Cytokine-Induced Increases in ADAMTS-4 Messenger RNA Expression Do Not Lead to Increased Aggrecanase Activity in ADAMTS-5-Deficient Mice

Objective. To compare the regulation of aggrecanase messenger RNA (mRNA) and enzyme activity by proinflammatory cytokines in primary mouse chondrocytes. Methods. Primary chondrocytes were isolated from knee epiphyses of 6-8-day-old mice and cultured as monolayers. The cells were incubated with tumor necrosis factor alpha (TNF alpha), oncostatin M (OSM), or interleukin-6 (IL-6)/soluble IL-6 receptor, and mRNA levels were measured by quantitative polymerase chain reaction at various time points. To measure aggrecanase activity, the cells were incubated with cytokine in the presence of exogenous aggrecan, and substrate cleavage was measured using antibodies to neoepitopes. Results. Expression of both ADAMTS-4 and ADAMTS-5 mRNA was up-regulated by TNF alpha and OSM. ADAMTS-5 mRNA expression was also upregulated by IL-6. Treatment of wild-type mouse chondrocytes with each of the 3 cytokines increased cleavage of aggrecan at Glu(373) down arrow (374)Ala and Glu(1670) down arrow (1671)Gly; in chondrocytes lacking ADAMTS-5 activity, there was negligible cleavage at either site despite increased expression of ADAMTS-4 mRNA in the presence of TNF alpha or OSM. None of the cytokines substantially altered mRNA expression of ADAMTS-1 or ADAMTS-9. Conclusion. Despite substantial increases in the expression of ADAMTS-4 mRNA induced by TNF alpha and OSM, these cytokines induced little if any increase in aggrecanolysis in ADAMTS-5-deficient mouse chondrocytes. Our data show a poor correlation between the level of cytokine-induced ADAMTS-4 mRNA expression and the level of aggrecan-degrading activity in cultured chondrocytes.