Phosphorylation of the transcriptional coregulator aNAC by DNA-PKcs regulates osteoblastogenesis

The αNAC (alpha chain of the Nascent polypeptide-Associated Complex) transcriptional coregulator is developmentally expressed in osteoblasts and regulates osteoblast differentiation in vitro and in vivo. αNAC can activate or repress gene transcription, a function that is dynamically regulated by post-translational modification. Phosphorylation of residue Ser132 stimulates the sumoylation of αNAC on Lys127 to repress gene expression. Using in vitro kinase assays, we show that Ser132 phosphorylation is mediated by the DNA-dependent protein kinase catalytic subunit (DNA-PKcs). Pharmacological inhibition of DNA-PKcs kinase activity or gene silencing of Prkdc (encoding DNA-PKcs) in murine osteoblastic MC3T3-E1 cells and human adipose-derived mesenchymal stromal cells markedly enhanced osteogenesis and the expression of osteoblast differentiation marker genes. ChIP-seq identified Ezh2 as a target of the αNAC/DNA-PKcs signaling pathway. Mechanistically, inhibition of DNA-PKcs repressed Ezh2 expression, induced cell cycle block, and increased osteogenesis by significantly enhancing the bone morphogenetic protein 2 (BMP-2) response in osteoblasts and other mesenchymal cells. Importantly, in vivo inhibition of the kinase enhanced bone biomechanical properties, and bones from osteoblast-specific conditional Prkdc-knockout mice exhibited increased stiffness. In conclusion, DNA-PKcs is a negative regulator of osteoblast differentiation, and therefore DNA-PKcs inhibitors may have therapeutic potential for bone regeneration and metabolic bone diseases. MC3T3-E1 cells were grown in αMEM (Gibco) supplemented with 10% FBS (Gibco) to confluency, followed by a treatment with either NU7441 (1 μM) (Selleckchem) or DMSO (Sigma) for 16h. Cells were harvested, and total RNA was isolated using mRNeasy Mini Kit (Qiagen) according to the manufacturer's protocol. Triplicates from each of the three independent experiments were pooled together, quantified, and assessed by RNA-Seq

αNAC（新生多肽相关复合体α链，alpha chain of the Nascent polypeptide-Associated Complex）是一种转录辅调节因子，在成骨细胞中呈发育依赖性表达，并可在体外（in vitro）及体内（in vivo）调控成骨细胞分化。αNAC可激活或抑制基因转录，这一功能可通过翻译后修饰实现动态调控。残基Ser132的磷酸化可促进αNAC在Lys127位点发生SUMO化修饰，进而抑制基因表达。通过体外激酶实验，本研究证实Ser132的磷酸化由DNA依赖蛋白激酶催化亚基（DNA-dependent protein kinase catalytic subunit，DNA-PKcs）介导。在小鼠成骨细胞MC3T3-E1及人脂肪来源间充质基质细胞中，对DNA-PKcs激酶活性进行药理学抑制，或对编码DNA-PKcs的Prkdc基因进行沉默，均可显著增强成骨作用及成骨细胞分化标记基因的表达。染色质免疫共沉淀测序（ChIP-seq）结果显示，Ezh2是αNAC/DNA-PKcs信号通路的靶基因。机制研究表明，抑制DNA-PKcs可下调Ezh2的表达、引发细胞周期阻滞，并通过显著增强成骨细胞及其他间充质细胞对骨形态发生蛋白2（bone morphogenetic protein 2，BMP-2）的应答反应，进而促进成骨。值得注意的是，体内抑制该激酶可改善骨骼的生物力学性能；而成骨细胞特异性条件性Prkdc敲除小鼠的骨骼则表现出刚度提升。综上所述，DNA-PKcs是成骨细胞分化的负调控因子，因此DNA-PKcs抑制剂有望在骨再生及代谢性骨病的治疗中展现应用潜力。MC3T3-E1细胞于添加10%胎牛血清（FBS，Gibco）的α-MEM培养基（Gibco）中培养至汇合状态，随后分别用NU7441（1 μM，Selleckchem）或二甲基亚砜（DMSO，Sigma）处理16小时。收集细胞后，依照制造商说明书使用mRNeasy Mini Kit（Qiagen）提取总RNA。将三次独立实验的三份生物学重复样本合并，经定量后通过RNA测序（RNA-Seq）进行检测。