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The extracellular matrix (ECM) is a complex mixture composed of fibrillar collagens as well as additional protein and carbohydrate components. Proteoglycans (PGs) contribute to the heterogeneity of the ECM and play an important role in its structure and function. While the small leucine rich proteoglycans (SLRPs), including decorin and lumican, have been studied extensively as mediators of collagen fibrillogenesis and organization, the function of large matrix PGs in collagen matrices is less well known. In this study, we showed that different matrix PGs have distinct roles in regulating collagen behaviors. We found that versican, a large chondroitin sulfate PG, promotes collagen fibrillogenesis in a turbidity assay and upregulates cell-mediated collagen compaction and reorganization, whereas aggrecan, a structurally-similar large PG, has different and often opposing effects on collagen. Compared to versican, decorin and lumican also have distinct functions in regulating collagen behaviors. The different ways in which matrix PGs interact with collagen have important implications for understanding the role of the ECM in diseases such as fibrosis and cancer, and suggest that matrix PGs are potential therapeutic targets.HighlightsSmall leucine rich proteoglycans (SLRPs) and large chondroitin sulfate (CS) proteoglycans (PGs) have distinct effects on collagen fibrous network behavior.Unlike other matrix proteoglycans, versican promotes collagen fibrillogenesis in anin vitrospectrophotometric (turbidity) assay.The versican core protein has a larger impact on collagen behavior in a fibrillogenesis assay than its glycosaminoglycan chains do.Versican increases the diameter of collagen fibers and the porosity of collagen fibrous networks, unlike aggrecan and SLRPs.The addition of versican to collagen does not alter fibroblast contractility but leads to enhanced cell-mediated collagen reorganization and contraction.

BACKGROUND: This study aimed to investigate differentially expressed proteins in the vitreous humor (VH) of pathological myopia (PM) and normal eyes. METHODS: VH samples were collected from patients undergoing surgical treatment for rhegmatogenous retinal detachment (RRD), idiopathic epiretinal membrane (ERM), myopic retinoschisis (MRS) or macular hole (MH). A label-free quantitative proteomic analysis was performed to detect the differentially expressed proteins, and expression of three differentially expressed proteins was confirmed by ELISA. RESULTS: In PM patients (MH-PM, MRS-PM and RRD-PM), the expression of prostaglandin-H2 D-isomerase (PGDS) and glutathione peroxidase 3 (GPX3) was significantly lower than in controls (MH, ERM, and RRD). The versican core protein expression decreased in the PM group. The vitreous concentrations of PGDS and GPX3 in patients with axial length (AL) of 26.5–29.0 mm were higher than in patients with AL >29.0 mm or AL 29.0 mm. CONCLUSIONS: Our study provides new evidence on the molecular changes in the VH of PM patients, and these molecules have the potential to become new targets for the therapy of PM.

Hepatocellular carcinoma (HCC) is one of the most aggressive tumors, and the treatment outcome of this disease is improved when the cancer is diagnosed at an early stage. This requires biomarkers allowing an accurate and early tumor diagnosis. To identify potential markers for such applications, we analyzed a patient cohort consisting of 50 patients (50 HCC and 50 adjacent nontumorous tissue samples as controls) using two independent proteomics approaches. We performed label-free discovery analysis on 19 HCC and corresponding tissue samples. The data were analyzed considering events known to take place in early events of HCC development, such as abnormal regulation of Wnt/b-catenin and activation of receptor tyrosine kinases (RTKs). 31 proteins were selected for verification experiments. For this analysis, the second set of the patient cohort (31 HCC and corresponding tissue samples) was analyzed using selected (multiple) reaction monitoring (SRM/MRM). We present the overexpression of ATP-dependent RNA helicase (DDX39), Fibulin-5 (FBLN5), myristoylated alanine-rich C-kinase substrate (MARCKS), and Serpin H1 (SERPINH1) in HCC for the first time. We demonstrate Versican core protein (VCAN) to be significantly associated with well differentiated and low-stage HCC. We revealed for the first time the evidence of VCAN as a potential biomarker for early-HCC diagnosis.

Adipose microenvironment is involved in signaling pathways that influence breast cancer. We aim to characterize factors that are modified: 1) in tumor and non tumor human breast epithelial cell lines when incubated with conditioned media (CMs) from human breast cancer adipose tissue explants (hATT) or normal breast adipose tissue explants (hATN); 2) in hATN-CMs vs hATT-CMs; 3) in the tumor associated adipocytes vs. non tumor associated adipocytes. We used hATN or hATT- CMs on tumor and non-tumor breast cancer cell lines. We evaluated changes in versican, CD44, ADAMTS1 and Adipo R1 expression on cell lines or in the different CMs. In addition we evaluated changes in the morphology and expression of these factors in slices of the different adipose tissues. The statistical significance between different experimental conditions was evaluated by one-way ANOVA. Tukey's post-hoc tests were performed within each individual treatment. hATT-CMs increase versican, CD44, ADAMTS1 and Adipo R1 expression in breast cancer epithelial cells. Furthermore, hATT-CMs present higher levels of versican expression compared to hATN-CMs. In addition, we observed a loss of effect in cellular migration when we pre-incubated hATT-CMs with chondroitinase ABC, which cleaves GAGs chains bound to the versican core protein, thus losing the ability to bind to CD44. Adipocytes associated with the invasive front are reduced in size compared to adipocytes that are farther away. Also, hATT adipocytes express significantly higher amounts of versican, CD44 and Adipo R1, and significantly lower amounts of adiponectin and perilipin, unlike hATN adipocytes. We conclude that hATT secrete a different set of proteins compared to hATN. Furthermore, versican, a proteoglycan that is overexpressed in hATT-CMs compared to hATN-CMs, might be involved in the tumorogenic behavior observed in both cell lines employed. In addition, we may conclude that adipocytes from the tumor microenvironment show a less differentiated state than adipocytes from normal microenvironment. This would indicate a loss of normal functions in mature adipocytes (such as energy storage), in support of others that might favor tumor growth. Fil: Fletcher, Sabrina Johanna. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina Fil: Sacca, Paula Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina Fil: Pistone Creydt, Mercedes. Instituto ; Argentina Fil: Coló, Federico Andrés. Instituto ; Argentina Fil: Serra, María Florencia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; Argentina Fil: Santiano, Flavia Eliana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; Argentina Fil: Sasso, Corina Verónica. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; Argentina Fil: López Fontana, Constanza Matilde. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; Argentina Fil: Caron, Ruben Walter. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; Argentina Fil: Calvo, Juan Carlos. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; Argentina Fil: Pistone Creydt, Virginia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina

Versican G1 domain-containing fragments (VG1Fs) have been identified in extracts from the dermis in which hyaluronan (HA)-versican-fibrillin complexes are found. However, the molecular assembly of VG1Fs in the HA-versican-microfibril macrocomplex has not yet been elucidated. Here, we clarify the role of VG1Fs in the extracellular macrocomplex, specifically in mediating the recruitment of HA to microfibrils. Sequential extraction studies suggested that the VG1Fs were not associated with dermal elements through HA binding properties alone. Overlay analyses of dermal tissue sections using the recombinant versican G1 domain, rVN, showed that rVN deposited onto the elastic fiber network. In solid-phase binding assays, rVN bound to isolated nondegraded microfibrils. rVN specifically bound to authentic versican core protein produced by dermal fibroblasts. Furthermore, rVN bound to VG1Fs extracted from the dermis and to nondenatured versican but not to fibrillin-1. Homotypic binding of rVN was also seen. Consistent with these binding properties, macroaggregates containing VG1Fs were detected in high molecular weight fractions of sieved dermal extracts and visualized by electron microscopy, which revealed localization to microfibrils at the microscopic level. Importantly, exogenous rVN enhanced HA recruitment both to isolated microfibrils and to microfibrils in tissue sections in a dose-dependent manner. From these data, we propose that cleaved VG1Fs can be recaptured by microfibrils through VG1F homotypical interactions to enhance HA recruitment to microfibrils.

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.

Versican/proteoglycan-mesenchymal (PG-M) is a large chondroitin sulfate (CS) proteoglycan of the extracellular matrix (ECM) that is constitutively expressed in adult tissues such as dermis and blood vessels. It serves as a structural macromolecule of the ECM, while in embryonic tissue it is transiently expressed at high levels and regulates cell adhesion, migration, proliferation, and differentiation. Knock-in mouse embryonic (Cspg2(Delta3/Delta3)) fibroblasts whose versican lack the A subdomain of the G1 domain exhibit low proliferation rates and acquire senescence. It was suspected that chondroitin sulfate on versican core protein would be altered when the A subdomain was disrupted, so fibroblasts were made from homozygous Cspg2(Delta3/Delta3) mouse embryos to investigate the hypothesis. Analysis of the resulting versican deposition demonstrated that the total versican deposited in the Cspg2(Delta3/Delta3) fibroblasts culture was approximately 50% of that of the wild type (WT), while the versican deposited in the ECM of Cspg2(Delta3/Delta3) fibroblasts culture was 35% of that of the WT, demonstrating the lower capacity of mutant (Cspg2(Delta3/Delta3)) versican deposited in the ECM. The analysis of CS expression in the Cspg2(Delta3/Delta3) fibroblasts culture compared with wild-type fibroblasts showed that the composition of the non-sulfate chondroitin sulfate isomer on the versican core protein increased in the cell layer but decreased in the culture medium. Interestingly, chondroitin sulfate E isomer was found in the culture medium. The amount of CS in the Cspg2(Delta3/Delta3) cell layer of fibroblasts with mutant versican was dramatically decreased, contrasted to the amount in the culture medium, which increased. It was concluded that the disruption of the A subdomain of the versican molecule leads to lowering of the amount of versican deposited in the ECM and the alteration of the composition and content of CS on the versican molecule.

The composition of extracellular matrix during growth and regression of the neointima was analyzed during healing in a baboon aortoiliac polytetrafluoroethylene graft. Graft neointimal thickening can be modulated by altering blood flow by construction of downstream arteriovenous fistulas. Normal flow with normal shear stress induces neointimal thickening, whereas high flow with high shear stress upstream of a fistula induces regression of established neointima. The neointima formed under normal shear stress is enriched in hyaluronan and proteoglycans, particularly versican. On the other hand, the neointima near the graft material is enriched in collagen and biglycan. Neointimal regression in response to high shear stress is associated with a loss of proteoglycans as detected by histochemical staining. Immunostaining with an antibody against an ADAMTS cleavage neoepitope of versican increases after switching to high flow, although immunostaining for versican core protein is not appreciably changed by high flow. The present data demonstrate that the graft neointima is enriched with proteoglycans, particularly versican and hyaluronan, as well as collagen, and there is a differential distribution of each. Neointimal atrophy occurs with an apparent loss of proteoglycans and evidence of versican degradation.

We previously showed the expression of PG-M/versican in embryonic chicken retina. In this study, we characterized the alternatively spliced forms of PG-M/versican and their developmental regulation to investigate the implication of PG-M/versican in neurite outgrowth from retinal cells during development. On day 5, the immunolocalization of PG-M was first observed at the inner surface of neural retina. On day 7, the pronounced staining was observed in the nerve fiber layer and inner plexiform layer where neural networks of ganglion cells were being formed. As the development proceeded, more intensive staining was observed in these layers. The staining peaked on day 14 and then decreased. Northern analysis and western blotting revealed the presence of a single-sized transcript (13 kb) and the PG-M/versican core protein (550 kDa) on day 14, but the absence of any transcripts or protein bands on day 20, indicating a transient expression of PG-M+ (VO), the alternatively spliced form with the most abundant sites for the chondroitin sulfate attachment. Taken together, it is likely that PG-M/versican is involved in neurite outgrowth from ganglion cells during retinal development, and antiadhesion activity of its chondroitin sulfate chains may be important for regulation.

The ability of the periodontal ligament (PDL) to rapidly remodel is the basis of orthodontic tooth movement. During the tooth movement, matrix proteoglycans (PGs) may play important roles in spatial, mechanical and biological aspects for the maintenance and repair of the PDL. The aim of this study was to characterize the distribution of a large hyaluronic acid (HA)-binding proteoglycan, versican, link protein (LP) and HA in the rat molar PDL during experimental tooth movement by histochemical and immunohistochemical methods. Experimental tooth movement was performed according to Waldo's method. Histologically, regressive changes, such as decrease of fibroblasts and collagen fibers and exudative change of edema were observed in the compressive side and progressive changes, such as proliferation of fibroblasts and collagen fibers, in the strain side one day after treatment. By 3 days after tooth movement, regressive or progressive changes were not observed in either side. Using monoclonal antibodies specific to versican core protein or LP, the positive immunoreactivity for both molecules was constantly observed throughout the PDL. After the experimental force was applied to the tooth, however, the immunostainings of versican and LP became significantly intense only in the compressive side but decreased in the strain side. The intensity in the compressive side was strongest one day after the force was applied and gradually diminished thereafter. HA of both sides did not change during experimental tooth movement. Since HA is present in the PDL, large amounts of versican and LP expressed in the compressive side may create large hydrated aggregates via their association with HA that dissipates the compressive force applied to this tissue.

O -linked N -acetylglucosamine ( O -GlcNAc) is a reversible posttranslational modification of Ser and Thr residues on cytosolic and nuclear proteins of higher eukaryotes catalyzed by O -GlcNAc transferase (OGT). O -GlcNAc has recently been found on Notch1 extracellular domain catalyzed by EGF domain-specific OGT. Aberrant O -GlcNAc modification of brain proteins has been linked to Alzheimer's disease (AD). However, understanding specific functions of O -GlcNAcylation in AD has been impeded by the difficulty in characterization of O -GlcNAc sites on proteins. In this study, we modified a chemical/enzymatic photochemical cleavage approach for enriching O -GlcNAcylated peptides in samples containing ∼100 μg of tryptic peptides from mouse cerebrocortical brain tissue. A total of 274 O -GlcNAcylated proteins were identified. Of these, 168 were not previously known to be modified by O -GlcNAc. Overall, 458 O -GlcNAc sites in 195 proteins were identified. Many of the modified residues are either known phosphorylation sites or located proximal to known phosphorylation sites. These findings support the proposed regulatory cross-talk between O -GlcNAcylation and phosphorylation. This study produced the most comprehensive O -GlcNAc proteome of mammalian brain tissue with both protein identification and O -GlcNAc site assignment. Interestingly, we observed O -β-GlcNAc on EGF-like repeats in the extracellular domains of five membrane proteins, expanding the evidence for extracellular O -GlcNAcylation by the EGF domain-specific OGT. We also report a GlcNAc-β-1,3-Fuc-α-1- O -Thr modification on the EGF-like repeat of the versican core protein, a proposed substrate of Fringe β-1,3- N -acetylglucosaminyltransferases.

The expression of the large chondroitin sulfate proteoglycan versican was studied in human adult skin. For this purpose, bacterial fusion proteins containing unique portions of the versican core protein were prepared. Polyclonal antibodies against the fusion proteins specifically reacted with versican from a proteoglycan fraction of MG63 osteosarcoma cells. In immunohistochemical experiments, the affinity-purified antibodies localized versican in the stratum basale of the epidermis, as well as in the papillary and reticular layers of the dermis. An apparent codistribution of versican with the various fiber forms of the elastic network of the dermis suggested an association of versican with microfibrils. Both dermal fibroblasts and keratinocytes expressed versican in culture during active cell proliferation. In line with the observation that versican is absent in the suprabasal layers of the epidermis where keratinocytes terminally differentiate, culture conditions promoting keratinocyte differentiation induced a down-regulation of versican synthesis. In Northern blots versican mRNA could be detected in extracts from proliferating keratinocytes and dermal fibroblasts. Comparison of RNA preparations from semi-confluent and confluent fibroblast cultures demonstrated decreasing amounts of versican mRNA at higher cell densities. This inverse correlation of versican expression and cell density was confirmed by indirect immunofluorescence staining of cultured fibroblasts and keratinocytes. The localization of versican in the basal zone of the epidermis as well as the density dependence of versican in cell cultures suggest a general function of versican in cell proliferation processes that may not solely be confined to the skin.

Protein post-translational modifications (PTMs) are regulated separately from protein expression levels. Thus, simultaneous characterization of the proteome and its PTMs is pivotal to an understanding of protein regulation, function and activity. However, concurrent analysis of the proteome and its PTMs by mass spectrometry is a challenging task because the peptides bearing PTMs are present in sub-stoichiometric amounts and their ionization is often suppressed by unmodified peptides of high abundance. We describe here a method for concurrent analysis of phosphopeptides, glycopeptides and unmodified peptides in a tryptic digest of rat kidney tissue with a sequence of ERLIC and RP-LC-MS/MS in a single experimental run, thereby avoiding inter-experimental variation. Optimization of loading solvents and elution gradients permitted ERLIC to be performed with totally volatile solvents. Two SCX and four ERLIC gradients were compared in details, and one ERLIC gradient was found to perform the best, which identified 2929 proteins, 583 phosphorylation sites in 338 phosphoproteins and 722 N-glycosylation sites in 387 glycoproteins from rat kidney tissue. Two hundred low-abundance proteins with important functions were identified only from the glyco- or phospho-subproteomes, reflecting the importance of the enrichment and separation of modified peptides by ERLIC. In addition, this strategy enables identification of unmodified and corresponding modified peptides (partial phosphorylation and N-glycosylation) from the same protein. Interestingly, partially modified proteins tend to occur on proteins involved in transport. Moreover, some membrane or extracellular proteins, such as versican core protein and fibronectin, were found to have both phosphorylation and Nglycosylation, which may permit an assessment of the potential for cross talk between these two vital PTMs and their roles in regulation. Published version

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.

The aim of the present study was to characterize the proteoglycans and catabolic products of proteoglycans present in the tensile region of ligament and explant cultures of this tissue, and to compare these with those observed in the tensile region of tendon. Approx. 90% of the total proteoglycans in fresh ligament was decorin, as estimated by N-terminal amino acid sequence analysis. Other species that were detected were biglycan and the large proteoglycans versican (splice variants V0 and/or V1 and/or V2) and aggrecan. Approx. 23% of decorin detected in the matrix was degraded. Intact decorin and decorin fragments similar to those observed in the matrix that retained the N-terminus were also observed in the medium of ligament cultures. Intact biglycan core protein was detected in the matrix and medium of ligament cultures, and two fragments originating from the N-terminal region of biglycan were observed in the matrix of cultured ligament. Versican and versican fragments that retained the N-terminus of versican core protein were detected in fresh matrix and medium of tendon cultures. Approx. 42% of versican present in the fresh ligament was degraded. Aggrecan catabolites appearing in the culture medium were derived from aggrecanase cleavage of the core protein. An intact link protein and a degradation product from the N-terminal region of type XII collagen were also detected in the medium of the ligament explant.

Splicing variation of the versican message and size heterogeneity of the versican core protein were analyzed in human articular cartilage and intervertebral disc. Splicing variation of the message was studied by PCR analysis to detect the presence or absence of exons 7 and 8, which encode large chondroitin sulfate attachment regions. At all ages in normal cartilage from the third trimester fetus to the mature adult, the presence of the versican isoform possessing exon 8 but not exon 7 (V1) could be readily detected. The message isoforms possessing neither exon 7 nor 8 (V3) or both exons 7 and 8 (V0) were only detectable in the fetus, and the isoform possessing only exon 7 (V2) was never detected. In osteoarthritic cartilage and in adult intervertebral disc the versican message pattern was the same as that observed in the normal adult with only the isoform possessing exon 8 being detected. Core protein heterogeneity was studied by immunoblotting following enzymic removal of the glycosaminoglycan chains from the proteoglycan, using an antibody recognizing the globular G1 region of versican. All articular cartilage extracts from the fetus to the mature adult contained multiple core protein sizes of greater than 200 kDa. The adult cartilage extracts tended to have an increased proportion of the smaller sized core proteins and osteoarthritic cartilage possessed similar core protein sizes to the normal adult. In contrast, intervertebral disc at all post-natal ages showed a greater range of size heterogeneity with a prominent component of about 50 kDa. The abundance of this component increased if the samples were treated with keratanase prior to analysis, suggesting that the G1 region of versican in disc can be substituted with keratan sulfate. The increased presence of versican in the disc relative to articular cartilage may suggest a more pronounced functional role for this proteoglycan, particularly in the nucleus pulposus.

To investigate the effects of lead on the formation of extracellular matrix in the vascular wall, we characterized proteoglycans synthesized by cultured vascular smooth muscle cells after exposure to the metal by biochemical techniques. Confluent cultures of bovine aortic smooth muscle cells were metabolically labeled with [(35)S]sulfate or [(35)S]methionine/cysteine in the presence of lead nitrate. The amount of glycosaminoglycans (GAGs) was evaluated by the incorporation of [(35)S]sulfate into GAGs by the cetylpyridinium chloride precipitation method. The labeled proteoglycans were characterized by DEAE-Sephacel ion exchange chromatography and Sepharose CL-2B molecular sieve chromatography. The GAG M(r) and composition were analyzed by Sepharose CL-6B chromatography, and the core protein M(r) was analyzed by SDS-polyacrylamide gel electrophoresis, before and after digestion with papain or chondroitin ABC lyase. Lead significantly decreased the [(35)S]sulfate incorporation into GAGs accumulated in the cell layer and the conditioned medium. [(35)S]Sulfate-labeled proteoglycans obtained from the cell layer and the conditioned medium were separated into three peaks on DEAE-Sephacel chromatography and only the peak with the highest charge density was decreased by lead. The highly charged peak was eluted near the void volume on Sepharose CL-2B molecular sieve chromatography and sensitive to chondroitin ABC lyase on Sepharose CL-6B chromatography, indicating that lead selectively inhibits the synthesis of large and highly charged chondroitin/dermatan sulfate proteoglycans (CS/DSPGs). However, the size of chondroitin/dermatan sulfate chains of the CS/DSPGs was M(r) approximately 47000 in both the control and lead-treated cultures. On the other hand, lead decreased the accumulation of a large CS/DSPG with a core protein of approximately 450 kDa in the cell layer and the conditioned medium; the core protein was identified as versican core by Western blot analysis. It is therefore suggested that lead inhibits the synthesis of the versican core protein in vascular smooth muscle cells without a change in length of chondroitin/dermatan sulfate side chains. As a result, versican-poor extracellular matrix would be induced by lead in vascular smooth muscle cells.

The extracellular matrix of human fetal skin differs substantially from that of adult skin. Fetal skin contains sparse amounts of fibrillar collagen enmeshed in a highly hydrated amorphous matrix composed of hyaluronan and sulfated proteoglycans. Both fetal and adult skin contain two major interstitial proteoglycans that are extracted by chaotrophic agents and detergents. These are the large chondroitin sulfate proteoglycan versican and the small dermatan sulfate proteoglycan decorin. For this study, proteoglycans extracted from fetal and adult skin were compared on Western blots to determine the relative amounts of versican. Decorin present in the same samples provided an internal standard for these studies. Fetal skin differed from adult skin in that it contained a significantly higher proportion of versican than did adult skin. Immunohistochemical studies compared early-fetal with mid-fetal skin and found that versican was a significant component of the interstitial extracellular matrix at both of these stages of skin development. However, by the mid-fetal period, interstitial versican became restricted to the upper half of the dermis, although versican also continued to be highly expressed around hair follicles, glands, and vasculature in the lower half of the dermis. Fetal skin extracts differed from an adult skin extract by the presence of a 66-kDa protein immunologically related to versican and by the absence of a 17-kDa core protein of a proteoglycan related to decorin. Both of these molecular species may represent degradation products of their respective proteoglycans. Monoclonal antibodies which detect epitopes in native chondroitin sulfate glycosaminoglycan chains recognized versican extracted from fetal skin. However, the tissue distribution of these antigens did not entirely conform to that for versican core protein, suggesting that versican in different regions of the skin may be substituted with glycosaminoglycan chains with different microchemistries. The results of these studies indicate that human fetal skin is structurally different from adult skin in terms of both the distribution and the composition of the large, aggregating chondroitin sulfate proteoglycan versican.

Platelet-derived growth factor (PDGF) stimulates not only the proliferation and migration of arterial smooth muscle cells (ASMCs), but also the transcription, translation, and posttranslational processing of versican, a large chondroitin sulfate proteoglycan present in the extracellular matrix of blood vessels. PDGF receptor tyrosine kinase activity is required for signaling events associated with mitogenic and motogenic stimulation of cells by PDGF. Therefore, we have asked if inhibiton of tyrosine kinase activity by genistein also blocks the stimulation of both versican core protein synthesis and glycosaminoglycan (GAG) chain modifications induced by PDGF in ASMCs. The tyrosine kinase inhibitor, genistein, in a dose-dependent manner, reversibly inhibits PDGF-stimulated ASMC cell proliferation and RNA and core protein expression of versican, without affecting the expression of decorin and biglycan. In contrast, genistein does not affect the increase in GAG chain elongation that is induced by PDGF. This suggests that different aspects of the biosynthesis of versican are differentially regulated. To determine if such differential regulation involves downstream activation of protein kinase C, ASMCs were treated with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) to directly activate this kinase. In comparison to PDGF stimulation, TPA has little effect on expression of versican mRNA expression, nor does TPA stimulate ASMC cell proliferation. However, like PDGF, TPA increases [35S]sulfate incorporation into proteoglycans and GAG chain elongation. These results indicate that PDGF-induced GAG chain elongation, which is not inhibited by genistein treatment and is stimulated by protein kinase C activation, involves signaling pathways different from those that regulate PDGF-stimulated versican mRNA and protein expression.

Summary The expression of PG-M in osteoarthritic cartilage was investigated. Cartilage from five hip joints with osteoarthritis (OA) and control cartilage from five knee joints with post-traumatic injury were obtained and analyzed with anti-PG-M antibodies. Control cartilage showed no staining, but in osteoarthritic cartilage there was strong staining of the cytoplasm of chondrocytes with abnormal morphology. The cytoplasm of inflammatory cells invading the osteoarthritic cartilage matrix was also strongly stained which led to determining the sequence of PG-M core protein. The deduced amino acid sequence and homology analysis indicated that PG-M had a complement regulatory protein-like domain, a lectin-like domain, two EGF-like domains from the carboxyl-terminal with an extremely high homology to the respective domains of versican, a large proteoglycan expressed by human fibroblasts. The anti-PG-M antibodies cross-reacted with Ver-27b fusion protein which was expressed by a cDNA clone coding the N-terminal portion of versican core protein. Thus, the immunological and sequencing data suggest that PG-M is a molecule similar to or identical with human versican, and that the material in cartilage reactive to the anti-PG-M antibodies is versican. These findings suggest the PG-M/versican is expressed in osteoarthritic cartilage.

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.

The primary structure of a large chondroitin sulfate proteoglycan expressed by human fibroblasts has been determined. Overlapping cDNA clones code for the entire 2389 amino acid long core protein and the 20-residue signal peptide. The sequence predicts a potential hyaluronic acid-binding domain in the amino-terminal portion. This domain contains sequences virtually identical to partial peptide sequences from a glial hyaluronate-binding protein. Putative glycosaminoglycan attachment sites are located in the middle of the protein. The carboxy-terminal portion includes two epidermal growth factor (EGF)-like repeats, a lectin-like sequence and a complement regulatory protein-like domain. The same set of binding elements has also been identified in a new class of cell adhesion molecules. Amino- and carboxy-terminal portions of the fibroblast core protein are closely related to the core protein of a large chondroitin sulfate proteoglycan of chondrosarcoma cells. However, the glycosaminoglycan attachment regions in the middle of the core proteins are different and only the fibroblast core protein contains EGF-like repeats. Based on the similarities of its domains with various binding elements of other proteins, we suggest that the large fibroblast proteoglycan, herein referred to as versican, may function in cell recognition, possibly by connecting extracellular matrix components and cell surface glycoproteins.