Furin在成骨细胞分化及骨形成中的调节机制研究

Osteoblasts are primary bone forming cells in adult. The cause of osteoporosis is often associated with reduced number and activity of osteoblasts. Our preliminary data suggested that Furin was expressed throughout osteoblast differentiation. Using osteoblast-specific Furin conditional knockout mice (Furin△OB), we found these mice displayed an increased bone mass phenotype. Consistent with Micro-CT data, histomorphometric analysis showed bone mass was significantly increased. In older Furin△OB mice, bone mass was also significantly increased as compared age matched Furin-flox littermate controls. In addition, bioinformatics analyses predicted that the propeptide of BMP3 was a candidate substrate of Furin in osteoblasts. Thus, we propose that Furin cleaves the propeptide of BMP3, therefore modulates osteoblast differentiation and bone formation. In order to address this, we therefore plan to: characterize the bone phenotype of Furin△OB; explore the role of Furin in osteoblast differentiation and bone formation in vitro; investigate the role of Furin in BMP3 protein cleavage in osteoblasts; further analyze the effect of Furin inhibitor on OVX-induced osteoporosis in mice. Overall, we aim to explore the function of Furin in osteoblast and understand the molecular mechanism underlying, which might provide a new strategy for treatment of osteoporosis in humans.

成骨细胞是人体内具有骨形成功能的细胞，其数量和功能的下降容易引起骨质疏松症。我们前期研究发现，Furin是成骨细胞分化过程表达明显的基因；通过构建在成骨细胞中条件性敲除Furin基因小鼠，发现小鼠呈骨量升高表型，并进一步在组织形态学分析验证了骨量增高的结果；且随着小鼠年龄的增大，与野生型相比，敲除小鼠依然表现骨量升高，提示Furin在成骨细胞中具有重要作用。此外，通过生物信息学预测BMP3是Furin在成骨细胞的作用底物。因此我们假设Furin通过调控BMP3蛋白从而干预成骨细胞分化及骨形成。本项目拟通过前期已建立的Furin条件性敲除小鼠，研究小鼠骨骼表型，成骨细胞分化及骨形成能力，Furin对其作用底物BMP3的调控，并针对成骨细胞研究Furin特异性抑制剂对去卵巢小鼠骨质疏松症的治疗作用，以明确Furin在成骨细胞的功能及分子调节机制，为骨质疏松症的治疗提供理论和实验依据。

成骨细胞是人体内具有骨形成功能的细胞，其功能的异常与骨质疏松、骨关节炎等骨相关疾病息息相关。因此，进一步加强成骨细胞的分子机制研究，寻求有意义的治疗靶点，提高临床治疗的疗效显得十分必要。本研究采用条件性敲除小鼠模型、去卵巢骨质疏松小鼠病理模型等研究对象，从骨骼形态表型层面、成骨细胞分化及功能层面、Furin调节作用机制层面、治疗骨质疏松层面进行了系统深入的探索，主要完成了：①成骨细胞中条件性敲除Furin基因小鼠呈现高骨量表型，并且与BMSC成骨分化有着密切联系。②为进一步研究Furin对BMSC成骨分化的影响，我们构建了MSC中条件性敲除Furin基因小鼠，结果显示小鼠也呈现骨量上升的表型，更为重要的是Furin基因的缺失影响体内成骨细胞-破骨细胞的偶联。③为进一步探究Furin的细胞学机制，通过细胞分子生物学方法，我们发现Furin基因的缺失抑制BMSC成骨细胞分化及骨形成功能；并且证实Furin能够作用MCSF前体蛋白从而调控MCSF的生物活性，从而间接抑制破骨细胞活性；此外，Furin基因的缺失还能够引起RANKL/OPG系统发生异常，进一步加剧破骨细胞活性的降低，最终引起骨量的变化。④在明确Furin调节骨重建过程的基础上，我们进一步通过药理学方法，发现Furin特异性抑制剂Dec-RVKR-CMK能够有效抑制去卵巢骨质疏松小鼠的骨量丢失。⑤基于上述研究，通过分子对接技术，以Furin作为潜在靶点筛选影响成骨细胞或破骨细胞的天然活性成分，以期发现疗效更好、毒性更低、价格低廉的治疗骨相关疾病候选药物。综上，本项目揭示了Furin能够通过调节成骨细胞从而间接影响破骨细胞，进而调节成骨细胞-破骨细胞的偶联，最终影响骨量的变化，为今后骨相关疾病的防治提供了新的策略。