探讨Tudor-SN蛋白参与调控软骨内成骨的作用与机制

本课题组多年来对Tudor-SN蛋白进行了系统研究，已成功构建Tudor-SN基因敲除（KO）小鼠，发现KO小鼠出生时呈现软骨发育不全和原发骨质疏松现象，提示Tudor-SN在软骨内成骨过程中发挥重要作用。软骨内成骨经历软骨细胞的增殖、分化和衰老等复杂过程，错综复杂的调控通路和分子机制备受关注。基于大量工作基础，本课题从几个层次进行探讨：Tudor-SN：Tudor-SN通过调控成骨关键因子Runx2和β-Catenin的蛋白水平，促进BMSC的分化能力；Tudor-SN通过与调控软骨细胞增殖的关键转录因子 Sox9共同作用；亦或作为新的TOP基因，通过mTOR通路调控另一软骨细胞增殖与分化的关键因子IHH活性，参与调控软骨内成骨。利用软骨细胞cKO小鼠，明确Tudor-SN缺失导致骨发育和骨内分泌功能异常，对靶器官功能的影响。本研究成果将为揭示原发性骨质疏松的发病机制提供理论依据。

The Tudor staphylococcal nuclease (Tudor-SN) protein is a multifunctional protein that involved in a variety of cellular processes, such as gene transcription, pre-mRNA splicing, DNA damage response and stress granule assembly. We have been focusing on investigating the physiologic function of Tudor-SN for nearly 10 years. We previously identified Tudor-SN as a novel regulator taking part in cell cycle regulation, and its expression level is closely related to the cell proliferation capacity that is highly expressed in proliferating cells while very low or undetectable in terminally differentiated cells. To further illustrate the n vivo function of Tudor-SN, we have successfully constructed Tudor-SN gene knockout mice (KO). The KO mice were born with cartilage dysplasia and primary osteoporosis. Comparing with the wild type mice, the bone density was significantly lower, the number of bone girders was significantly reduced, and the thickness of the cortical bone was very thin. All these suggest that Tudor-SN deficiency affected bone development in mice. The endochondral ossification forms most of the skeleton, including vertebrae, ribs and long bones. It involves an initial cartilage anlagen within which chondrocytes undergo an elaborate and well-controlled differentiation process. We therefore, hypothesize that Tudor-SN protein plays essential roles in endochondral ossificition. .Our research goal is to investigate the fundamental function of Tudor-SN in endochondral ossificition by regulating chondrocyte proliferation and differentiation. Linear growth is largely a function of endochondral bone elongation. It is accounted for by the complex regulation of chondrocyte proliferation, differentiation and senescence. The mesenchymal precursors first condense and commit to chondrocyte lineage under master regulator Sox9, Ihh by coordinating chondrocyte proliferation and differentiation. Runx2 and β-Catenin are essential factors for osteoblast. We revealed that in the bone tissue of new born KO-mice, the expression of Runx2 and β-Catenin was significantly lower than the WT-mice. In addition, using Data Base of Transcription Start Sites (DBTSS), we revealed that Tudor-SN is a terminal oligo-pyrimidine (TOP) gene. And we further confirmed the translation efficiency of Tudor-SN was controlled by mammalian target of rapamycin complex1 (mTORC1) pathway by using Torin 1 to inhibit the activated mTORC1 pathway cells. .All these observations prompted us to speculate that Tudor-SN plays essential roles in regulating chondrocyte proliferation and differentiation in endochondral ossificition. Aim 1: To determine the molecular mechanism by which Tudor-SN regulates the expression of Runx2 and β-Catenin in in the differentiation of BMSC to chondrocyte and osteoblast; Aim2: To characterize the function of Tudor-SN in collaborating with transcription factor Sox9 in regulating chondrocyte proliferation and differentiation; Aim3: To determine the involvement of Tudor-SN, as a new TOP gene, in regulating The Indian Hedgehog (IHH) activation through the mTOR signaling pathway; Aim4:To define the impact of Tudor-SN on primary osteoporosis, as well as secondary bone endocrine dysfunction and the consequence effect on the function of other target organs. The experiments will be carried out by using the conditional mice (cKO) with knockout of Tudor-SN gene in chondrocyte. .Significances: The goal of this study is to provide molecular insights and determine the important roles by which Tudor-SN regulates chondrocyte proliferation and differentiation. It may provide novel insights into the therapeutic strategies for primary osteoporosis.