Oxidative Stress-Induced Sulfenylation of SIRT6 IS Associated with Enhanced NF-κB Signaling in Human Chondrocytes

Purpose: Increases in reactive oxygen species (ROS) lead to oxidative stress conditions that can disrupt homeostatic cell signal transduction through protein thiol oxidation. The pro-longevity protein sirtuin 6 (SIRT6) has been shown to undergo sulfenylation (cysteine oxidation to sulfenic acid, -SOH) in the presence of H2O2, which could disturb normal SIRT6-mediated downstream signaling pathways to promote OA. Active SIRT6 contributes to transcriptional repression of the NF-κB transcription factor which regulates pro-catabolic signaling proteins implicated in aging and OA. The purpose of this study was to investigate the redox sensitivity of SIRT6 and SIRT6-mediated downstream cell signaling in human chondrocytes. Methods: Primary chondrocytes isolated from normal human articular cartilage (age range 21-70 yrs), or from chondrocytes isolated from OA cartilage (age range 47-74 yrs) were treated with H2O2 (0-50 μM), fibronectin fragments (FN-f, 1 μM), or menadione (25 μM) to generate cellular ROS. Intracellular H2O2 was measured using the redox biosensor, Orp1-roGFP. SIRT6 and p65 protein levels were measured by immunoblotting. SIRT6 gene expression was analyzed by quantitative PCR. S-sulfenylation of SIRT6 was detected using dimedone-based probes (DCP-Bio1) that react selectively with cysteines oxidized to sulfenic acid. The activity of recombinant SIRT6 was measured using a SIRT6 deacetylation activity assay. To assess the effect of oxidative stress on nuclear p65 translocation (needed for transcriptional regulation), chondrocytes were treated with menadione and nuclear and cytosolic fractions were isolated and analyzed by immunoblotting. To examine the effect of SIRT6 overexpression on p65 signaling and oxidative stress levels, chondrocytes were transduced with an adenoviral vector encoding SIRT6. Hyperoxidized PRXs resulting from excessive H2O2 were detected using an antibody that reacts with PRX 1-3 when it is in the PRX-SO2/3 state. Results: In unstimulated chondrocytes, basal p65 protein levels were increased in OA chondrocytes when compared to younger chondrocytes (Fig. 1A). SIRT6 protein levels (Fig. 1B) and gene expression did not change with age or OA. Menadione treatment generated a rapid increase in H2O2 levels that oxidized Orp1-roGFP within 20 seconds. Menadione-induced H2O2 generation led to a significant increase in SIRT6 DCP-Bio1 labeling compared to control cells (Fig. 1C) suggesting that SIRT6 undergoes sulfenylation in response to high levels of ROS. Recombinant SIRT6 activity was significantly inhibited within 5 min of 50 μM H2O2 treatment and remained inhibited over the time course studied (Fig. 1D). At time points that elicit menadione-induced SIRT6 oxidation (30 min), a significant increase in nuclear phosphorylated p65 was observed. Overexpression of SIRT6 led to a reduction in p65 nuclear localization as well as a decrease in nuclear PRX-SO2/3 formation. Conclusions: We propose that high levels of H2O2, such that occur in aging and OA tissues, lead to oxidation of SIRT6, which inhibits SIRT6 activity and SIRT6-regulated cell signaling. Our findings support this hypothesis and demonstrate that SIRT6 undergoes sulfenylation in response to H2O2 in chondrocytes, an effect which correlates with inhibition of SIRT6 activity in an in vitro system. SIRT6 oxidation was associated with enhanced nuclear localization of phosphorylated p65, which was abrogated by SIRT6 overexpression. Targeted therapies aimed at maintaining SIRT6 activity could represent a novel strategy to slow or stop the progression of OA through maintenance of physiological redox signaling.