Synthesis and extracellular deposition of fibronectin in chondrocyte cultures. Response to the removal of extracellular cartilage matrix.

Abstract

Fibronectin, the major cell surface glycoprotein of fibroblasts, is absent from differentiated cartilage matrix and chondrocytes in situ. However, dissociation of embryonic chick sternal cartilage with collagenase and trypsin, followed by inoculation in vitro reinitiates fibronectin synthesis by chondrocytes. Immunofluorescence microscopy with antibodies prepared against plasma fibronectin (cold insoluble globulin [CIG]) reveals fibronectin associated with the chondrocyte surface. Synthesis and secretion of fibronectin into the medium are shown by anabolic labeling with [3~S]methionine or [3H]glycine, and identification of the secreted proteins by immunoprecipitation and sodium dodecyl sulfate (SDS)-disc gel electrophoresis.When chondrocytes are plated onto tissue culture dishes, the pattern of surfaceassociated fibronectin changes from a patchy into a strandlike appearance. Where epithelioid clones of polygonal chondrocytes develop, only short strands of fibronectin appear preferentially at cellular interfaces. This pattern is observed as long as cells continue to produce type II collagen that fails to precipitate as extracellular collagen fibers for some time in culture. Using the immunofluorescence double-labeling technique, we demonstrate that fibroblasts as well as chondrocytes which synthesize type I collagen and deposit this collagen as extracellular fibers show a different pattern of extracellular fibronectin that codistributes in large parts with collagen fibers. Where chondrocytes begin to accumulate extracellular cartilage matrix, fibronectin strands disappear.From these observations, we conclude (a) that chondrocytes synthesize fibronectin only in the absence of extracellular cartilage matrix, and (b) that fibronectin forms only short intercellular "stitches" in the absence of extracellular collagen fibers in vitro. KEY

Extracellular matrix formation by chondrocytes in monolayer culture.

“…No extracellular type II collagen matrix is observed during the first days in monolayer culture when cells have flattened, as demonstrated previously (5,29,32) . It is unlikely that the failure of extracellular deposition of type II collagen in early stages of the culture resulted from lack of ascorbate (8,24), for fresh sodium ascorbate was added with each medium change.…”

“…Type II collagen reappears in the extracellular space after chondrocytes in the center of epithelioid colonies reassume spherical shape and accumulate highly refractile extracellular matrix (5) . This matrix stains with antibodies to both CSPG and type II collagen (Fig.…”

“…Indirect immunofluorescence was carried out as described previously (5,29,30) . For intracellular fluorescence labeling of chondrocytes in monolayer culture, culture dishes were rinsed with phosphate-buffered saline, treated for 5 min with 70% ethanol, and air-dried before application of the antibodies.…”

“…Although synthesis and secretion of type II collagen continued, extracellular deposition of type II collagen was not observed until cells reassumed spherical shape. In the newly formed cartilage matrix, fibronectin disappeared (5).…”

Role of anchorin CII, a 31,000-mol-wt membrane protein, in the interaction of chondrocytes with type II collagen.

“…Immunohistochemical and clectronmicroscopical studies on isolated chondrocytes (15)(16)(17)(18)(19) have demonstrated a close association of type II collagen with the chondrocyte surface . The precise nature ofthis cell:matrix association, however, is still unclear .…”