营养感应信号mTORC1促进软骨衰老及其调控机制

Mechanistic target of rapamycin (mTOR) is a conserved serine/threonine protein kinase that sense nutrients and growth factors availability to regulate cell growth, proliferation, metabolism and ageing. However, the roles and regulatory mechanisms of mTOR signaling in maintaining cartilate homeostasis and cartilage ageing remains to be elucidated. Our preliminary results suggest that mTOR complex 1 (mTORC1) activities and the expression of its endogenous inhibitor, FKB38, ubiquitin ligase F-box and WD repeat domain-containing 7 (FBW7) dynamically altered during cartilage aging and chondrocyte proliferation and differentiation in vitro as well as in mouse growth plates in vivo. Mice with chondrocyte-specific mTORC1 activation or inhibition exhibit accelerated ageing cartilage and spontaneous arthritis phenotypes, implicating the essential roles of mTORC1 signaling in controlling endochondral bone development and cartilage ageing. The current project aims to identify the roles and regulatory mechanisms of mTORC1 signaling in cartilage senescence. The clinical cases and specimens, and the inducible and cell-specific knockout mice and cellular models will be used, in which the FKBP38, FBW7, ubiquitin associated protein 2-like (UBAP2L, a newly identified ubiquitination-related protein by our group which interacts with and controls mTORC1 activity) and mTORC1 in chondorcyte in mice at different ages could be deleted or specifically activated or inhibited. How FBW7, UBAP2L and FKBP38 expression are regulated during chondrocyte proliferation, differentiation and senescence and their roles in mTORC1 activation will be explored. The effects of mTORC1 on the expression of chondrocyte-related proteins, secretion of cytokines and inflammatory factors in these mice and cellular models will also be investigated. This study will identify novel function of mTORC1 in cartilage development and senescence, provide novel mechanisms for cartilage aging and evidences for prevention and treatment of chondrodysplasia and arthritis.

雷帕霉素靶蛋白(mTOR)是机体感受营养、调节代谢的主要信号通路，但mTOR在维持软骨代谢稳态与软骨衰老中的作用及其上、下游调控机制未阐明。我们前期工作发现软骨发育与衰老过程中，mTORC1活性与其抑制分子FKBP38、泛素连接酶FBW7表达呈动态变化并负相关，软骨细胞特异mTORC1活化能促进小鼠软骨衰老与自发骨关节炎(OA)，提示mTORC1在软骨衰老与OA发生中起关键作用。本项目将通过病例标本、可诱导软骨细胞基因敲除小鼠与细胞等模型，深入研究调控软骨细胞mTORC1活性的分子机制，诱导不同年龄小鼠软骨细胞FBW7、FKBP38、UBAP2L(我们新鉴定的、泛素化相关mTORC1调节分子)敲除与mTORC1激活或抑制，探讨泛素化对mTORC1的调节及其对软骨细胞衰老的影响，阐明营养感应信号在维持软骨稳态与软骨衰老中的作用及其调控机制，为软骨生物学提出新观点，为OA发生提供新机制。

雷帕霉素靶蛋白(mTOR)是机体感受营养、调节代谢的主要信号通路，但mTOR在维持软骨代谢稳态与软骨衰老中的作用及其上、下游调控机制未阐明。本项目通过病例标本、可诱导软骨细胞基因敲除小鼠与细胞等模型，深入研究调控软骨细胞mTORC1活性的分子机制，并探讨其对软骨细胞衰老的影响，阐明营养感应信号在维持软骨稳态与软骨衰老中的作用及其调控机制。我们发现：(1)软骨发育过程中mTORC1通过调节PTHrP分泌，协调软骨细胞增殖与分化的耦联。mTORC1的下游激酶S6K1可结合并磷酸化Hedgehog下游转录因子Gli2，并促进其入核转录激活PTHrP，最终影响软骨细胞增殖与肥大分化。(2)关节炎发展过程中滑膜巨噬细胞mTORC1活化，促使其向M1型极化并分泌炎症因子与Rspo2促进软骨破坏与骨关节炎进展，使用雷帕霉素抑制mTORC1可使巨噬细胞向M2极化，抑制滑膜炎症并延缓小鼠骨关节炎进展。(3)初步阐明软骨细胞mTORC1促进软骨衰老与骨关节炎发生的机制。软骨细胞mTORC1活化通过下调P73和FGFR3促进软骨细胞增殖、通过下调PPR促进软骨细胞肥大分化，共同作用诱发并促进骨关节炎的发生发展。关节软骨细胞mTORC1活化可促进其分泌促血管形成因子，引起软骨下骨血形成，进而带来更多的营养，进一步活化mTORC1，形成一个正反馈调控机制。(4)发现成骨细胞mTORC1调节骨髓血管形成及其机制。成骨细胞mTORC1可通过上调转录因子STAT1表达，同时增强STAT1与Cxcl9基因启动子区的结合，增加Cxcl9转录与分泌，后者与VEGF相互作用并阻止其与血管内皮细胞膜上的受体(VEGFR)结合，从而抑制血管生成。(5)揭示机械应力通过FBXW7调节mTORC1活性促进关节软骨衰老的机制。我们发现过度机械应力通过下调FBXW7促进软骨细胞衰老和骨关节炎发展，高强度机械应力能够促进软骨细胞FBXW7表达下降，分解代谢增强，细胞衰老，敲除软骨细胞FBXW7能够明显诱导软骨细胞衰老，进而加重OA的发展。.因此，我们的研究证明了mTORC1活化促进关节软骨衰老，mTORC1活化参与骨关节炎中多个关节组织的病理改变，mTORC1是防治OA的理想靶点。