AMPK-SIRT1-PGC-1α signaling regulates mitochondrial function in human articular chondrocytes

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Purpose:Mitochondrial dysfunction in chondrocytes is associated with osteoarthritis (OA), and impacts pathways implicated in cartilage degradation, for example, by promoting oxidative stress and increasing inflammatory cytokine-induced chondrocyte inflammation and matrix catabolism. AMPK is a master regulator of cellular energy homeostasis. We have previously reported that AMPK activity is reduced in human knee OA articular chondrocytes. IL-1b, TNFa, and biomechanical injury decrease AMPK activity in chondrocytes. In addition, AMPK pharmacologic activators suppress chondrocyte catabolic responses to inflammatory cytokines. Recent studies have demonstrated that SIRT1, a nicotinamide adenine dinucleotide (NAD)-dependent protein deacetylase as another fuel-sensing molecule, also plays an important role in cartilage matrix homeostasis. AMPK and SIRT1 share some common target molecules involved in mitochondrial function including PGC-1a, a master regulator of mitochondrial biogenesis. Therefore, we tested the hypothesis that AMPK and SIRT1 coordinate to regulate mitochondrial function in human articular chondrocytes, which contributes to regulation of matrix metabolism. Methods: Primary human knee chondrocytes treated with AMPK pharmacologic activators AICAR (1 mM) and A-769662 (0.5 mM), primary mouse chondrocytes isolated from femoral head cartilages of AMPKa1 knockout (KO) and wild type (WT) mice, or primary human knee chondrocytes transfected with SIRT1 or PGC-1a siRNA (Invitrogen) were subjected to Western blot analyses for phosphorylation and expression of AMPKa, expression of SIRT1 and PGC-1a. NAD/NADH ratio and mitochondrial biogenesis such as expression of PGC-1a and mitochondrial transcriptional factor A (TFAM), a major transcriptional factor governing mitochondrial DNA replication and transcription during mitochondrial biogenesis) were assessed in chondrocytes stimulated with AICAR and A-769662 by a NAD/NADH quantification assay (Biovision) and RT-PCR for mRNA expression of PGC-1a and TFAM, respectively. The conditionedmedia of human knee chondrocytes in the presence or absence of AICAR or A-769662, or the PGC-1a knockdown chondrocytes stimulated with IL-1b (10 ng/ml) and TNFa (20 ng/ml) was used for measuring nitric oxide (NO) generation and release of MMP-3 and -13 by Griess reaction and ELISA, respectively. Results: AMPK pharmacologic activators (AICAR or A-769662) prevented oxidative stress by up-regulating expression of the anti-oxidant enzymes SOD2 and catalase, effects dependent on PGC-1a. We observed induction of NAD+/NADH ratio and SIRT1 expression in human chondrocytes stimulated with AICAR or A-769662. Conversely, inhibition of SIRT1 expression was seen in AMPKa1 KO mouse chondrocytes. However, knockdown of SIRT1 in human chondrocytes did not alter phosphorylation and expression of AMPKa, but impaired PGC-1a protein expression, suggesting that SIRT1 was downstream of AMPK but upstream of PGC-1a. In addition, AICAR or A769662 promoted mitochondrial biogenesis, as evidenced by increased PGC-1a and TFAM mRNA expression. These effects were linked to near complete inhibition of catabolic responses, including >90% decreased release of NO and MMP-3 andMMP-13 in chondrocytes (P< 0.01 for all) in response to IL1b and TNFa. Opposite results, with significantly increased release of NO, MMP-3 and MMP-13 (P<0.01) were observed in the PGC-1a knockdown chondrocytes. Conclusions: AMPK-SIRT1-PGC-1a signaling regulates mitochondrial function by promoting mitochondrial biogenesis, decreasing oxidative stress in chondrocytes. These effects contribute to attenuation, by AMPK, of catabolic chondrocyte responses to inflammatory cytokines. These findings provide a novel molecular mechanism by which pharmacologic activation of AMPK has translational potential to inhibit progression of cartilage degradation in OA.