HIF1α与Runx2在调控BMP9促进间充质干细胞成骨分化与血管形成双向效应中的作用和分子机制研究

Investigating the molecular mechanism of osteogenic differentiation has important theoretical and practical significance for developing new bone tissue engineering materials and treatment of related orthopedic diseases. We found that BMP9 not only could induce the osteogenic differentiation of MSCs through the classic Smads signaling pathway, but also could up-regulate the expression of HIF1α via phosphorylated Smad1/5/8 to promote vascularization, which means BMP9 has two-way effects to accelerate both osteogenesis and vascularization, the paper was published in J Cell Sci (IF 6.1). However, the molecular mechanism underlying the two effects remains to be explored. Based on the existing data and literature analysis, we hypothesized that the key molecule HIF1α and Runx2 could positively regulate each other and play an important role in coordinating the two-way effects of BMP9-induced osteogenesis and vascularization in mesenchymal stem cells. In this project, experiments both in vitro and in vivo will be carried out to deeply investigate the regulatory effects between HIF1α and Runx2, as well as their effects on regulating BMP9 to promote osteogenesis and vascularization. This study has essential academic significance in developing the basic theory of skeletal development. Moreover, it could provide new perspective and theoretical foundation for developing new tissue-engineered bone and new therapies for related orthopedic diseases.

深入研究成骨分化的分子机制对于研发新型骨组织工程材料和治疗骨科相关疾病具有重要理论与现实意义。BMP9是诱发成骨分化的重要分子。我们前期发现BMP9不仅通过激活经典Smads通路诱发MSCs成骨分化，还能通过磷酸化Smad1/5/8上调HIF1α而诱发血管生成，具有同时促进成骨与血管形成的双向效应，论文发表于J Cell Sci(IF6.1)。但这两种效应之间偶联的分子机制仍不清楚。结合前期数据和文献，我们提出学术假说：关键分子HIF1α与Runx2之间存在相互正向调控关系，在协调BMP9促进成骨与血管形成双向效应中具有重要作用。本项目拟采用体内外实验，对HIF1α与Runx2的相互调控作用及其在调控BMP9促进成骨与血管形成双向效应中的作用和分子机制进行深入研究。本研究不仅对于拓展骨发育生物学基础理论具有重要学术意义，而且对于研发新的组织工程骨和治疗相关骨科疾病提供新的视角和理论依据。

深入研究成骨分化分子机制对于研发新型骨组织工程材料和治疗骨科相关疾病，具有重要理论与现实意义。我们前期发现BMP9是诱发成骨分化的重要分子，磷酸化Smad1/5/8上调HIF1α而诱发血管生成，具有促进成骨与血管形成的双向效应。但这两种效应之间偶联的分子机制仍不清楚。. 我们发现：（1）HIF1α与Runx2促进BMP9诱导MEFs骨形成、矿化和成血管因子表达。HIF1α具有增强成骨分化与血管形成作用；Runx2调控成骨分化，也提高BMP9诱导表达成血管因子，增加血管长入，促进成骨分化。两者具有一定重叠效应。（2）ChIP提示HIF1α结合Runx2外显子前方-250-700bp的DNA片段，调节Runx2启动转录。HIF1α调控BMP9诱导MEFs的Smad1/5/8、Erk1/2、p38及JNK磷酸化。Runx2调控BMP9诱导MEFs的Erk1/2磷酸化与蛋白水平。（3）HIF1α作为BMP9-HIF1α-Runx2通路的调节核心，促进BMP-Smads1/5/8信号通路；Runx2也通过间接稳定HIF1α，保持BMP信号通路稳定传导。. 此外，RNA干扰沉默Runx2，并不能完全抑制HIF1α协同BMP9促进成骨和血管形成效应。我们探寻了新的分子靶标代偿Runx2沉默：（1）沉默 Shn3体内外促进 BMP9诱导的早期和晚期成骨分化以及血管生成，通过增强BMP/Smads信号通路和BMP/MAPK信号传导活性来介导。Shn3抑制作用在调节Runx2中起偶联作用，Runx2激活VEGF促进BMP9诱导hAMSCs成骨和血管生成。（2）RNA测序、基因分析图、Pathway和Go富集分析，进一步发现BMP9和HIF1α显著上调SLIT3表达。ChIP提示HIF1α与SLIT3启动子具有结合能力。验证了SLIT3协同BMP9促进成骨分化和小管形成。获得2020年国家自然科学基金面上项目（HIF1a正向调控SLIT3在BMP9促进MSCs成骨分化与血管形成中的作用与机制研究，8207091740）。. 本研究不仅对于拓展骨发育生物学基础理论具有重要学术意义，而且对于研发新的组织工程骨和治疗相关骨科疾病提供新的视角和理论依据。