Sirtuin 6 (SIRT6) overexpression regulates antioxidant status and redox signaling in human chondrocytes

Purpose: An imbalance between the generation of reactive oxygen species (ROS) and the antioxidant capacity of the cell leads to oxidative stress conditions, which can promote OA by disturbing physiological cell signal transduction. Recent evidence suggests that the pro-longevity protein sirtuin 6 (SIRT6) can contribute to redox homeostasis by upregulating the antioxidant response, but the effects of SIRT6 on oxidative stress and downstream redox signaling events in chondrocytes are not well defined. The purpose of this study was to investigate the role of SIRT6 overexpression on antioxidant status and downstream cell signaling events in human chondrocytes. Methods: Primary chondrocytes isolated from normal human articular cartilage were transduced with an adenoviral vector encoding SIRT6 (ad-SIRT6) or a null empty vector control (ad-Null) for 48 hours. Cells were treated with the H2O2-generating redox cycling oxidant, menadione (25 μM) for 0-30 mins to generate intracellular ROS. Histone 3 lysine 9 (H3K9) is a major SIRT6 substrate and its acetylation, detected by immunoblotting, was a marker of SIRT6 activity. Effects of oxidative stress and SIRT6 overexpression on p65 nuclear translocation was determined by immunoblotting cytosolic and nuclear fractions. As a marker of oxidative stress, hyperoxidized peroxiredoxins (Prxs) were measured using an antibody that reacts with Prx1-4 in the Prx-SO2/3 state. Basal antioxidant and antioxidant-related protein levels from cytosolic, nuclear, and total cell extracts were assessed by immunoblotting. These included Prxs 1-3, thioredoxin (Trx) isoforms 1 and 2, thioredoxin reductase (TrxR) isoforms 1 and 2, sulfiredoxin (Srx) and thioredoxin-interacting protein (TXNIP). The effect of SIRT6 overexpression on Prx1, TXNIP, RELA (p65) and Trx gene expression was analyzed by Real Time PCR (RT-PCR). Results: ROS generation by menadione led to a 6-fold increase in acetylation of H3K9 (15 mins p=0.0005; 30 mins p=0.0491), indicative of decreased chondrocyte SIRT6 activity under oxidative stress conditions (Fig1A). Decreased SIRT6 activity was associated with enhanced Prx hyperoxidation and increased phosphorylation and nuclear translocation of p65 when compared to control conditions. Overexpression of SIRT6 inhibited menadione-induced acetylation of H3K9 (Fig1A) and this was associated with decreased Prx hyperoxidation and p65 nuclear localization. Overexpression of SIRT6 had no effect on the basal protein levels of Prx2, Prx3, Trx1, Trx2, TrxR1, TrxR2 or Srx. However, overexpression of SIRT6 led to a significant increase in basal Prx1 protein levels measured in total cell lysates (↑50%) (Fig1B) as well as in the cytosol (↑320%), and nucleus (↑30%), when compared to null vector controls. A significant reduction in basal TXNIP protein levels in total (↓40%) (Fig1B), cytosolic (↓61%), and nuclear (↓36%) lysates was also observed compared to null vector controls. Overexpression of SIRT6 led to a significant increase in Prx1 mRNA levels (46%) and a significant decrease in TXNIP (54%) mRNA levels. Conclusions: These findings add strength to the hypothesis that maintenance of SIRT6 activity contributes to enhanced redox balance. We provide evidence that overexpression of SIRT6 associates with upregulation of Prx1 and downregulation of TXNIP, which correlates with decreased markers of oxidative stress and reduced activation of catabolic p65. TXNIP is a negative of regulator of Trx. Trx plays a crucial role in regenerating the Prx family of antioxidant proteins. Through decreasing TXNIP levels and concomitantly increasing Prx1, SIRT6 may represent an important regulator of redox homeostasis. Targeted therapies aimed at maintaining SIRT6 activity could represent a novel strategy to slow or stop the progression of OA through maintenance of redox balance and physiological redox signaling.