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：
10191890
负责人：
Michael J Econs
金额：
$ 20.92万
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 2023-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10191890
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.

我们之前的全基因组关联研究确定了 56 个与骨矿物质相关的位点男性和女性的密度 (BMD) 和骨折风险。染色体 12q23.3 上的一个基因座含有该基因。 TMEM263（跨膜蛋白 263）与髋部 BMD 密切相关 (p<9.6x10-10)。编码未知家族的多次跨膜蛋白以鉴定该基因在骨中的作用。生物学方面，我们测试了 TMEM263 基因中与髋部 BMD 相关的 SNP (rs1053051) 的等位基因特异性我们使用人类股骨样本观察到了单向等位基因的表达（ASE）差异。该 SNP 的 mRNA 表达不平衡，表明 TMEM263 中 ASE 的变异可能导致与非骨骼组织相比，小鼠 Tmem263 基因在骨骼组织中高表达。骨骼组织、成骨细胞和骨细胞表达的 Tmem263 mRNA 水平比对照组高 6 倍此外，在已建立的成骨细胞系 OB6 中，用特定的 shRNA 进行基因沉默。 Tmem263揭示了对骨形成重要的基因的表达减少，以及骨形成重要基因的表达增加我们最近制作了成骨细胞特异性 Tmem263 KO 小鼠的初步数据。表明这些小鼠出现骨折，并且全身 aBMD、BMC 和小梁显着降低与 WT 小鼠相比，骨量和骨微结构受损因此，我们发现： 1）Tmem263基因在骨量的获得和维持中发挥重要作用；2）成骨细胞- 与对照组相比，小鼠 Tmem263 的特异性缺失将导致骨量和强度降低 3) KO 小鼠的 BMD 和强度较低是由于增殖减少、分化受损和/或我们将在整体和成骨细胞特异性 Tmem263 中测试这些假设。我们将测量全身、股骨和 Tmem263 过表达 Tg 小鼠的面积 BMD。通过体内 DXA 检测脊柱，并通过体内 μCT 确定股骨皮质骨和小梁骨的体积 BMD。股骨的骨强度将通过三点弯曲进行测试，此外，我们将确定分子和细胞的强度。通过测量骨转换的血清标志物导致骨表型变化的机制，量化骨形成和吸收中重要基因的表达，并进行静态和动态分析我们将使用两种方法来鉴定 Tmem263 的蛋白质-蛋白质相互作用伙伴。我们还将在 KO 和过表达中使用 RNA 测序技术。 Tmem263 新生幼崽识别整体基因表达变化的蛋白质组和 RNA 测序。研究将确定 Tmem 在骨骼健康中发挥作用的具体途径和网络。提议使用新型小鼠模型来了解独特基因产物对骨骼和矿物质的功能成功完成拟议的研究可能会开辟骨生物学的新领域，并可能。为骨疾病药物开发提供重要的分子靶点。