromatography. The versican component was characterized by Western analysis with antibodies to the N-terminal peptide (LF99), the N-terminal globular domain (12C5) and the two GAG (glycosaminoglycan) attachment regions (anti-GAG-alpha and anti-GAG-beta). The results indicated that versican V2 is the major variant in all brain samples, and that it exists as the full-length form and also as at least six C-terminally truncated forms. The major immunoreactive species present is a 64 kDa product, which we identified by biochemical and immunological analysis as the brain protein previously termed GHAP (glial hyaluronate binding protein) [Perides, Lane, Andrews, Dahl and Bignami (1989) J. Biol. Chem. 264, 5981-5987]. Immunological analysis of purified human GHAP using a new anti-neoepitope antiserum (JSCNIV) showed that its C-terminal sequence is NIVSFE(405), and digestion of human cerebellum proteoglycans with ADAMTS4 (aggrecanase-1, where ADAMTS, a disintegrin and metalloproteinase with thrombospondin-1-like motifs) indicated that GHAP is a product of cleavage of versican V0 or V2 at the Glu(405)-Gln(406) bond. Since human cerebellum extracts contained multiple forms of ADAMTS4 protein on Western analysis, these data suggest that one or more members of the 'aggrecanase' group of the ADAMTS family (ADAMTS 1, 4, 5 and 9) are responsible for turnover of versican V2 in the adult human brain.

ADAM Proteins↗
PubMed1993

Versican gene expression in human articular cartilage and comparison of mRNA splicing variation with aggrecan.

The chondrocytes in human articular cartilage from subjects of all ages express mRNAs for both of the aggregating proteoglycans aggrecan and versican, although the level of expression of versican mRNA is much lower than that of aggrecan mRNA. Aggrecan shows alternative splicing of the epidermal growth factor (EGF)-like domain within its C-terminal globular region, but there is no evidence for a major difference in situ in the relative expression of this domain with age. At all ages studied from birth to the mature adult, a greater proportion of transcripts lacked the EGF domain. The relative proportions of the two transcripts did not change upon culture and passage of isolated chondrocytes. In contrast, the neighbouring complement regulatory protein (CRP)-like domain was predominantly expressed irrespective of age, but cell culture did result in variation of the splicing of this domain. Versican possesses two EGF-like domains and one CRP-like domain, but at all ages the three domains were predominantly present in all transcripts. This situation persisted upon culture and passage of the chondrocytes. Thus, unlike aggrecan, the versican expressed by human articular cartilage does not appear to undergo alternative splicing of its C-terminal globular region, either in cartilage in situ or in chondrocytes in culture.

Adult↗
PubMed1994

Glomerular mesangial cells in vitro synthesize an aggregating proteoglycan immunologically related to versican.

Recent studies have shown that mesangial cells derived from human adult glomeruli synthesize a number of 35S-labelled proteoglycans including a large chondroitin sulphate proteoglycan (CSPG), two dermatan sulphate proteoglycans (biglycan and decorin) and two heparan sulphate proteoglycans [Thomas, Mason and Davies (1991) Biochem. J. 277, 81-88]. In the present study we have examined the interaction of these proteoglycans with hyaluronan (HA) using associative gel chromatography. Only the large CSPG bound to HA, with 60% of those molecules in the medium and 80% of those in the cell layer being able to interact. Reduction and alkylation, or treatment of the monomer CSPG with proteinases, prevented the formation of aggregates, suggesting that the core protein was involved. The aggregates formed between purified CSPG and HA could be dissociated in the presence of HA-oligosaccharides of at least 10 monosaccharides in length. The inclusion of link protein with CSPG and HA promoted the formation of aggregates. Experiments with 3H-labelled mesangial-cell proteoglycans confirmed that only the large CSPG, with core protein molecular masses of 400 kDa and 500 kDa, interacted with HA. After chondroitin ABC lyase treatment of CSPG isolated from conditioned culture medium, several bands similar to those observed with 3H-labelled core proteins were identified using a polyclonal antiserum that recognizes versican. A monoclonal antibody recognizing the 1-C-6 epitope in the G1 and G2 globular regions of aggrecan did not recognize either mesangial-cell CSPG or bovine aortic versican. Northern-blot analysis confirmed that human mesangial cells express versican. Thus human mesangial large CSPG is a member of the versican family of proteoglycans. The interaction of CSPG and HA within the glomerulus may be important in glomerular cell migration and proliferation.

Adult↗
PubMed1995

Glial hyaluronate-binding protein: a product of metalloproteinase digestion of versican?

Glial hyaluronate-binding protein (GHAP) is a 60 kDa glycoprotein with an amino acid sequence identical to that of the hyaluronate-binding region of versican, a large fibroblast aggregating proteoglycan found in the brain. Both GHAP and versican were identified by immunoblot in bovine brain extracts prepared only minutes after death. Human recombinant collagenase, stromelysin, mouse gelatinase and gelatinases isolated from human brain by affinity chromatography digest versican and give rise to a polypeptide with electrophoretic mobility identical to GHAP. Immunoblot analysis, peptide mapping and C-terminal amino acid sequencing indicate that the polypeptide generated by digestion with human brain gelatinases is identical to GHAP. We suggest that GHAP is a naturally occurring versican degradation product.

Amino Acid Sequence↗
PubMed2006

Versican V0 and V1 guide migratory neural crest cells.

We previously showed the selective expression of the chondroitin sulfate proteoglycans versican V0 and V1 in barrier tissues that impede the migration of neural crest cells during embryonic trunk development (Landolt, R. M., Vaughan, L., Winterhalter, K. H., and Zimmermann, D. R. (1995) Development 212, 2303-2312). To test for an active involvement of these isoforms in the guidance process, we have now established protocols to isolate intact versican V0 and V1 in quantities sufficient for functional experiments. Using stripe choice assays, we demonstrate that pure preparations of either a mixture of versican V0/V1 or V1 alone strongly inhibit the migration of multipotent Sox10/p75NTR double-positive early neural crest stem cells on fibronectin by interfering with cell-substrate adhesion. We show that this inhibition is largely core glycoprotein-dependent, as the complete removal of the glycosaminoglycan chains has only a minor effect on the inhibitory capacity. Our findings support the notion that versican variants V0 and V1 act, possibly in concert with other inhibitory molecules such as aggrecan and ephrins, in directing the migratory streams of neural crest cells to their appropriate target tissues.

Animals↗
PubMed2006

Versican G3 domain regulates neurite growth and synaptic transmission of hippocampal neurons by activation of epidermal growth factor receptor.

Versican is one of the major extracellular matrix (ECM) proteins in the brain. ECM molecules and their cleavage products critically regulate the growth and arborization of neurites, hence adjusting the formation of neural networks. Recent findings have revealed that peptide fragments containing the versican C terminus (G3 domain) are present in human brain astrocytoma. The present study demonstrated that a versican G3 domain enhanced cell attachment, neurite growth, and glutamate receptor-mediated currents in cultured embryonic hippocampal neurons. In addition, the G3 domain intensified dendritic spines, increased the clustering of both synaptophysin and the glutamate receptor subunit GluR2, and augmented excitatory synaptic activity. In contrast, a mutated G3 domain lacking the epidermal growth factor (EGF)-like repeats (G3deltaEGF) had little effect on neurite growth and glutamatergic function. Treating the neurons with the G3-conditioned medium rapidly increased the levels of phosphorylated EGF receptor (pEGFR) and phosphorylated extracellular signal-regulated kinase (pERK), indicating an activation of EGFR-mediated signaling pathways. Blockade of EGFR prevented the G3-induced ERK activation and suppressed the G3-provoked enhancement of neurite growth and glutamatergic function but failed to block the G3-mediated enhancement of cell attachment. These combined results indicate that the versican G3 domain regulates neuronal attachment, neurite outgrowth, and synaptic function of hippocampal neurons via EGFR-dependent and -independent signaling pathway(s). Our findings suggest a role for ECM proteolytic products in neural development and regeneration.

Animals↗
PubMed2004

Expression of extracellular matrix components versican, chondroitin sulfate, tenascin, and hyaluronan, and their association with disease outcome in node-negative breast cancer.

PURPOSE: The purpose is to determine whether the levels of expression of extracellular matrix components in peritumoral stroma are predictive of disease outcome for women with node-negative breast cancer. EXPERIMENTAL DESIGN: Tumor tissue from 86 patients with node-negative breast cancer was examined by immunohistochemical staining for the expression of versican, chondroitin sulfate (CS), tenascin, and hyaluronan (HA). With the exception of HA, the expression of the extracellular matrix components was measured by video image analysis. Statistical correlation of the immunohistochemical data with clinicopathological characteristics and disease outcome was performed. RESULTS: All of the extracellular matrix components were present in the peritumoral stroma of the entire study cohort. In contrast, immunoreactivity within the cancer cell was observed in 82% of tumors for HA, 12% for CS, and 4% for tenascin; no immunostaining of cancer cells for versican was observed for any of the tumors. Cox regression and Kaplan-Meier analyses indicated that elevated expression of stromal versican predicted increased risk and rate of relapse in this cohort. Elevated expression of tenascin was predictive of increased risk and rate of death only. Although neither CS nor HA were predictive of disease outcome in this cohort, tumor size was predictive of increased risk and rate of both relapse and survival. CONCLUSIONS: Elevated expression within peritumoral stromal matrix of versican and tenascin was predictive of relapse-free and overall survival, respectively, in women with node-negative breast cancer.

Adult↗
PubMed2004

Role of ADAMTS-1 in atherosclerosis: remodeling of carotid artery, immunohistochemistry, and proteolysis of versican.

OBJECTIVE: We investigated the potential role of ADAMTS-1 (a disintegrin and metalloprotease with thrombospondin motif type I) in atherogenesis. METHODS AND RESULTS: ADAMTS-1 is expressed at the highest levels in the aorta when compared with other human tissues examined. Immunolocalization studies in human aorta and coronary artery indicate that ADAMTS-1 expression is mainly seen at low levels in the medial layer, but upregulated in the intima when plaque is present. We found that ADAMTS-1 mRNA levels are significantly higher in proliferating/migrating cultured primary aortic vascular smooth muscle cells (VSMCs) compared with resting/confluent cells. Using the mouse carotid artery flow cessation model, we show that there are differences in vessel remodeling in ADAMTS-1 transgenic/apoE-deficient mice compared with apoE deficiency alone, particularly a significant increase in intimal hyperplasia. We show that ADAMTS-1 can cleave the large versican containing proteoglycan population purified from cultured human aortic VSMCs. Finally, using versican peptide substrates, we show data suggesting that ADAMTS-1 cleaves versican at multiple sites. CONCLUSIONS: We hypothesize that ADAMTS-1 may promote atherogenesis by cleaving extracellular matrix proteins such as versican and promoting VSMC migration.

ADAM Proteins↗
PubMed2006

The versican gene and the risk of intracranial aneurysms.

BACKGROUND AND PURPOSE: The proteoglycan versican is an excellent candidate gene for intracranial aneurysms (IAs) because it plays an important role in extracellular matrix assembly and is localized in a previously implicated locus for IAs on chromosome 5q. METHODS: We analyzed all the common variations using 16-tag single nucleotide polymorphisms (SNPs) and haplotypes in the versican gene using a 2-stage genotyping approach. For stage 1, 16 SNPs were genotyped in 307 cases and 639 controls. For stage 2, the two SNPs yielding the most significant associations (P<0.01) were genotyped in a second independent cohort of 310 cases for confirmation of the associations. RESULTS: In stage 1, we found several SNPs in strong linkage disequilibrium and haplotypes constituting these SNPs associated with IAs in the Dutch population (strongest SNP association for rs173686 with odds ratio=1.34, 95% CI=1.09 to 1.65, P=0.004). In stage 2, we confirmed association for the 2 SNPs with the most significant associations (strongest SNP association for rs173686 with odds ratio=1.36, 95% CI=1.11 to 1.67, P=0.003). CONCLUSIONS: SNPs in strong linkage disequilibrium and haplotypes constituting these SNPs in the versican gene are associated with IAs suggesting that variation in or near the versican gene plays a role in susceptibility to IAs.

Adult↗
PubMed1993

Extracellular matrix in regenerating rat sciatic nerve: a comparative study on the localization of laminin, hyaluronic acid, and chondroitin sulfate proteoglycans, including versican.

Using a glial hyaluronate-binding protein as a probe, monoclonal antibodies against versican and ABC digested chondroitin sulfate proteoglycans, and polyclonal antibodies against laminin, we localized these extracellular matrix (ECM) components in the endoneurium of the adult rat sciatic nerve. During Wallerian degeneration caused by nerve crushing, the staining pattern of these ECM elements changed dramatically. In the first stages and up to 5 days after injury, the tubular endoneurial structures remained the same as in control nerves. Ten days after crush, the bands of Büngner formed by proliferating Schwann cells in the distal stump of crushed nerves stained diffusely for hyaluronate, laminin, and chondroitin sulfate. Regenerating axons were demonstrated in this location by double-labeling experiments with neurofilament antibodies. Conversely, staining with antibodies against versican, a hyaluronate binding proteoglycan, was reduced and the bands of Büngner were not stained. After 30 days the endoneurial tubes made their reappearance and, as in normal nerve, they stained for hyaluronate, laminin, versican, and chondroitin sulfate. It is concluded that proliferating Schwann cells in peripheral nerve undergoing Wallerian degeneration are capable of producing several endoneurial ECM constituents, versican being a notable exception.

Animals↗
PubMed2003

Expression of the proteoglycans versican and mel-CSPG in dysplastic nevi.

Nevi with architectural disorder and cytologic atypia of melanocytes (NAD) (also called dysplastic nevi) have been controversial with regard to their relationship with melanoma risk and to their gradation in 3 degrees of atypia. Versican and the melanoma-associated proteoglycan (mel-CSPG) are 2 major proteoglycans expressed by malignant melanoma, and they have a role in the regulation of cell adhesion, migration, and differentiation. We evaluated the differences in versican and mel-CSPG expression in nevi, NAD with several degrees of atypia, and primary malignant melanoma. Immunoreactivity for versican was negative in benign melanocytic nevi, positive in NAD (ranging from weakly to intensely positive), and intensely positive in malignant melanoma. Immunostaining for mel-CSPG was negative in benign melanocytic nevi and mild to moderately positive in NAD and melanoma. Our results suggest that versican expression may be of value for distinguishing NAD from benign melanocytic nevi and for distinguishing severe NAD from mild and moderate NAD.

Aggrecans↗
PubMed1991

Differential regulation of extracellular matrix proteoglycan (PG) gene expression. Transforming growth factor-beta 1 up-regulates biglycan (PGI), and versican (large fibroblast PG) but down-regulates decorin (PGII) mRNA levels in human fibroblasts in culture.

Proteoglycans (PGs) comprise a group of extracellular matrix macromolecules which play an important role in matrix biology. In this study, normal human skin and gingival fibroblast cultures were incubated with transforming growth factor-beta 1 (TGF-beta 1), and the expression of three PGs, viz. biglycan (PGI), decorin (PGII), and versican (a large fibroblast proteoglycan) was examined. The results indicate that TGF-beta 1 (5 ng/ml) markedly increased the expression of biglycan (up to 24-fold) and versican (up to 6-fold) mRNAs and the enhancement of biglycan expression was coordinate with elevated type I procollagen gene expression in the same cultures. In contrast, the expression of decorin mRNA was markedly (up to approximately 70%) inhibited by TGF-beta 1. The response to TGF-beta 1 was similar in both skin and gingival fibroblasts, although the gingival cells were clearly more responsive to stimulation by TGF-beta 1 with respect to biglycan gene expression. Analysis of 35S-labeled proteoglycans in the culture media of skin and gingival fibroblasts also revealed stimulation of biglycan and versican production, and reduction in decorin production. Quantitation of both [35S]sulfate and [3H]leucine-labeled decorin in cell culture media by immunoprecipitation revealed a 50% reduction in decorin production in cell cultures treated with TGF-beta 1. This TGF-beta 1-elicited reduction was accompanied by an apparent increase in the size of the decorin molecules, although the size of the core protein was not altered, as judged by Western immunoblotting following chondroitinase ABC digestion. Analysis of the proteoglycans in the matrix and membrane fractions also revealed increased amounts of versican in cultures treated with TGF-beta 1. These results indicate differential regulation of PG gene expression in fibroblasts by TGF-beta 1, and these observations emphasize the role of PGs in the extracellular matrix biology and pathology.

Biglycan↗
PubMed1993

cDNA cloning of PG-M, a large chondroitin sulfate proteoglycan expressed during chondrogenesis in chick limb buds. Alternative spliced multiforms of PG-M and their relationships to versican.

We have isolated cDNA clones encoding the core protein of PG-M, a large chondroitin sulfate proteoglycan that has been shown to be expressed in the prechondrogenic condensation area of the developing chick limb buds (Shinomura T., Jensen, K. L., Yamagata, M., Kimata, K., and Solursh, M. (1990) Anat. Embryol. 181, 227-233). The amino acid sequence deduced from the cDNA analysis revealed the presence of a hyaluronic acid binding domain at the amino-terminal side and two epidermal growth factor-like domains, a lectin-like domain, and a complement regulatory protein-like domain at the carboxyl-terminal side. These domains show an extremely high homology to corresponding domains of a human fibroblast large chondroitin sulfate proteoglycan, versican. Such evolutionally conserved structures in the PG-M core protein might be involved in important biological functions of this molecule. On the other hand, the chondroitin sulfate attachment domain at the middle region of the PG-M core protein shows no significant amino acid sequence homology to the corresponding domain of the versican core protein. Further, the chondroitin sulfate attachment domain of PG-M core protein is about 100 kDa larger than that of versican core protein. The finding of alternatively spliced forms of the PG-M core protein suggests that versican might be one of the multiple forms of PG-M.

Alternative Splicing↗
PubMed1996

Versican.

Proteoglycans have long been recognized as participating in a number of biological activities ranging from structural roles to regulation of transcription. Versican is a large chondroitin sulfate proteoglycan expressed in several tissues, including the nervous system. Significant progress has been made in the understanding of the molecular structure and biological activity of versican. The progress is largely due to the application of recombinant DNA methodology and the generation of domain-specific anti-versican antibodies. In the central and peripheral nervous system, versican is expressed by glial cells and is implicated in the regulation of cell adhesion, migration, pattern formation, and regeneration.

Animals↗
PubMed1997

Selective deposits of versican in the extracellular matrix of restenotic lesions from human peripheral arteries.

Although a large percentage of the volume of human restenotic arterial lesions is occupied by extracellular matrix (ECM), the composition and organization of this ECM are not well characterized. In this study, restenotic segments taken from 30 human peripheral arteries by directional atherectomy at times ranging from 13 days to 36 months after angioplasty were analyzed for specific patterns of ECM composition and organization by light and electron microscopic histochemistry and immunohistochemistry. Histochemical analysis revealed the presence of distinct zones, enriched either in proteoglycans or fibrillar collagen. Most sections contained these regions juxtaposed to each other. The frequency of these two distinct ECMs did not change as a function of time after angioplasty. The collagen-rich zone usually contained elongated smooth muscle cells spaced close together except in regions resembling fibrous plaques. The proteoglycan-rich ECM contained both elongated and stellate-shaped smooth muscle cells randomly arranged and separated by wide distances. This region resembled the loose-connective-tissue-containing myxoid region typical of restenotic lesions. Immunohistochemical analysis of these regions revealed that the proteoglycan-containing zone stained intensely for versican, a large interstitial chondroitin sulfate proteoglycan, whereas the collagen-containing areas were mostly negative for versican but positive for type I collagen. The versican-positive regions also immunostained for biglycan, a small leucine-rich dermatan sulfate proteoglycan, and sparsely for elastin. However, both of these ECM molecules were present in the versican-negative type I collagen-positive regions of the lesions. These results suggest that the development of restenotic lesions involves localized deposits of specific ECM molecules that may play a role in the asymmetric renarrowing of this tissue after angioplasty.

Angioplasty, Balloon↗
PubMed1998

The soft tissue cover of the mandibular condyle. Differentiation in histological forms and age-related changes of aggrecan- and versican-like proteoglycans.

Age-related changes of the composition of the extracellular matrix of the soft tissue cover of the mandibular condyle (STC), especially of the large proteoglycans, have been investigated. Proteoglycans were extracted from the STC of neonatal, juvenile and adult domestic pigs, fractionated by density gradient centrifugation and analyzed by electrophoresis/Western blotting. Experiments revealed firstly that a large CS/KS proteoglycan (aggrecan) is an essential constituent of the STC at all ages. This proteoglycan is required for nutrition of avascular tissues, and age-related changes in its average size and substitution with KS (keratan sulfate) may be a response to altered functional loading and tissue architecture of the STC. Secondly it was shown that a large CS/DS (chondroitin sulfate/dermatan sulfate) proteoglycan characterized by a doublet of core proteins at 200 and 250 kDa, thereby resembling perlecan, is present in the tissue of adults, but not of neonates and juveniles. Thirdly a large CS/DS proteoglycan characterized by core proteins at 350, 450 and 550 kDa, thereby resembling versican, was present in juveniles. It was detectable only weakly in neonates and not in adults. Results of core protein analysis were confirmed by results of agarose gel electrophoresis/Western blotting of the undigested proteoglycans isolated directly from the tissue extracts. Versican is believed to destabilize cell-matrix interactions required for cell proliferation and differentiation. In this context, presence of versican-like proteoglycans in the STC of growing individuals and its disappearance in adults appears to be related to the growth potential of the mandibular condyle.

Aggrecans↗
PubMed2004

Lp3/Hapln3, a novel link protein that co-localizes with versican and is coordinately up-regulated by platelet-derived growth factor in arterial smooth muscle cells.

Link proteins (LPs) belong to the link-module superfamily, which can stabilize and enhance the binding of lecticans to hyaluronan. We report here the identification and characterization of a novel rat link protein gene (Lp3/Hapln3). The deduced protein sequence shares the typical modular elements of link proteins and has an estimated mass of 39 kDa. Examination of the rat genomic DNA sequence revealed that Lp3/Hapln3 and aggrecan genes were paired on chromosome 1q31. Another LP gene and the lectican gene were also paired at a different locus, as they are in the human and mouse genomes. Immunohistochemical analysis showed the prominent expression of Lp3/Hapln3 in the smooth muscle tissues of the vascular wall and gastrointestinal tract. Further comparative studies revealed that Lp3/Hapln3 was well co-localized with versican around the smooth muscle cells of blood vessels but not around endothelial cells. In vitro experiments using primary cultured rat arterial smooth muscle cells (ASMCs) demonstrated the coordinated up-regulation of Lp3/Hapln3 and versican by platelet-derived growth factor (PDGF). These data were supported by in vivo studies of a mechanical vascular injury model in mice. Altogether, our results suggest that Lp3/Hapln3 is involved, together with versican and hyaluronan, in the formation of the pericellular matrix of vascular smooth muscle cells.

Amino Acid Sequence↗