组蛋白去甲基化酶KDM7A通过经典Wnt通路及C/EBPα调控骨髓微环境成骨-成脂分化平衡和骨稳态

A few histone demethylases have recently been reported to play key roles in osteogenic differentiation from marrow stromal stem cells (MSCs), however their in vivo roles remain to be validated. Our preliminary data showed that histone demethylase KDM7A negatively regulated osteogenic differentiation and positively regulated adipogenic differentiation from murine ST2 stromal cell and primary marrow stromal cells. Further mechanism investigations demonstrated that KDM7A increased the expression of canonical Wnt inhibitor secreted frizzled-related protein 1 (SFRP1) by removing histone methylation marks from the promoter of Sfrp1. KDM7A also promoted CCAAT/enhancer binding protein α (C/EBPα) expression by removing histone methylation marks. These suggest that KDM7A might regulate reciprocal commitment of MSCs to osteoblasts and adipocytes, and bone homeostasis through interacting with canonical Wnt signaling and C/EBPα. In the present study we will further investigate the involvement of SFRP1/Wnt signaling and C/EBPα in detail in the reciprocal regulation of osteogenic and adipogenic differentiation by KDM7A using primary marrow stromal cells. Then we will develop Kdm7a-floxed mice and make osteogenic progenitor-specific knockout mice by crossing the Kdm7a-floxed mice with Osx-Cre mice. The phenotypes of the conditional knockout (cKO) mice will be analyzed to demonstrate if the Kdm7a deletion in osteoblast progenitor cells results in altered differentiation of osteoblasts and adipocytes and activity of Wnt/β-Catenin signaling in vivo and then lead to changes in bone homestasis and bone mass. If the above mentioned studies give us positive results, we will further investigate if overexpression of Sfrp1 and/or C/EBPα in mice will rescue the phenotypes observed in the Kdm7a cKO mice. Our proposed studies will provide novel insights into the mechanisms of osteoblast development and bone homeostasis, and implicate potential novel approach for bone loss recovery.

近年研究提示组蛋白去甲基化酶调控骨代谢，但鲜有体内实验证据。本课题组前期研究发现组蛋白去甲基化酶KDM7A抑制骨髓基质细胞成骨分化而促进其成脂分化，此作用依赖于其脱甲基活性。机制研究提示KDM7A通过移除组蛋白甲基化标记促进Wnt通路抑制因子SFRP1及脂肪分化转录因子C/EBPα表达。因此提出KDM7A可能通过抑制经典Wnt通路及促进C/EBPα表达而调控骨髓微环境成骨-成脂分化平衡，影响骨稳态。本项目将完善KDM7A通过调节Wnt通路活性及C/EBPα而调控骨髓基质干细胞和前体细胞定向分化的机制研究；构建成骨前体细胞条件性Kdm7a基因敲除鼠，采用影像学、生物力学、组织形态学结合细胞分子生物学等实验，观察Kdm7a敲除对小鼠骨形成/骨吸收和骨稳态的影响；探索Sfrp1和C/EBPα过表达能否逆转Kdm7a敲除鼠生物表型。本研究将深化对骨发育和骨稳态机制的认识，为骨质疏松防治提供新线索。

前期研究发现组蛋白去甲基化酶KDM7A抑制骨髓基质细胞成骨分化而促进其成脂分化，此作用依赖于其脱甲基活性。机制研究提示KDM7A可能通过上调SFRP1而抑制经典Wnt通路及促进 C/EBPα表达而调控骨髓微环境成骨-成脂分化平衡。本项目在此基础上，完善了KDM7A 调节体外骨髓基质干细胞定向分化的分子机制，发现敲减Sfrp1水平可以抑制基质细胞向脂肪细胞分化而促进其向成骨细胞分化，且有效缓解KDM7A对脂肪细胞分化的促进作用和对成骨细胞分化的抑制作用。建立了成骨前体细胞Kdm7a基因敲除鼠（Kdm7a cKO）模型，进行生物表型分析。分离颅盖骨细胞进行成骨/成脂诱导，与对照小鼠细胞相比，Kdm7a cKO小鼠的颅盖骨细胞成骨分化明显增强而成脂分化明显减弱，与体外研究结论一致。与同周龄Kdm7afx/fx对照小鼠相比，24周龄Kdm7a cKO小鼠胫骨干骺端表现出明显的骨量增加，小梁骨成骨细胞数量增加，破骨细胞数量减少，骨髓脂肪细胞减少。此外，骨组织矿化沉积率增加，血清中骨形成标志物I型前胶原氨基端原肽（PINP）增加而骨吸收标志物I型胶原交联C-末端肽（CTX-1）减少。这些表明Kdm7a cKO小鼠骨形成增加而骨吸收减弱。分离Kdm7a cKO小鼠与对照小鼠颅盖骨细胞进行RNA-seq分析，测序报告并未揭示Sfrp1 及 C/ebpα表达变化，但发现Kdm7a缺失后成纤维细胞激活蛋白α（Fap）和Rankl基因表达显著下降。FAP是新近研究报道的骨代谢调节因子，可以在小鼠体内抑制成骨细胞骨形成而影响骨量。我们的测序结果提示FAP在小鼠体内介导KDM7A调节骨髓基质干细胞分化的作用可能较之SFRP1和C/EBPα更加显著。进一步通过ChIP-qPCR实验证明KDM7A 可通过移除Fap和Rankl启动子区组蛋白 H3K9me2 和 H3K27me2 甲基化修饰而促进FAP和RANKL基因表达。进一步机制研究发现KDM7A上调FAP表达而抑制Wnt信号通路；重组FAP可有效缓解 Kdm7a缺失后对脂肪细胞分化的抑制作用和对成骨细胞分化的促进作用。此外，KDM7A促进成骨谱系RANKL基因表达而间接促进单核巨噬细胞向破骨细胞分化。分别建立Kdm7a cKO小鼠与对照小鼠OVX模型，发现Kdm7a缺失能有效缓解雌激素缺乏引起的骨丢失。本研究为骨质疏松防治提供新线索。