Integrin expression by primary and immortalized human chondrocytes: evidence of a differential role for alpha1beta1 and alpha2beta1 integrins in mediating chondrocyte adhesion to types II and VI collagen.

OBJECTIVE: Chondrocytes have been shown to express beta1-containing integrins both in vitro and in situ, but their role in regulating chondrocyte function is poorly understood. The objective of this study was to determine how the relative expression of different integrins may be modulated in relation to the differentiated state and proliferative capacity of the chondrocyte. DESIGN: Integrin expression by four different cell lines of human chondrocytes immortalized with Simian virus 40 large T-antigen (SV40-TAg) was studied and compared to primary chondrocytes. Differences in alpha1 and alpha2 integrin subunit expression were utilized to further study the role of these integrins in mediating adhesion to types II and VI collagen. RESULTS: The overall cell-surface levels of beta1-containing integrins were higher on all four immortalized cell lines which expressed over 10-fold higher levels of alpha2 and alpha3 integrin subunits compared to primary cells. However, primary cells expressed higher levels of the alpha1 integrin subunit which was not expressed by T/C28a4 cells and expressed at variable and lower levels in the other lines. Levels of the alpha3 integrin subunit were significantly greater on the highly proliferative juvenile costal chondrocyte lines (T/C-28a4, C-2812, and C-20a4) compared to primary articular chondrocytes and tsT/AC-62 cells which were derived from adult articular chondrocytes. Expression of alpha5 was similar among primary cells and cell lines except on C-20/A4 cells which had an average of over 4-fold higher levels. None of the primary or immortalized chondrocytes tested expressed significant levels of alpha4. Cell adhesion assays revealed that both alpha1beta1 and alpha2beta1 could serve as chondrocyte adhesion receptors for types II and VI collagen. In cell lines expressing both integrins, alpha1beta1 was the preferential receptor for type VI collagen while alpha2beta1 was the preferential receptor for type II collagen. Rather than inhibiting adhesion, incubation with the alpha3 blocking antibody P1B5 increased adhesion of C-28/12 cells to both fibronectin and type II collagen by 67% and 100% respectively. CONCLUSIONS: Immortalization with SV40-TAg results in altered integrin expression by chondrocytes. Changes in the relative levels of alpha1, alpha2, and alpha3 subunits may significantly alter the manner in which chondrocytes interact with types II and VI collagen in the extracellular matrix.

DNA sequence and muscle-specific expression of human sarcosin transcripts.

We have characterized two abundant human cDNAs which, through Northern hybridization analysis, are selectively expressed in human sarcomeric muscle. DNA sequencing was performed and the two cDNAs were found to share sequence identity, with the exception of a 3' UTR extension present on the longer transcript. Our data suggest that the two transcripts are generated through alternative use of two poly(A) addition signals. The cDNAs encode a large open reading frame encompassing at least 435 codons. Through sequence comparisons, both at the DNA and predicted amino acid sequence level, we have been unable to find significant sequence similarity to any other characterized sequence. Consequently, we have termed this novel human sequence sarcosin. Although novel, Southern hybridization analysis demonstrated that the sarcosin sequence has been conserved in several mammalian species.

Human chondrocyte expression of growth-arrest-specific gene 6 and the tyrosine kinase receptor axl: potential role in autocrine signaling in cartilage.

OBJECTIVE: To determine if human articular chondrocytes express the axl tyrosine kinase receptor and its ligand Gas-6, a protein product of growth-arrest-specific gene 6, and to determine if Gas-6 and axl function in the regulation of chondrocyte growth and survival. METHODS: The presence of Gas-6 and axl was examined in situ in human articular cartilage by immunohistochemistry and in vitro in cell culture studies using primary human chondrocytes and immortalized human chondrocytes. The ability of recombinant Gas-6 to mediate adhesion of chondrocytes and to stimulate chondrocyte axl phosphorylation was determined. Studies of the role of Gas-6 and axl in cell proliferation and survival were also performed. RESULTS: Both Gas-6 and axl were detected in cartilage by immunohistochemical staining. Gas-6 and axl messenger RNA (mRNA) and protein were also detected in cultures of primary and immortalized human chondrocytes. Compared with cells cultured in medium containing 10% serum, Gas-6 mRNA levels were increased in immortalized chondrocytes cultured in serum-free medium, while axl expression decreased. Chondrocytes attached to Gas-6-coated plastic, and the attachment was blocked by a soluble Ig fusion protein containing the axl extracellular domain. Recombinant human Gas-6 and serum-free conditioned medium from primary and immortalized human chondrocyte cultures stimulated chondrocyte axl tyrosine phosphorylation. A mitogenic effect was noted both when immortalized chondrocytes were stimulated with recombinant Gas-6 or when they were made to overexpress axl by transfection. Addition of recombinant Gas-6 to serum-free medium resulted in increased survival of primary chondrocytes cultured at low density in agarose. CONCLUSION: These findings present evidence for an autocrine signaling pathway in cartilage involving Gas-6 and the axl tyrosine kinase adhesion receptor. Stimulation of axl by Gas-6 may play an important role in the control of chondrocyte growth and survival.