Identification of Proteins Important for Male Osteoporosis

骨质疏松症是一种主要的公共卫生问题，主要表现为低骨密度（BMD）。骨密度的变异很大程度上是由遗传决定的（遗传率约为60%）。一些骨密度基因/基因组区域是性别特异性的。尽管女性骨密度低于男性，但男性骨质疏松性骨折的死亡率明显高于男性。然而，对骨质疏松症的研究主要集中在女性身上。男性骨质疏松症的遗传流行病学研究很少，蛋白质组学流行病学研究尚未开展。骨髓间充质干细胞（BMMSCs）和外周血单核细胞（PBMs）分别是成骨细胞（骨形成细胞）和破骨细胞（骨吸收细胞）的前体。蛋白质组学是一种强大的先进的策略，在复杂疾病的遗传解剖，如骨质疏松症。然而，影响目前大多数蛋白质组学研究的一个主要问题是低丰度蛋白质和具有极端等电点、分子量和疏水性的蛋白质，特别是膜蛋白的检测有限。解决这一问题的一种新方法是亚细胞蛋白质组提取，从膜、细胞质、细胞核和细胞骨架E组分中逐步分离蛋白质，然后采用灵敏的2D-nanoLC-ESI-MS/MS进行分离和鉴定重要蛋白质。我们的假设是bmmsc和PBMs中蛋白表达谱的变化是BMD变异的分子机制的基础，并与男性骨质疏松症有关。我们的主要目标是鉴定bmmsc和PBMs在高骨密度和低骨密度男性中的差异表达蛋白，从而鉴定bmmsc和PBMs中与男性骨质疏松症相关的蛋白（及其基因）。我们将招募120名健康的白种人男性，骨量峰值年龄在25-50岁之间，其中骨密度低和高分别为60名和60名（骨密度最低和最高分别为年龄匹配人群的20%）。一半的样本（30名低骨密度和30名高骨密度受试者）将作为“发现队列”，另一半（30名低骨密度和30名高骨密度受试者）将作为“复制队列”。我们将从每个男性受试者身上采集新鲜骨髓和外周血样本，就像我们在正在进行的女性受试者的NIH SCOR项目中所做的那样。BMMSCs和PBMs将通过亚细胞蛋白质组提取膜蛋白以及细胞质、核和细胞骨架部分的蛋白质来分离。将使用2D-nanoLC-ESI-MS/MS对发现E队列中分离的蛋白质样品进行蛋白质组学分析实验和分析。鉴定出的显著差异表达蛋白将使用复制队列中的样品进行Western blotting验证。本研究的主要结果（特别是从PBMs获得的结果）可用于设计用于男性骨质疏松症预后的常规诊断蛋白抗体芯片和/或蛋白质标记物。结合我们正在进行的通过全基因组DNA关联扫描和成骨细胞全基因组mRNA表达研究鉴定骨质疏松危险基因的项目，本研究将有力有效地鉴定男性骨质疏松的基因及其部分功能。公共卫生相关性：骨质疏松症是一种严重骨质流失和低创伤性骨折风险显著增加的疾病。男性骨质疏松症与女性骨质疏松症相比，骨折后死亡率更高。该项目旨在确定对男性骨质疏松症有重要作用的蛋白质。研究结果将为有效预防、诊断和治疗该病提供重要的科学依据。

DESCRIPTION (provided by applicant): Identification of Proteins Important for Male Osteoporosis Osteoporosis is a major public health problem, mainly characterized by low bone mineral density (BMD). Variation of BMD is largely genetically determined (heritability of > 60%). Some BMD genes/genomic regions are gender specific. Although women have lower BMD than men, men suffer significantly higher mortality rate upon osteoporotic fractures. However, studies on osteoporosis have largely been focused on women. Few genetic epidemiological studies and no proteomic epidemiological study on osteoporosis have been performed on men. Bone marrow mesenchymal stem cells (BMMSCs) and peripheral blood monocytes (PBMs) are precursors for osteoblasts (bone formation cells) and osteoclasts (bone resorption cells), respectively. Proteomics is a powerful state-of-the-art strategy in genetic dissection of complex diseases, such as osteoporosis. However, a major problem affecting the power of current most proteomic studies is the limited detection of low abundance proteins and proteins with extreme isoelectric point, molecular weight, and hydrophobicity, especially membrane proteins. A NOVEL approach to this problem is subcellular proteome extraction to stepwise isolate proteins from membrane, cytosol, nucleus, and cytoskeleton E fractions followed by sensitive 2D-nanoLC-ESI-MS/MS for fractioning and identifying significant proteins. Our hypothesis is that changes in the protein expression profiles in BMMSCs and PBMs underlie molecular mechanisms of BMD variation and are associated with osteoporosis in men. Our major goals here are to identify proteins differentially expressed in BMMSCs and PBMs in men with high vs. low BMD and thus identify proteins (and their genes) associated with male osteoporosis in BMMSCs and PBMs. We will recruit 120 otherwise healthy Caucasian males at peak bone mass ages of 25-50, including 60 subjects with low and 60 with high BMD (age matched population bottom or top 20% respectively in terms of BMD). Half of the sample (30 low vs. 30 high BMD subjects) will serve as the "discovery cohort" and the other half (30 low vs. 30 high BMD subjects) will serve as the "replication cohort". We will take fresh bone marrow and peripheral blood samples from each male subject, as we do in our ongoing NIH SCOR projects for female subjects. BMMSCs and PBMs will be isolated by subcellular proteome extraction for membrane proteins together with proteins of cytosolic, nuclear, and cytoskeletal fractions. Proteomic profiling experiments and analyses will be performed on the isolated protein samples from the discovery E cohort using 2D-nanoLC-ESI-MS/MS . Significant differentially expressed proteins identified will be verified by Western blotting using samples from the replication cohort. The major results (particularly those obtained from PBMs) of this study may be used to design customary diagnostic protein antibody chips and/or protein markers for prognosis of male osteoporosis. In combination with our ongoing projects for identifying risk genes of osteoporosis through genome-wide DNA association scan and genome-wide mRNA expression study of osteogenic cells, this study will powerfully and efficiently identify genes and some of their functions for male osteoporosis. PUBLIC HEALTH RELEVANCE: Osteoporosis is a disease with severe bone loss and a significantly increased risk of low trauma fractures. Male osteoporosis, compared with female osteoporosis, has higher mortality rate upon fractures. This project aims to identify proteins that are important to male osteoporosis. The findings will provide essential scientific basis for effective prevention, diagnosis and treatment of the disease.