The role of Tmem263 in regulation of bone mass and strength

Tmem263 在骨量和骨强度调节中的作用

• 批准号: 10401449
• 负责人: Michael J Econs
• 金额: $ 17.26万
• 依托单位: INDIANA UNIV-PURDUE UNIV AT INDIANAPOLIS
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-05-05 至 2024-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10401449
• 关键词: Adult; Affinity Chromatography; Age; Alkaline Phosphatase; Alleles; Allelic Imbalance; Architecture; Area; Biochemistry; Biology; Bone Density; Bone Resorption; Bone Tissue; Bone structure; C-terminal; Calvaria; Cell Line; Cells; Cellular Membrane; Chromosomes; Crosslinker; Cytolysis; Data; Developmental Bone Diseases; Dual-Energy X-Ray Absorptiometry; Family; Female; Femur; Fracture; Gene Expression; Gene Silencing; Genes; Genotype; Goals; Heterozygote; Homozygote; Human; Impairment; Integral Membrane Protein; Investigation; Knock-out; Knockout Mice; Maintenance; Mass Spectrum Analysis; Measures; Membrane; Messenger RNA; Metabolism; Methods; Minerals; Molecular; Molecular Target; Mus; Newborn Infant; Osteoblasts; Osteocalcin; Osteoclasts; Osteocytes; Osteogenesis; Pathway interactions; Permeability; Phenotype; Play; Proteins; Proteomics; RNA; Regulation; Roentgen Rays; Role; Sampling; Serum; Serum Markers; Site; Streptavidin; Sulfoxide; Surface; TRANCE protein; Techniques; Testing; Transgenic Mice; Transgenic Organisms; Variant; Vertebral column; Woman; base; bone; bone health; bone mass; bone strength; bone turnover; conditional knockout; cortical bone; drug development; fracture risk; gene product; genetic manipulation; genome wide association study; hip bone; in vivo; mRNA Expression; male; member; men; mineralization; molecular drug target; mouse model; novel; overexpression; promoter; protein protein interaction; pup; skeletal tissue; small hairpin RNA; substantia spongiosa; transcriptome; transcriptome sequencing

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.

我们先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， 骨密度（BMD）和男性和女性的骨折风险。染色体12q23.3上的一个位点携带该基因 TMEM 263（跨膜蛋白263）与髋部BMD密切相关（p<9.6x10-10）。TMEM263 编码未知家族的多通道跨膜蛋白。为了确定这种基因在骨骼中的作用， 在生物学方面，我们检测了TMEM 263基因中与髋部BMD相关的SNP（rs 1053051）， 使用人股骨样品测定ASE表达（ASE）差异。我们观察到一个单向等位基因 该SNP mRNA表达不平衡，表明TMEM 263中ASE的变化可能有助于 髋部BMD的变化。小鼠Tmem 263基因在骨骼组织中的表达高于非骨骼组织。 骨骼组织、成骨细胞和骨细胞表达的Tmem 263 mRNA水平是对照组的6倍。 破骨细胞此外，在一个已建立的成骨细胞系OB 6中，用特异性的shRNA进行基因沉默， tmem 263揭示了对骨形成重要的基因表达降低， 骨吸收相关基因我们最近制造了成骨细胞特异性Tmem 263 KO小鼠。初步数据 显示这些小鼠显示骨折和显著降低的全身aBMD、BMC和骨小梁 骨量和受损的骨微结构。因此，我们假设： 1)Tmem 263基因在骨量的获得和维持中起重要作用; 2）成骨细胞- 与对照相比，小鼠中Tmem 263的特异性缺失将导致骨量和强度降低; 3） KO小鼠中较低的BMD和力量将由增殖减少、分化受损和/或 成骨细胞活性降低。我们将在整体和成骨细胞特异性Tmem 263中测试这些假设。 敲除小鼠和Tmem 263过表达Tg小鼠。我们将测量全身、股骨和 通过体内DXA测定脊柱骨密度，通过体内μCT测定股骨皮质骨和松质骨的体积BMD。 将通过3点弯曲测试股骨的骨强度。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， 定量在骨形成和再吸收中重要的基因的表达，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鉴定Tmem 263的蛋白质-蛋白质相互作用伴侣。 互补蛋白质组学技术。我们还将在KO和过表达中使用RNA测序， tmem 263新生幼崽，以确定全球基因表达变化。蛋白质组和RNA测序 研究将确定特定的途径和网络参与TdR的作用，在骨骼健康。总之，我们 我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作用 新陈代谢.拟议研究的成功完成可能会开辟骨生物学的新领域，并可能 为骨疾病的药物开发提供了重要的分子靶点。