We previously identified that both Sirt1 and autophagy were important regulators in cartilage development, growth and health including endochondral ossification and aging-related OA. In this work, we aimed to investigate the role of Sirt1 in the regulation of chondrocyte autophagy in the pathogenesis of OA. The OA cartilage samples showed reduced Sirt1-FoxO1 expression. Based on this observation we teased the Sirt1's role in chondrocyte autophagy using a novel Col2-ERT-Cre; Sirt1flox/flox mice. SIRT1 remarkably decreased in the advanced OA stage of mouse OA model. Meanwhile, the expression levels of FoxO1 and autophagy also reduced. Our data further revealed that Sirt1 regulated autophagy via nucleo-cytoplasmic shuttling of FoxO1.
FoxO1, as a transcription factor, mediates an auto-feedback loop regulating SIRT1 in vitro[16], while FoxO1 antagonist suppresses autophagy and lipid droplet growth in adipocytes[20]. Moreover, FoxO1-mediated autophagy imbalanced anabolic and catabolic in human OA-like changes chondrocytes[21]. However, the potential mechanism of autophagy activity in arthritis has not been fully elucidated. Autophagy played a diphasic role in the pathogenesis of articular cartilage degeneration [22]. It was reported that autophagy may protect cartilage from matrix degradation[23]. Previous literature that SIRT1, FoxO1 and autophagy are highly expressed in normal cartilage, while those decrease in severely degenerated cartilage respectively [24, 25]. While the direct relationship between FoxO1 and autophagy is not clearly defined. We observed increased cartilage-degrading enzymes, hypertrophic proteins and damaged cartilage-anabolism in human and mice. One possible mechanism for these observations is the decrease autophagy regulated by Sirt1 and FoxO1.
Sirt1 deacetylase is an important regulator of autophagy both in vivo and in vitro[14, 26]. Increased Sirt1 expression is beneficial for OA osteoblasts and attenuates the progression of osteoarthritis in mice OA models [27, 28]. To strengthen our hypothesis that Sirt1 is chondro-protective, the Sirt1 activator Res and inhibitor EX-527 were utilized to test the benefits on primary human OA-like chondrocytes. Our results suggested that Sirt1 was involved in cellular stress, especially in the early cartilage degeneration process through SIRT1/P53/P21 pathway[19]. To further answer the question that how Sirt1 modulates the autophagy to achieve the preventive effects in the OA development and progression, our previous work showed that Sirt1 or Atg7 deletion in cartilage delay the progress of endochondral ossification[3, 12]. These findings consist with precious studies that cartilage-specific FoxO1 or autophagy deficiency impaired[29, 30]. Moreover, our results revealed that FoxO1 and autophagy decreased in OA patients and Sirt1-CKO mice after surgery. Taken our results and previous studies together, FoxO1 may bridge the gap of how SIRT1 regulates autophagy in OA. Thus, decreased autophagy activity in Sirt1-CKO mice might be related to the Sirt1 deficiency or FoxO1 inhibition.
We planned to determine whether Sirt1 regulated autophagy through FoxO1 in chondrocytes. Given that there is an interaction between SIRT1 and ATG7 in Hela cells and mouse embryonic fibroblasts [14], and FoxO1 was significantly mediated by SIRT1 and other histone deacetylases in neutrophils [31, 32]. We also investigated the interaction between ATG7, SIRT1 and FoxO1 in chondrocytes, unlike in the cancer cells or liver, and the involvement of autophagy and FoxO1 in OA progress. Previous study shows FoxO1 and FoxO3 deletion exhibit more severity cartilage degradation than FoxO4 [29, 31, 32], which suggested that FoxO transcription factors modulate autophagy and proteoglycan 4 in cartilage homeostasis and OA. By using C28 cell lines, it is confirmed that ATG7, FoxO1 and SIRT1 have an apparent interaction, which is supported by precious studies in other organs[14, 33, 34]. Our study demonstrated that Sirt1 regulated FoxO1 transcriptional activity, and then FoxO1 mediated autophagy to maintain cartilage homeostasis via cartilage hypertrophy and apoptosis.
To further verify the direct connection between Sirt1 and autophagy, it is important to determine the function of autophagosome formation in Sirt1 deleted mice. To target the Sirt1-FoxO1 axis in an autophagy-dependent manner in arthritis, several potential drugs could be beneficial to arthritis treatment. Except Resveratrol used in this study, the old drug metformin may alleviate oxidative stress and enhances autophagy in diabetic kidney disease via AMPK/SIRT1-FoxO1 pathway[35], and by enhancing autophagy flux and decreasing the release of vWF and P-selectin. The Sirt1/FoxO1 pathway is also a promising target to prevent atherosclerosis (AS) and arterial thrombosis [36]. Similar to aging and mechanical stress-induced chronic inflammatory diseases in kidney and cardiovascular system, our previous reports[19] and current observations suggested that Sirt1 possibly protected against injury-induced OA with aging via SIRT1/FoxO1/autophagy during OA progress.
There are some limitations to our study. The connection between autophagy and OA progression, and Sirt1's role in this mechanism was reported by other groups to some extent, while we attempted to make a leap conceptually and serves to validate previous findings regarding Sirt1's protective role in cartilage supporting autophagy in normal chondrocytes. To examine whether deletion of Sirt1 in mice is dependent on age and/ or the surgical method, we selectively delete Sirt1 gene in cartilage. We further explored the role of SIRT1 in tamoxifen-induced cartilage-specific deletion of Sirt1 male mice with trauma-induced OA followed by a 12-month duration, which differed from that the small sample size (n = 4) in young mice used for the surgical induction of OA[5, 17]. Another concern of previous reports may not represent OA pathology in humans because the observation lasted only for 8 weeks after surgery with insignificant OA scores between Sirt1-CKO and control mice. Lastly, the direct association between Sirt1/FoxO1/autophagy requires further investigation.