Endothelin-1 in osteoarthritic chondrocytes triggers nitric oxide production and upregulates collagenase production

Background

Cartilage degradation in osteoarthritis (OA) and rheumatoid arthritis constitutes a major structural change in the joint, which may severely impair its function causing pain and disability. This degradation is accompanied by the release in the synovial fluid of degraded matrix constituents that primarily result from an increased matrix catabolism. Various factors are directly involved in this process. Endothelin-1 (ET-1), a potent vasoconstrictor and promitogen peptide for many cell types, including chondrocytes, was recently identified as one such factor.

Objective

We previously demonstrated that ET-1 induces matrix metalloproteinase (MMP)-1 and MMP-13 synthesis, secretion and activation. Here, we investigated the mechanism by which ET-1 induces the production of these two MMPs.

Methods

Human OA chondrocytes were cultured in the presence of ET-1 with or without inhibitors of protein kinase or LY83583 (an inhibitor of soluble guanylate cyclase and of cGMP) and then MMP-1, MMP-13 and nitric oxide (NO) levels were measured by ELISA and Griess reaction, respectively. Additionally, inducible nitric oxide synthase (iNOS) and activated forms of p38 mitogen-activated protein kinase, p44/42, SAP/JNK and serine threonine kinase Akt were determined by western blot, Bad and Bcl2 proteins by immunocytochemistry and apoptosis by TUNEL.

Results

ET-1 greatly increased MMP-1 and MMP-13 production, NO release and iNOS expression. LY83583 decreased the production of both MMPs below basal levels, whereas the inhibitor of p38 kinase, SB202190, suppressed ET-1-stimulated production only. Similarly, the ET-1-induced NO production was partially suppressed by the p38 kinase inhibitor and completely suppressed by the PKA kinase inhibitor, KT5720, and LY83583, suggesting the involvement of these enzymes in ET-1 signaling pathways. ET-1 does not induce apoptosis and could even have a protective effect through the induction of Akt phosphorylation.

Conclusions

In human OA chondrocytes, ET-1 controls the production of MMP-1 and MMP-13. ET-1 also induces NO release via iNOS induction. Thus, ET-1 and NO should become important target molecules for future therapies aimed at stopping cartilage destruction.