The role of Tmem263 in regulation of bone mass and strength

我们之前的全基因组关联研究确定了56个与骨矿物质相关的基因座 男性和女性的骨密度(BMD)和骨折风险。染色体12q23.3上含有该基因的一个基因座 TMEM263(跨膜蛋白263)与髋部骨密度密切相关(p&lt；9.6x10-10)。TMEM263 编码一种未知家族的多通道跨膜蛋白。以确定该基因在骨骼中的作用 生物学方面，我们测试了TMEM263基因中与髋部骨密度相关的SNP(Rs1053051)的等位基因特异性 使用人股骨样本的差异表达(ASE)。我们观察到一种单向等位基因 该SNP的mRNA表达不平衡，提示TMEM263酶的变异可能参与了 髋部骨密度的变异。小鼠Tem263基因在骨骼组织中高表达，而在非骨骼组织中表达 骨骼组织、成骨细胞和骨细胞表达的Tmem263的mRNA水平是对照组的6倍 到破骨细胞。此外，在已建立的成骨细胞系OB6中，用针对 Tem263显示骨形成重要基因的表达降低，而骨形成相关基因的表达增加 与骨吸收相关的基因。我们最近培育了成骨细胞特异性的Tem263 KO小鼠。初步数据 显示这些小鼠出现骨折，并显著降低全身骨密度、骨密度和骨小梁 与WT小鼠相比，骨量和受损的骨微结构。因此，我们假设： 1)Tem263基因在骨量的获得和维持中起重要作用；2)成骨细胞- 与对照组相比，小鼠中特定缺失的Tem263会导致骨量和强度降低；3) KO小鼠的骨密度和强度降低的原因是增殖减少、分化受损和/或 成骨细胞活性减弱。我们将在全局和成骨细胞特异性的Tem263中测试这些假设 基因敲除小鼠和Tem263过表达甘油三酯小鼠。我们将测量全身、股骨和 用体内DXA测量脊柱骨密度，用体内μCT测量股骨皮质骨和松质骨的体积骨密度。 骨骼强度将通过三点弯曲在股骨进行测试。此外，我们将确定分子和细胞 通过测量骨转换的血清标志物来解释骨表型变化的机制， 量化骨形成和骨吸收中的重要基因的表达，并进行静态和动态 骨组织形态计量学。我们将使用两种方法来确定Tem263的蛋白质-蛋白质相互作用伙伴 互补的蛋白质组学技术。我们还将在KO和过度表达中使用RNA测序 Tem263新生幼崽识别全球基因表达的变化。蛋白质组学和RNA测序 研究将确定TMEM在骨骼健康中扮演的角色所涉及的具体途径和网络。总而言之，我们 建议使用新的小鼠模型来了解一种独特的基因产物对骨骼和矿物质的功能 新陈代谢。拟议研究的成功完成可能开辟骨生物学的一个新领域，并可能 为治疗骨病的药物开发提供了重要的分子靶点。

Our previous genome-wide association study identified 56 loci that were associated with bone mineral density (BMD) and fracture risk in men and women. One locus on chromosome 12q23.3 harboring the gene TMEM263 (Transmembrane protein 263) was strongly associated with hip BMD (p<9.6x10-10). TMEM263 encodes a multi-pass transmembrane protein of unknown family. To identify the role of this gene in bone biology, we tested the SNP (rs1053051) associated with hip BMD in the TMEM263 gene for allele-specific expression (ASE) differences using human femoral bone samples. We observed a unidirectional allelic imbalance in mRNA expression for this SNP, suggesting that variation in ASE in TMEM263 may contribute to variation in hip BMD. The mouse Tmem263 gene was highly expressed in skeletal tissue compared to non- skeletal tissues, and osteoblasts and osteocytes expressed 6-fold higher mRNA levels of Tmem263 compared to osteoclasts. Also, in an established osteoblast cell line, OB6, gene-silencing with shRNA specific for Tmem263 revealed decreased expression of genes important for bone formation and increased expression of genes related to bone resorption. We recently made osteoblast-specific Tmem263 KO mice. Preliminary data show that these mice display broken bones and significantly lower whole body aBMD, BMC and trabecular bone mass and compromised bone micro-architecture compared to WT mice. Therefore, we hypothesize that: 1) the Tmem263 gene plays an important role in the acquisition and maintenance of bone mass; 2) osteoblast- specific deletion of Tmem263 in mice will lead to reduced bone mass and strength compared to controls; 3) lower BMD and strength in KO mice will result from decreased proliferation, impaired differentiation and/or diminished activity of osteoblasts. We will test these hypotheses in global and osteoblast-specific Tmem263 knockout mice and Tmem263 overexpressing Tg mice. We will measure areal BMD of whole body, femur and spine by in-vivo DXA and determine volumetric BMD for cortical and trabecular bone in femur by in-vivo μCT. Bone strength will be tested in femur by 3-point bending. Further, we will determine the molecular and cellular mechanisms responsible for changes in bone phenotype by measuring serum markers of bone turnover, quantifying expression of genes important in bone formation and resorption, and performing static and dynamic bone histomorphometry. We will identify protein-protein interaction partners of Tmem263 using two complementary proteomics techniques. We will also use RNA sequencing in KO and overexpressing Tmem263 newborn pups to identify global gene expression changes. The proteomic and RNA sequencing studies will identify specific pathways and networks involved in Tmem’s role in bone health. In summary, we propose to use novel mouse models to understand the function of a unique gene product on bone and mineral metabolism. Successful completion of the proposed studies could open a new area of bone biology and may provide an important molecular target for drug development for bone disorders.