Objective
Hypoxia-inducible factor-2a (HIF-2a) transcriptionally upregulates Nampt in articular chondrocytes. NAMPT, which exhibits nicotinamide phosphoribosyltransferase activity, in turn causes osteoarthritis (OA) in mice by stimulating the expression of matrix-degrading enzymes. Here, we sought to elucidate whether HIF-2a activates the NAMPT-NAD+-SIRT axis in chondrocytes and thereby contributes to the pathogenesis of OA.

Methods
Assays of NAD levels, SIRT activity, reporter gene activity, mRNA, and protein levels were conducted in primary cultured mouse articular chondrocytes. Experimental OA in mice was induced by intra-articular (IA) injection of adenovirus expressing HIF-2a (Ad-Epas1) or NAMPT (Ad-Nampt). The functions of SIRT in OA were examined by IA co-injection of SIRT inhibitors or adenovirus expressing individual SIRT isoforms or shRNA targeting specific SIRT isoforms.

Results
HIF-2a activated the NAMPT-NAD+-SIRT axis in chondrocytes by upregulating NAMPT, which stimulated NAD+ synthesis and thereby activated SIRT family members. The activated NAMPT-SIRT pathway, in turn, promoted HIF-2a protein stability by negatively regulating its hydroxylation and 26S proteasome-mediated degradation, resulting in increased HIF-2a transcriptional activity. Among SIRT family members (SIRT1–7), SIRT2 and SIRT4 were positively associated with HIF-2a stability and transcriptional activity in chondrocytes. This reciprocal regulation was required for the expression of catabolic matrix metalloproteinases (MMP3, MMP12, and MMP13) and OA cartilage destruction caused by IA injection of Ad-Epas1 Ad-Nampt.

Conclusion
The reciprocal regulation of HIF-2a and the NAMPT-NAD+-SIRT axis in articular chondrocytes is involved in OA cartilage destruction caused by HIF-2a or NAMPT.

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