Portal de Periódicos da CAPES

Immortalized human adult articular chondrocytes maintain cartilage-specific phenotype and responses to interleukin-1β

2000; Wiley; Volume: 43; Issue: 10 Linguagem: Inglês

10.1002/1529-0131(200010)43

1529-0131

James R. Robbins, Beatrice C. Thomas, Lujian Tan, Bob K. Choy, Jack L. Arbiser, Françis Berenbaum, Mary B. Goldring,

Cell Adhesion Molecules Research

Objective To develop a reproducible immortalized human chondrocyte culture model for studying the regulation of chondrocyte functions relevant to arthritic diseases in adult humans. Methods Primary adult articular chondrocytes were immortalized with a retrovirus expressing a temperature-sensitive mutant of SV40–large T antigen (tsTAg). The established tsT/AC62 chondrocyte cell line was examined in monolayer and alginate culture systems. The levels of messenger RNA (mRNA) encoding cartilage matrix proteins and interleukin-1β (IL-1β)–inducible mRNA were analyzed by reverse transcriptase–polymerase chain reaction. Matrix protein synthesis was analyzed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis of 35S-sulfate–labeled proteoglycans and Western blotting of type II collagen and aggrecan. Type II collagen (COL2A1)–luciferase reporter gene expression was analyzed by transient transfection. Phosphorylated stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK), p38 mitogen-activated protein kinase (p38 MAPK), and activating transcription factor 2 (ATF-2) were detected by Western blotting. Results The tsT/AC62 cells expressed TAg at the permissive temperature (32°C), and the loss of TAg at 37°C and 39°C correlated with decreased cell proliferation. Cells in alginate culture deposited abundant alcian blue–stainable matrix and continued to proliferate at 32°C. Preferential retention of aggrecan was observed in the cell-associated matrix, while biglycan and decorin were secreted into the medium of monolayer and alginate cultures. The levels of COL2A1 and aggrecan mRNA were increased after transfer from monolayer to alginate culture at 32°C. Treatment with IL-1β decreased COL2A1 and aggrecan mRNA levels and increased the levels of matrix metalloproteinases 1, 3, and 13 mRNA, as well as those of cyclooxygenase 2, type I collagen, and secretory phospholipase A2 type IIA mRNA, but not those of inducible nitric oxide synthase mRNA. IL-1β also stimulated phosphorylation of p38 MAPK, SAPK/JNK, and ATF-2. The p38 MAPK–selective inhibitor, SB203580, partially reversed IL-1β–induced inhibition of COL2A1 mRNA levels and COL2A1–luciferase reporter gene expression. Conclusion The tsT/AC62 cells provide a reproducible model that mimics the adult articular chondrocyte phenotype, particularly in alginate culture, and demonstrates characteristic responses to IL-1β. These studies also show, for the first time, that p38 MAPK is one of the signals required for IL-1β–induced inhibition of COL2A1 gene expression. Availability of this model will permit identification of signals that regulate cytokine responses, and will also provide rational strategies for targeting these pathways.