Roquin在骨质疏松症中的分子机制研究

Pathological bone loss induced by excessive osteoclast formation is the major mechanism underlying many debilitating bone diseases, including osteoporosis, Paget’s disease, lupus, and aseptic loosening of orthopaedic implants. To understand the root cause of bone destruction as well as its molecules involved is vital for an improved therapeutic strategy against osteolytic bone diseases. To gain insights into the molecular genetics and mechanisms of bone loss, we have screened for bone phenotypes in chemical (ENU)-induced mutant mice to identify key molecules that regulate bone homeostasis. We have uncovered one mouse line which carries a M199R mutation in the Roquin gene, and exhibits severe bone loss. Preliminary data show that the Roquin mutant mice display substantial bone loss with reduced trabecular and cortical bone volume; an increase of TRACP positive cells in vivo and osteoclastogenesis in vitro; and an increase of RANKL gene expression in T cells. Based on these intriguing and novel observations we hypothesize that Roquin plays a critical role in bone homeostasis by intrinsic and extrinsic mechanisms. In order to test this hypothesis, three specific aims are proposed: 1) To characterize the bone phenotype of Roquin mice；2) To determine the physiological role of Roquin in osteoclast differentiation and bone resorption, and identify the molecular regulation of Roquin as a RING-type E3 ubiquitin ligase in RANKL signaling pathways；3) To investigate the extrinsic mechanism of bone loss in Roquin mutant mice via activated T cells in osteoimmunology. This research may provide a new way for the treatment of osteoporosis by investigating the molecular mechanism of Roquin in bone mineral density, osteoclastogenesis, and osteoclast fuction.

破骨细胞增生过度是导致骨质丢失的主要原因，临床上常见于骨质疏松症、Paget’s病等疾病。从分子机制研究骨破坏的潜在原因是治疗溶骨性疾病至关重要的策略。我们通过筛查乙酰基亚硝基脲（ENU）诱导的突变小鼠骨骼表型，进而确认其是调节骨质平衡的关键分子。前期研究实验证实ENU诱变小鼠Roquin的新型突变，表现出严重病理性骨丢失、破骨细胞生成异常增多、T细胞RANKL表达增加。通过这些新发现，本研究假设Roquin蛋白在骨代谢中起到至关重要的作用。为了证实该假设，提出3个具体研究目标：Roquin突变小鼠骨骼系统组织形态学改变和生物力学特性；Roquin在破骨细胞分化、骨重吸收中的生理学作用和对RANKL细胞通路的调节作用； Roquin突变致骨丢失的骨免疫学机制。探讨Roquin影响骨密度的机制，尤其是其在破骨细胞形成和功能的分子调节机制，将为临床治疗骨质疏松症提供新的治疗思路。

破骨细胞过度分化和活化是导致骨质丢失的主要原因，临床上常见于骨质疏松症、Paget’s病等疾病。骨质疏松症是以骨量下降导致骨折风险增高的严重危害老年人健康的慢性代谢性疾病，而长期药物治疗给家庭及社会造成了严重的经济负担，是亟待解决的临床痛点和难点。本研究首先通过X线和micro-CT分析ENU诱导的Roquin基因突变小鼠（Sanroque小鼠）的下肢骨、脊柱骨及尾骨，发现Sanroque小鼠骨量较野生型小鼠显著下降；通过组织形态学进一步定量分析发现Sanroque小鼠骨小梁明显减少，而破骨细胞参数（数量和细胞贴附骨小梁面积）则显著上升。由此初步证实了破骨细胞异常分化和活化是导致Sanroque小鼠全身骨量的降低的主要原因。为进一步探讨Sanroque小鼠破骨细胞异常功能的机制，本课题组从细胞水平和分子生物学水平展开研究，结果显示Sanroque小鼠来源的破骨细胞前体细胞（骨髓单核/巨噬细胞）在体外经核因子κB受体活化因子配体（RANKL）和巨噬细胞集落刺激因子（MCSF）诱导后与野生型小鼠来源的破骨细胞前体细胞相比出现破骨细胞形成增多、特异性基因表达增加、破骨细胞吸收能力增强等，同时破骨细胞中的Roquin基因突变导致RANKL诱导的NF-κB、MAPK等信号通路增强。为探究Sanroque小鼠骨微环境中成骨细胞是否对破骨细胞有影响，本课题组通过突变小鼠和野生小鼠长骨来源的成骨细胞与骨髓细胞共培养发现Roquin基因突变的成骨细胞明显促进了破骨细胞的形成；通过Western Blot技术发现Sanroque小鼠来源的成骨细胞分泌的RANKL显著提高，从而促进了破骨细胞的分化和活化。总体来说，本项目通过骨组织形态学、细胞生物学和分子生物学证明了Roquin基因在骨微环境中调控骨代谢的重要作用：Roquin通过直接作用影响了破骨细胞分化和功能的主要信号通路并且可以间接地干预了成骨细胞分泌RANKL，为运用Roquin分子靶向治疗骨质疏松症奠定前期研究基础。