FoxO3a转录因子调控Neurochondrin对软骨分化的作用及机制研究

Neurochondrin (NCDN) ,a protein rich in leucine in the cytoplasm , is highly expressed in nervous system, bone and cartilage system, which is involved in neuronal differentiation, hydroxyl phosphorus absorption and chondrogenic differentiation,respectively.FoxO transcription factor is a transcription factors that are very important in the process of cartilage formation , the lack of FOXO leading to chondrocyte hypertrophy and the abnormal of cartilage .But whether FoxO are involved in chondrogenic differentiation are unkown. Our preliminary results have shown that NCDN as a new direct target of FOXO3a plays an important role in NGF-induced neuronal differentiation. Recently we found that the expression of FoxO3a/NCDN were regulated by phosphatidylinositol-3-kinase (PI3K)-protein kinase B (PKB, Akt) and MAPK signaling pathways in ITS-induced chondrogenic differentiation.In this project, we will use ATDC5 cell as the classic cell model of chondrogenic differentiation ,and together with chondrogenic progenitor cells to study the effect and underlying mechanisms of FoxO transcription factor on chondrogenic differentiation genes like NCDN. The aims of this study(FoxO3a as example) are to investigate (i) the effect of FoxO3a on the expression of NCDN and its underlying machanism and to verify if NCDN is a new target of FoxO3a (ii) the role of NCDN in chondrogenic differentiation and its underlying mechanisms and (iii) to determine if NCDN is involved in the inhibitory effect of FoxO3a on the differentiation of ATDC5 induced by ITS. Our study will deepen the understanding of the mechanisms of neuronal differentiation and provide new evidence for FoxO and its downstream targets as new therapeutic targets for osteoarthritis(OA) .

神经软骨蛋白(neurochondrin,NCDN)是胞浆内一种富含亮氨酸的蛋白质，高表达于神经系统、骨组织及软骨系统，分别参与神经细胞分化、羟磷再吸收及软骨分化。FoxO转录因子是软骨形成过程中重要的转录因子，该基因的缺失会导致软骨细胞肥大及软骨发育的异常。但FoxO参与软骨分化调控的研究报道十分罕见。我们前期对FoxO3a调控神经细胞分化的分子机制进行了深入研究，发现了NCDN是FoxO3a的一个新的下游靶基因参与NGF诱导的神经分化。近期在ITS诱导的软骨细胞ATDC5分化中发现PI3K/Akt和MAPK通路调控FoxO3a/NCDN表达。本课题拟采用ATDC5，原代培养软骨干细胞(CPC)及转基因鼠，研究FoxO3a转录因子调控NCDN对软骨细胞分化的作用及机制，为确立FoxO及其下游靶标作为退行性骨关节疾病的治疗新靶点及软骨细胞分化机制提供依据。

FoxO转录因子(FoxOs)最近被证明可以防止软骨细胞功能障碍并调节骨关节炎中的软骨稳态。FoxOs的调控软骨细胞分化机制仍不清楚。RUNX1介导软骨细胞分化和成骨细胞分化。我们发现FoxO3a和RUNX1共同高表达于ATDC5细胞和未分化MCSs，并随着软骨细胞中从增生向肥大分化过程逐渐增加。在ATDC5中细胞或小鼠MSCs过表达FoxO3a诱导软骨细胞分化标记基因表达，而敲低FoxO3a或RUNX1显著抑制软骨细胞分化基因的表达。免疫共沉淀证明FoxO3a结合RUNX1协同调控软骨分化基因转录。免疫组化显示FoxO3a和RUNX1高度共表达在新生小鼠后肢生长板的增殖软骨细胞。综上，我们发现FoxO3a与RUNX1通过促进早期软骨形成和软骨祖细胞的末端肥大进而协同促进软骨细胞分化，提示FoxO3a与RUNX1相互作用可能是骨关节炎及其他骨骼疾病的治疗靶点。. 前期研究发现FoxO3a调控神经软骨蛋白（NCDN)和神经肽W(NPW)表达，我们深入研究发现NPW具有促进软骨干细胞增殖和定向分化形成软骨细胞的能力，并且其对细胞软骨分化过程的诱导分化作用主要通过与细胞膜表面GPR7受体结合，进而激活细胞内PKA和PKC信号途径的传导。因此，该研究表明NPW对软骨损伤修复具有一定的意义，并获得具有创新性的研究成果。. 采用CBA对照小鼠和超愈合能力的MRL/MpJ (MRL)小鼠模型为研究对象，系统分析了CBA对照小鼠和超愈合能力的MRL/MpJ (MRL)小鼠模型软骨干细胞(CPC)功能差异，重点探讨CBA和MRL小鼠软骨干细胞来源外泌体在治疗骨关节炎中的差异，发现MRL-CPC和对照CBA小鼠CPC来源的外泌体在体外显著促进软骨细胞增殖和迁移，体内具有修复软骨损伤作用，并且MRL-CPC来源外泌体体外促进软骨增殖/迁移能力及体内的软骨损伤修复作用更强。通过两种小鼠CPC来源外泌体miRNA测序分析。发现miR-148a-3p, miR-221-3p, miR-222-3p 在MRL小鼠CPC细胞来源外泌体中显著增加，而let-7b-5p, miR-22-3p , miR-125a-5p , and miR-26a-5p 显著下降，以上研究可能对阐明自发性骨关节炎的发病机制具有重要意义。