Ding Yong, Yang Hongmei, Wang Yucai, Chen Jun, Ji Zhenwei, Sun Honghui
Department of Orthopedic, Tangdu Hospital, Fourth Military Medical University, #569 Xinsi Road, Xi'an 710038, China.
Department of Orthopedic, BaoJi Centre Hospital, #8 Jiangtan Road, Bao ji 721008, China.
Int J Biol Sci. 2017 Feb 12;13(2):254-264. doi: 10.7150/ijbs.17053. eCollection 2017.
Osteogenic differentiation is crucial for the maintenance of bone homeostasis. Sirtuin 3 (SIRT3), a member of sirtuins family, functions as a critical deacetylase that regulates many key proteins. In the current study, we aimed to clarify the role of SIRT3 in osteogenic differentiation and the possible mechanisms, using mouse pre-osteoblastic MC3T3-E1 cells. Expression of SIRT3 was substantially increased in differentiated MC3T3-E1 cells. Knock down of SIRT3 significantly decreased alkaline phosphatase (ALP) staining, and mRNA expression of runt-related transcription factor 2 (Runx2) and collagen type I ɑ 1 (Col1ɑ1), and osteocalcin in differentiated MC3T3-E1 cells. Overexpression of wild type but not mutant SIRT3 could reverse SIRT3 knockdown-resulted decrease of ALP staining. Complex I, II, III, IV, and V activities, oxygen consumption and mitochondrial membrane potential were significantly decreased by SIRT3 knockdown. Moreover, SIRT3 knockdown reduced mitochondrial density, increased mitochondrial size and decreased the expression of NRF1 and TFAM. Knock down of SIRT3 decreased mRNA and protein expression of SOD2 and increased ROS level. Overexpression of SOD2 significantly suppressed SIRT3 knockdown-induced decrease of mitochondrial function and osteogenic differentiation. SIRT3 knockdown resulted in a significant decrease of PGC-1ɑ protein expression but not mRNA expression. Overexpression of wild type but not mutant SIRT3 could reverse SIRT3 knockdown-resulted decrease of PGC-1ɑ protein expression. Moreover, we detected a direct interaction between SIRT3 and PGC-1ɑ and SIRT3 knockdown reduced SIRT3 and PGC-1ɑ interaction, resulting in a reduction of PGC-1ɑ protein stability and PGC-1ɑ-binding in the promoters of SOD2. Overexpression of PGC-1ɑ blocked SIRT3 knockdown-induced decrease of SOD2 expression, increase of ROS level, and decrease of mitochondrial function and biogenesis, leading to improvement of osteogenesis. Overall, the data provide a better understanding of the role of SIRT3 in osteogenic differentiation.
成骨分化对于维持骨稳态至关重要。沉默调节蛋白3（SIRT3）是沉默调节蛋白家族的成员之一，作为一种关键的去乙酰化酶，可调节许多关键蛋白。在本研究中，我们旨在利用小鼠前成骨细胞MC3T3-E1细胞阐明SIRT3在成骨分化中的作用及其可能机制。在分化的MC3T3-E1细胞中，SIRT3的表达显著增加。敲低SIRT3可显著降低分化的MC3T3-E1细胞中的碱性磷酸酶（ALP）染色、 runt相关转录因子2（Runx2）和I型胶原ɑ1（Col1ɑ1）的mRNA表达以及骨钙素水平。野生型而非突变型SIRT3的过表达可逆转SIRT3敲低导致的ALP染色减少。SIRT3敲低显著降低了复合体I、II、III、IV和V的活性、氧气消耗以及线粒体膜电位。此外，SIRT3敲低降低了线粒体密度，增加了线粒体大小，并降低了NRF1和TFAM的表达。敲低SIRT3可降低SOD2的mRNA和蛋白表达，并增加活性氧（ROS）水平。SOD2的过表达显著抑制了SIRT3敲低诱导的线粒体功能和成骨分化降低。SIRT3敲低导致PGC-1ɑ蛋白表达显著降低，但mRNA表达未受影响。野生型而非突变型SIRT3的过表达可逆转SIRT3敲低导致的PGC-1ɑ蛋白表达降低。此外，我们检测到SIRT3与PGC-1ɑ之间存在直接相互作用，SIRT3敲低减少了SIRT3与PGC-1ɑ的相互作用，导致PGC-1ɑ蛋白稳定性降低以及PGC-1ɑ在SOD2启动子中的结合减少。PGC-1ɑ的过表达阻断了SIRT3敲低诱导的SOD2表达降低、ROS水平升高以及线粒体功能和生物发生减少，从而改善了成骨作用。总体而言，这些数据有助于更好地理解SIRT3在成骨分化中的作用。
