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

基本信息

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
项目类别：
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

项目摘要

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.

我们以前的全基因组关联研究确定了与骨矿物质相关的56个基因座男性和女人的密度（BMD）和骨折风险。染色体12q23.3上的一个基因座携带该基因 TMEM263（跨膜蛋白263）与髋关节BMD密切相关（p <9.6x10-10）。 TMEM263 编码未知家族的多通跨膜蛋白。确定该基因在骨骼中的作用生物学，我们测试了与TMEM263基因中与髋关节BMD相关的SNP（RS1053051）使用人股骨样品的表达（ASE）差异。我们观察到一个单向等位基因该SNP的mRNA表达不平衡，表明TMEM263中ASE的变化可能有助于臀部BMD的变化。小鼠TMEM263基因在骨骼组织中高度表达骨骼组织，成骨细胞和骨细胞表达的TMEM263的mRNA水平高6倍到破骨细胞。同样，在已建立的成骨细胞系中，OB6，具有特异性shRNA的基因沉默 TMEM263显示，基因表达降低了对骨形成重要的基因表达和增加的表达与骨骼分辨率有关的基因。我们最近制作了成骨细胞特异性TMEM263 KO小鼠。初步数据表明这些小鼠表现出骨折的骨骼，并显着降低全身ABMD，BMC和小梁与WT小鼠相比，骨骼质量和骨微体系结构受损。因此，我们假设： 1）TMEM263基因在骨骼的获取和维持中起重要作用； 2）成骨细胞 - 与对照组相比，小鼠TMEM263的特异性缺失将导致骨骼质量和强度降低。 3）较低的BMD和KO小鼠的强度将是由于增殖减少，分化受损和/或导致的成骨细胞活性减少。我们将在全球和成骨细胞特异性TMEM263中检验这些假设敲除小鼠和TMEM263过表达TG小鼠。我们将测量全身，股骨和通过体内DXA的脊柱，并确定股骨中皮质和小梁骨的体积BMD通过体内μCT。骨骼强度将通过三分弯曲在股骨中测试。此外，我们将确定分子和细胞通过测量骨骼更新的血清标志物来导致骨表型变化的机制，量化在骨形成和分辨率中重要的基因表达，并执行静态和动态骨骼组态法。我们将使用两个完全蛋白质组学技术。我们还将在KO中使用RNA测序和过表达 TMEM263新生幼崽确定全球基因表达的变化。蛋白质组学和RNA测序研究将确定与TMEM在骨骼健康中作用有关的特定途径和网络。总而言之，我们建议使用新型鼠标模型来了解独特基因产品对骨骼和次要的功能代谢。成功完成拟议的研究可能会开放一个新的骨骼生物学领域，可能为骨骼疾病的药物发育提供了重要的分子靶标。