IGF-1 does not moderate the time-dependent transcriptional patterns of key homeostatic genes induced by sustained compression of bovine cartilage

Objective To determine changes in chondrocyte transcription of a range of anabolic, catabolic and signaling genes following simultaneous treatment of cartilage with Insulin-like growth factor-1 (IGF-1) and ramp-and-hold mechanical compression, and compare with effects on biosynthesis. Methods Explant disks of bovine calf cartilage were slowly compressed (unconfined) over 3-min to their 1 mm cut-thickness (0%-compression) or to 50%-compression with or without 300 ng/ml IGF-1. Expression of 24 genes involved in cartilage homeostasis was measured using qPCR at 2, 8, 24, 32, 48 h after compression ±IGF-1. Clustering analysis was used to identify groups of co-expressed genes to further elucidate mechanistic pathways. Results IGF-1 alone stimulated gene expression of aggrecan and collagen II, but simultaneous 24h compression suppressed this effect. Compression alone up-regulated expression of matrix metalloproteinase (MMP)-3, MMP-13, a disintegrin and metalloproteinase with thrombospondin motif (ADAMTS)-5 and transforming growth factor (TGF)-β, an effect not reversed by simultaneous IGF-1 treatment. Temporal changes in expression following IGF-1 treatment were generally slower than that following compression. Clustering analysis revealed five distinct groups within which the pairings, tissue inhibitor of metalloproteinase (TIMP)-3 and ADAMTS-5, MMP-1 and IGF-2, and IGF-1 and Collagen II, were all robustly co-expressed, suggesting inherent regulation and feedback in chondrocyte gene expression. While aggrecan synthesis was transcriptionally regulated by IGF-1, inhibition of aggrecan synthesis by sustained compression appeared post-transcriptionally regulated. Conclusion Sustained compression markedly altered the effects of IGF-1 on expression of genes involved in cartilage homeostasis, while IGF-1 was largely unable to moderate the transcriptional effects of compression alone. The demonstrated co-expressed gene pairings suggest a balance of anabolic and catabolic activity following simultaneous mechanical and growth factor stimuli.National Institutes of Health (U.S.) (grant R01-AR33236)National Institutes of Health (U.S.) (grant R01-HG003352)National Institutes of Health (U.S.) (grant P42-ES04699)National Institutes of Health (U.S.) (grant T32-EB006348

Co-culture of mechanically injured cartilage with joint capsule tissue alters chondrocyte expression patterns and increases ADAMTS5 production

We studied changes in chondrocyte gene expression, aggrecan degradation, and aggrecanase production and activity in normal and mechanically injured cartilage co-cultured with joint capsule tissue. Chondrocyte expression of 21 genes was measured at 1, 2, 4, 6, 12, and 24 h after treatment; clustering analysis enabled identification of co-expression profiles. Aggrecan fragments retained in cartilage and released to medium and loss of cartilage sGAG were quantified. Increased expression of MMP-13 and ADAMTS4 clustered with effects of co-culture, while increased expression of ADAMTS5, MMP-3, TGF-β, c-fos, c-jun clustered with cartilage injury. ADAMTS5 protein within cartilage (immunohistochemistry) increased following injury and with co-culture. Cartilage sGAG decreased over 16-days, most severely following injury plus co-culture. Cartilage aggrecan was cleaved at aggrecanase sites in the interglobular and C-terminal domains, resulting in loss of the G3 domain, especially after injury plus co-culture. Together, these results support the hypothesis that interactions between injured cartilage and other joint tissues are important in matrix catabolism after joint injury

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