OLFM4在老年性骨质疏松发生中的作用和机制研究

Senile osteoporosis (SOP) is closely correlated to reduced osteoblastic differentiation of bone marrow mesenchymal stem cell (BMMSC). However, its precise mechanism is unclear. An extracellular protein - olfactomedin 4 (OLFM4) has been revealed to be markedly upregulated in femoral cancellous bone of old male C57B6 mice and BMMSC of SAMP6 mice, the two well-known mice model for SOP, by a proteomic approach and verified by Western Blot in our preliminary study. While OLFM4 suppressed osteoblastic differentiation of MSC and osteoprogenitor cell lines, it promoted adipogenic differentiation of MSC. Additionally, OLFM4 suppressed the activity of mechanistic target of rapamycin complex 2 (mTORC2), a novel kinase complex recently revealed to be key for BMMSC osteoblastic differentiation. Moreover, while OLFM4 repressed the protein expression of β-Catenin, a key osteogenic transcriptional factor, it promotes the expression of Ebf1, an important adipogenic transcriptional factor. Taken together, our results indicated OLFM4 may modulate the balance between key osteogenic or adipogenic transcriptional factors via mTORC2 or other pathways, suppressing the osteogenic differentiation of BMMSC and thus reducing bone formation. In our current project, the mechanism by which OLFM4 inhibits BMMSC osteogenic differentiation and reduces bone formation, as well as its functional role in the development of SOP will be further studied at molecular, cellular, and whole-body level, using cell lines, OLFM4-knockout (KO) mice, and primary cells from OLFM4-KO mice. Moreover, the therapeutic effects of targeting OLFM4 in SOP mice models will be evaluated. Collectively, our study will provide novel insights into the theory of bone metabolism and a novel rationale for targeting OLFM4 to prevent and treat SOP.

老年性骨质疏松(SOP)与骨髓间充质干细胞(BMMSC)的成骨分化减少密切相关，但其具体机制未阐明。课题组利用蛋白组学技术筛选和验证出SOP模型雄鼠股骨松质骨和BMMSC中表达量显著升高的胞外基质蛋白OLFM4，并在MSC和成骨祖细胞系中发现其有抑制成骨分化和促进成脂分化的作用。进一步研究提示OLFM4可抑制雷帕霉素靶蛋白复合物2(mTORC2)活性和成骨关键因子β-Catenin的表达，同时上调成脂因子Ebf1的表达，提示OLFM4可能通过mTORC2等途径调控成骨成脂分化关键调控因子的平衡，从而抑制BMMSC的成骨分化与骨形成。本项目拟构建OLFM4基因敲除的小鼠与细胞模型，在分子、细胞和整体水平进一步明确OLFM4在SOP发生中的作用，阐明其抑制成骨分化与骨形成的机制，并探讨靶向抑制其表达对SOP的治疗效果，为骨代谢的调节机制提出新观点，并为SOP的防治提供新靶点。

老年性骨质疏松(SOP)与骨髓间充质干细胞(BMSC)的成骨分化减少密切相关，但其具体机制未阐明。课题组前期研究利用蛋白组学技术筛选出SOP模型-雄性老年小鼠股骨松质骨中表达量显著升高的胞外基质蛋白OLFM4，并在MSC和成骨祖细胞系中发现其有抑制成骨分化和促进成脂分化的作用。OLFM4可抑制雷帕霉素靶蛋白复合物2(mTORC2)活性和成骨关键因子β-Catenin的表达，同时上调成脂因子Ebf1的表达，提示OLFM4可能通过mTORC2等途径调控成骨成脂分化关键调控因子的平衡，从而抑制BMMSC的成骨分化与骨形成。本项目通过OLFM4 BMSC(在肢芽间充质干细胞中敲除OLFM4), OLFM4 POB (在成骨前体细胞中敲除OLFM4)的小鼠模型与细胞模型，探讨了OLFM4通过mTORC2抑制成骨分化的作用和机制。. 本研究发现，OLFM4BMSC KO和OLFM4POB KO小鼠不仅股骨远端/胫骨近端骨量增加，其BMSC和头盖骨成骨前体细胞的成骨分化增强。此外，无论在OLFM4BMSC KO和OLFM4POB KO小鼠中，或是在OLFM4敲低的MSC细胞系中，mTORC2活性增强而mTORC1活性降低。成骨分化时OLFM4定位于细胞-胞外基质连接处抑制黏着斑的形成，且OLFM4可与mTORC2关键成员Rictor结合，提示OLFM4可能通过抑制mTOR2抑制黏着斑的形成以抑制成骨分化。本研究还初步发现，OLFM4可结合于成骨关键因子TMEM119，抑制其表达并抑制成骨分化。此外，间充质干细胞的OLFM4可抑制巨核细胞分化和血小板形成。. 此外，平面细胞极性信号通路的核心蛋白Vangl2也在老年鼠松质骨中表达增强，提示Vangl2表达增强也与SOP相关。Vangl2在极性的上皮细胞中定位于质膜。本研究首次发现， MSC的Vangl2高度富集于分子伴侣介导的自噬（CMA）定向的溶酶体膜的脂筏中，并促进CMA受体LAMP-2A的降解，从而自我限制此类溶酶体的CMA活性。成骨分化进程中Vangl2缺失则导致此类溶酶体的激活，选择性降解关键成骨抑制因子ZNF423、TLE3及SOX9的表达，从而促进成骨分化。而在增龄性骨丢失阶段，Vangl2表达升高，LAMP2A表达-CMA活性随之降低，进而抑制成骨分化。