Hedgehog通路在调控BMSCs成骨-成脂分化和老年性骨质疏松症发病中的作用机制及干预策略

The age-dependent shift of osteogenic differentiation to adipogenic differentiation of BMSCs is an important cause of age-related osteoporosis. However, its underlying molecular mechanism is still under investigation. In our preliminary study, we found that the activity of Hedgehog (Hh) signaling decreased with aging, and that inhibition of Hh signaling led to reduced bone mass and marrow fat accumulation accompanied by reduced activity of mTORC2 signaling, suggesting that Hh signaling regulates commitment of BMSCs into osteoblast versus adipocyte lineages, and may be involved in age-related osteoporosis, however, the role and underlying mechanism of Hh signaling in these aspects is still unclear. In this project, we will: 1) investigate the role of Hh signaling in regulating bone metabolism, cell fate determination of BMSCs, and activity of mTORC2 signaling by inhibiting its activity in BMSCs through conditional knockout approach; 2) confirm that the above functions of Hh signaling is mediated by mTORC2 signaling using mice and cells over-expressing Rictor gene; 3) explore the role and mechanism of Hh signaling in the pathogenesis of age-related osteoporosis, and test the intervention strategy by investigating the effect of aging on Hh signaling and the therapeutic effect of activating Hh signaling on age-related osteoporosis in mice. This project will not only contribute to reveal the underlying mechanism of age-related osteoporosis, but also provide new thoughts and approaches for prevention and treatment of this disease.

骨髓间充质干细胞(BMSCs)成骨和成脂分化此消彼长是老年性骨质疏松症发生的重要原因,但其分子机制仍有待研究。我们前期研究发现，随年龄增加骨组织中调控细胞分化命运的重要通路Hedgehog(Hh)的活性减弱，而抑制Hh通路引起骨量减少和骨髓脂肪累积，伴随mTORC2通路活性下调，提示Hh通路在体内调控BMSCs成骨-成脂分化，且与老年性骨质疏松症的发病有关，但其作用机制尚不明确。本项目拟通过条件性基因敲除技术阻断BMSCs中Hh通路，研究其对骨代谢、BMSCs定向分化及mTORC2的作用；通过Rictor过表达小鼠及细胞，验证Hh通路的上述作用是否经由mTORC2途径介导；通过分析衰老对Hh通路的影响，以及靶向激活Hh通路对小鼠老年性骨质疏松症的治疗作用，探讨Hh通路在该病发生中的作用机制及干预策略。本项目将不仅有助于揭示老年性骨质疏松症的发病机制，而且有望为该病的防治提供新思路和新手段。

骨髓间充质干细胞成骨和成脂分化此消彼长是老年性骨质疏松症发生的重要原因, 但其分子机制仍有待研究。在本项目中：1）我们利用遗传标记和免疫组化相结合的方法，发现干骺端骨髓间充质干细胞（mSSCs）和软骨细胞具有高Hh通路活性，且Ihh是激活上述细胞中Hh通路的主要配体；2）利用Hh通路报告小鼠揭示了骨骼细胞中的Hh通路活性随年龄增加而降低；3）利用Osx-Cre在mSSCs中敲除Hh通路的关键组分Smo，发现Smo敲除可以阻断mSSCs中的Hh通路活力，并导致小鼠出现老年性骨质疏松症表型（即骨量减少伴随着骨髓脂肪量增加），表明Hh通路在骨稳态维持和老年性骨质疏松症发病中发挥着重要的生理和病理作用；4）在细胞学层面，我们发现mSSCs中Hh通路主要通过促进mSSCs的成骨分化和抑制其成脂分化参与调节骨形成，而对破骨细胞分化和骨吸收则没有明显作用；5）在分子机制研究层面，我们利用全转录组测序技术（RNA-seq）筛选出236个被Hh通路激活的下游基因，并对其中的Wnt10b和Foxc2等Wnt/β-catenin通路相关基因进行了深入研究，结果发现Hh通路通过诱导Wnt10b和Foxc2等基因的表达激活Wnt/β-catenin通路，而抑制Wnt信号通路则缓解Hh对mSSCs的促成骨和抑成脂作用，表明Hh通路通过Wnt/β-catenin通路调控mSSCs定向分化；6）此外，我们还发现通过腹腔注射Hh激动剂PM全身性激活Hh通路对骨质疏松症无明显疗效，而在mSSCs中半敲除Hh通路的负调控因子Sufu可促进小鼠骨量，表明骨特异性而非全身性适度激活Hh通路对骨质疏松症具有一定的治疗作用。本项目的研究结果不仅揭示了Hh在mSSCs成骨-成脂分化方向调节和老年性骨质疏松症发病中的作用机制，而且为该病的防治提供了新思路和潜在的干预靶点。