Identification and Characterization of Versican/PG-M Aggregates in Cartilage

Abstract

Versican/PG-M is a large chondroitin sulfate proteoglycan of the extracellular matrix with a common domain structure to aggrecan and is present in cartilage at low levels. Here, we characterized cartilage versican during development and growth. Immunostaining showed that versican was mainly localized in the interterritorial zone of the articular surface at 2 weeks in mice, whereas aggrecan was in the pericellular zone of prehypertrophic and hypertrophic cells of the growth plate. Although its transcription level rapidly diminished during growth, versican remained in the articular cartilage. Biochemical analysis of normal articular cartilage and aggrecan-null cartilage from cmd (cartilage matrix deficiency)/cmd mice revealed that versican was present as a proteoglycan aggregate with both link protein and hyaluronan. Chondroitin sulfate chains of versican digested with chondroitinase ABC contained 71% nonsulfated and 28% 4-sulfated unsaturated disaccharides, whereas those of aggrecan contained 25% nonsulfated and 70% 4-sulfated. Link protein overexpression in chondrocytic N1511 cells at the early stage of differentiation, in which versican is expressed, enhanced versican deposition in the matrix and prevented subsequent aggrecan deposition. These results suggest that versican is present as an aggregate distinct from the aggrecan aggregate and may play specific roles in the articular surface.Versican/PG-M (1, 2) is a large chondroitin sulfate proteoglycan of the extracellular matrix (ECM) 2 that exhibits two distinct expression patterns. In various developing embryonic tissues (3), including the nervous system, versican is transiently expressed and plays important roles in cell adhesion (4), migration (5), proliferation, and differentiation (6). In some adult tissues, such as the heart, blood vessels, and brain, it is constitutively expressed and serves as a structural macromolecule of the ECM.In developing cartilage, versican is transiently expressed at a high level in the mesenchymal condensation area and rapidly disappears during cartilage development (7,8). Recent immunohistochemical studies on developing limb bud cartilage revealed that an area positive for versican gradually shifts out of the diaphysis and is replaced by an area positive for aggrecan. Differentiating chondrocytic N1511 and ATDC5 cells showed similar expression patterns of these molecules (9). These reciprocal patterns of versican and aggrecan expression suggest that versican serves as a temporary framework in developing cartilage matrix. Although the aggrecan aggregate (10) is the major component of cartilage ECM and versican has not been detected by immunohistochemical studies (3), constitutive low level transcription of the versican gene is observed in cartilage (11) and chondrocytes (11,12). In addition, extracts of human adult articular cartilage contain versican (13), suggesting its distinct role there.The core protein of versican consists of N-and C-terminal globular domains (G1 and G3) and two chondroitin sulfate (CS) domains (CS-␣ a...

Versican Facilitates Chondrocyte Differentiation and Regulates Joint Morphogenesis

“…By chondroitinase ABC treatment of Prx1-Cre/Vcan ϩ/ϩ micromass for 48 h before fixation, both versican and TGF-␤ retained essentially the same localization patterns as the untreated micromass, eliminating a direct function of CS chains in accumulating TGF-␤ in the ECM. In addition, CS chains attached to versican in cartilage are undersulfated compared with those of aggrecan (41). Furthermore, gene-trapped mice of C4st1 (chondroitin-4-sulfotranferase 1), C4st1 gt/gt , with a substantial decrease in CS amounts, show no abnormalities at E11.5 and E13.5 when mesenchymal condensation occurs in the limb bud (42).…”

Versican/PG-M Assembles Hyaluronan into Extracellular Matrix and Inhibits CD44-mediated Signaling toward Premature Senescence in Embryonic Fibroblasts

“…The chamber slides were immunostained with the following specific antibodies: rabbit polyclonal anti-versican CS-␣ and CS-␤ (a mixture of ϫ1000 dilutions) (27), rabbit polyclonal anti-laminin (ϫ1000) (28), rabbit polyclonal anti-fibronectin (ϫ1000) (29), and rat monoclonal anti-CD44 (clone IM7.8.1, Acris, 5 g/ml). An anti-rabbit IgG conjugated with horseradish peroxidase (ϫ1000; Dako) was used as the secondary antibody.…”

“…An equal amount of proteoglycan was dissolved in chondroitinase ABC buffer (20 mM Tris-HCl, pH 8.0, 20 mM sodium acetate, 0.02% bovine serum albumin), digested with chondroitinase ABC (5 milliunits/ml) at 37°C for 3 h, and then the reaction was stopped by boiling at 100°C for 5 min. The unsaturated CS disaccharide product was analyzed by fluorometric postcolumn high performance liquid chromatography as described previously (27).…”

Chondroitin Sulfate N-Acetylgalactosaminyltransferase-1 Plays a Critical Role in Chondroitin Sulfate Synthesis in Cartilage

“…For chondroitin 4-sulfate detection, mouse monoclonal anti-chondroitin 4-sulfate (LY111, ϫ200; Seikagaku) and Alexa Fluor TM 488-conjugated anti-mouse IgM (ϫ1000) were used as the primary and secondary antibodies, respectively. For aggrecan core protein detection, rabbit polyclonal anti-aggrecan (ϫ1000) (26) and Alexa Fluor TM 594-conjugated anti-rabbit IgG (ϫ1000) were used as the primary and secondary antibodies, respectively. The specimen was observed using a Zeiss LSM 5 Pascal laser confocal microscope.…”