Effects of adipokines on the degradation of OA cartilage matrix The effect of the adiponectin on matrix degradation in OA cartilage explants was evaluated ex vivo. IL 1b served as a positive control. On days 4 and 8, the levels of C1 2C were significantly increased in the super natants of cartilage explants cultures by 5 ngml of IL 1b. In the meantime, C1 2C concentrations were significantly elevated on day 8 with 30 ugml adiponectin. Effect of protein kinase inhibitors on adiponectin induced production of MMPs and NO Because adiponectin was a potential player in cartilage degradation in vitro and ex vivo, we assessed signaling pathways involved in adipokine induced upregulation of NO and MMPs. After plating OA chondrocytes in wells coated with poly HEMA, protein kinases were added to the media 1 hour before adiponectin treatment, and cells were incubated for 24 hours.
Adi ponectin induced total NO production was significantly suppressed a cool way to improve by inhibitors of NF B, AMPK, and JNK. In addition, MMP 1 secretion was inhibited by p38, AMPK, or JNK inhibitors, MMP 3 by ERK, AMPK, and JNK inhibitors, and MMP 13 by all but NF B inhibitor. Espe cially AMPK and JNK inhibitors significantly suppressed production of total NO and all three MMPs by 40% or more, suggesting that AMPK JNK axis is the major pathway involved in adiponectin induced biologic actions. When examined with immunoblotting, increased phospho AMPK and phospho JNK levels were observed in adiponectin stimulated OA chondrocytes.
Effect of NOS inhibitors on adiponectin induced production of MMPs Because adiponectin markedly enhanced NO produc tion in OA chondrocytes in the present study and because NO has been previously suggested to selelck kinase inhibitor affect the expression of MMPs, the effects of NOS inhibi tors on adiponectin induced MMPs production were evaluated by using a nonselective NOS inhibitor, L NMMA, and a selective iNOS inhibitor, L NIL. Inter estingly, when the NOS inhibitors were added to chondrocytes 24 hours before adiponectin stimulation, both inhibitors significantly augmented adiponectin induced secretion of the three MMPs. Especially the levels of MMP 13 were increased by an average of 3. 3 fold with L NMMA and by an aver age of 2. 8 fold with L NIL. Discussion The present study demonstrates that adiponectin increased NO and three MMPs production in human OA chondrocytes mainly via the AMPK JNK pathway in vitro and that adiponectin induced NO and MMPs lead to accelerated degradation of OA cartilage matrix ex vivo.
Our in vitro findings indicate that adiponectin is a potential catabolic mediator in OA. This is in line with the previous findings that adiponectin induces iNOS, MMP 3, MMP 9, and MCP 1 in murine chondrocytes. More important, increased cartilage degradation products after adiponectin treatment further supports that in vitro catabolic activity induced by adiponectin is relevant to cause cartilage degradation.