Depletion of SIRT6 causes cellular senescence, DNA damage, and telomere dysfunction in human chondrocytes

Abstract

Purpose: Recently, it has been suggested that chondrocyte senescence plays an important role in the pathogenesis and development of Osteoarthritis (OA). SIRT6 is a member of sirtuin family of NAD+-dependent protein deacetylase. Recently, SIRT6 has been implicated in DNA repair, telomere maintenance, attenuation of inflammation and glucose homeostasis. Notably studies in mice have shown that SIRT6 deficiency produces premature aging phenotypes, whereas SIRT6 overexpression causes a moderate increase in the lifespan of male mice, strongly suggesting an important role of SIRT6 in aging and aging-associated diseases. However, its role in chondrocytes has so far not been explored. The purpose of this study is to examine the role of SIRT6 in human chondrocytes by inhibiting SIRT6 with a RNA interference (RNAi) technique. The hypothesis of the present study is that SIRT6 has a protective function toward human chondrocytes Methods: OA cartilage tissues were obtained from varus knee OA during total knee arthroplasty. Lateral and medial femoral condyles were used as cartilage with mild and severe OA, respectively. Articular cartilage tissues without OA were obtained from the patients underwent surgery for femoral neck fracture. These were used for histological experiments. Next, SIRT6 was depleted by RNAi and examined the influence of SIRT6 depletion on gene expression changes, proliferation, and senescence in normal human chondrocytes. Normal Human Articular Chondrocytes-knee (NHAC-kn) was used as a normal human chondrocytes. RNA or proteins were extracted 48 hours after the lipofection, and real-time PCR and western blotting analysis were respectively performed. The proliferation activity was examined using a Cell Counting Kit-8. Optical density was measured at 0, 24, 48, 72, 96 hours after the lipofection. Senescence associated β-Galactosidase (SA-β-Gal) staining was examined using a SA-β-Gal staining kit. Furthermore, to detect DNA damage and telomere dysfunction, the γH2AX foci and telomere dysfunction-induced foci (TIFs) where γH2AX foci co-localized with telomere repeat binding factor-1 (TRF-1) were examined using an immunofluorescence confocal microscopy. Results: Immunohistochemical analysis showed that SIRT6-positive cells were observed in normal cartilage, and mild and severe OA cartilage. In real-time PCR analysis, the depletion of SIRT6 significantly increased expression of matrix metalloproteinase (MMP) -1 and 13 mRNA. The proliferation assay showed that the absorbance was significantly decreased by the depletion of SIRT6 at 72 and 96 hours after lipofection, indicating reduced proliferation by the depletion of SIRT6. SA-β-Gal assay showed that the percentage of SA-β-Gal positive cells was significantly increased by the depletion of SIRT6, indicating that the depletion of SIRT6 induced premature senescence. Immunofluorescence microscopic analysis showed that γH2AX foci and TIFs was significantly increased in the SIRT6-depleted chondrocytes compared with control. Furthermore, to examine the downstream signaling pathways that mediate the induction of senescence by the depletion of SIRT6, the protein level of mediators for DNA damage induced-senescence, p16 and p21, was examined by western blotting. p16 protein level was increased while p21 protein level was reduced in the in SIRT6-depleted chondrocytes compared with control. Conclusions: The present study demonstrated that the depletion of SIRT6 in human chondrocytes caused increased expression of MMP-1 and 13, reduced proliferation and increased senescent cells. In addition, the depletion of SIRT6 caused accumulation of γH2AX and TIFs. These observations suggested that the depletion of SIRT6 induced premature senescence associated with a secretory phenotypic change and that the induction of the premature senescence may be caused by increased DNA damages and telomere dysfunction. Moreover, we observed p16 was increased by SIRT6 depletion. The p16-pRB pathway has been reported to be a major pathways that mediate premature senescence and proliferation arrest. Therefore taken together, these observations suggest the depletion of SIRT6 in human chondrocytes caused DNA damage and telomere dysfunction, followed by a premature senescence possibly via the p16-pRB pathway. SIRT6 may play an important role to protect chondrocytes from premature senescence, DNA damage, and telomere dysfunction.