Role of Darc-Chemokine Interaction in the Trafficking of Osteoclast Precursors

描述（由申请人提供）：骨质疏松症是一种以低骨矿物质密度（BMD）为特征的疾病，是骨折风险的最强预测因子。现在众所周知，BMD方差是由遗传和环境因素决定的。因此，在人类和动物模型中的研究已经揭示了几个数量性状基因座（QTL）的存在的证据，有助于在人类和实验动物的BMD变化。然而，BMD QTL基因的鉴定仍然是一个很大的挑战。在我们最近的研究中，我们已经确定达菲抗原趋化因子受体（Darc）作为BMD QTL基因在染色体1，通过增加破骨细胞分化负调控BMD。Darc与已知参与破骨细胞分化的趋化因子结合。在Darc调节破骨细胞的机制方面，我们的初步研究表明：1）来自Darc敲除（Darc-KO）小鼠的M-CSF依赖性骨髓细胞表现出与趋化因子（如MCP-1和RANTES）的结合减少; 2）与对照小鼠相比，从Darc-KO小鼠分离的股骨显示TRAP阳性骨吸收表面减少。相比之下，两种小鼠系中的骨形成参数相似; 3）与用空载体转染的RAW细胞相比，过表达Darc的小鼠单核细胞RAW 264.6细胞表现出朝向趋化因子RANTES的跨内皮迁移（TEM）的显著增加。这些数据表明，破骨细胞TEM对趋化因子的调节部分由Darc。基于这些和其他初步数据，Darc-KO小鼠表现出更高的BMD相比，野生型小鼠和已知的作用Darc在中性粒细胞/单核细胞TEM，我们提出的假设，Darc调节破骨细胞前体（OCP）细胞的迁移，通过趋化因子为后续的融合和分化。为了检验这一假设，我们将1）确定内皮细胞或OCP细胞中Darc表达的过表达和阻断是否会影响OCP细胞向MCP-1和RANTES趋化因子的迁移; 2）在Darc-KO中诱导趋化因子表达，通过脂多糖处理仅在内皮细胞中过表达Darc的转基因小鼠和对照小鼠，并评估趋化因子表达增加对体内OCP募集和骨吸收。我们预计，这项研究的成功完成将导致更多的了解Darc-趋化因子的相互作用，在调节破骨细胞的发展和炎症诱导的骨丢失的作用。我们假设Darc通过与趋化因子结合来调节破骨细胞活性，这一假设的证实可能会导致诊断筛选的发展，以识别骨质疏松症风险增加的患者，以及治疗趋化因子诱导的高骨转换的骨质疏松患者的治疗策略。 公共卫生相关性：低峰值骨矿物质密度（BMD）已被确定为骨质疏松症的一个重要危险因素。由于峰值骨密度的变化>70%是由遗传决定的，因此识别导致峰值骨密度变化的基因及其机制非常重要。在这方面，我们最近发现，达菲抗原趋化因子受体（Darc）的遗传变异，通过影响骨密度骨吸收负调控BMD。这项研究将测试一个新的假设，即DARC调节破骨细胞的流动性，通过调节趋化因子的活性。我们对Darc通过与趋化因子结合来调节破骨细胞功能这一假设的证实可能会导致未来诊断筛查的发展，以识别有骨质疏松症风险的患者，以及治疗趋化因子诱导的高骨转换的骨质疏松患者的治疗策略。

DESCRIPTION (provided by applicant): Osteoporosis is a disease characterized by low bone mineral density (BMD), the strongest predictor of fracture risk. It is now well known that BMD variance is determined by both genetic and environmental factors. Accordingly, studies in human and animal models have revealed evidence for the presence of several quantitative trait loci (QTL) that contribute to BMD variations in humans and experimental animals. However, the identification of BMD QTL genes remains a big challenge. In our recent study, we have identified duffy antigen receptor for chemokines (Darc) as a BMD QTL gene in chromosome 1 which regulates BMD negatively by increasing osteoclast differentiation. Darc binds to chemokines known to be involved in osteoclast differentiation. In terms of mechanism by which Darc regulates osteoclasts, our preliminary studies have shown that 1) M-CSF dependent bone marrow cells derived from Darc-knockout (Darc-KO) mice exhibit reduced binding to chemokines such as MCP-1 and RANTES; 2) femurs isolated from Darc-KO mice showed reduced TRAP positive bone resorbing surface compared to control mice. In contrast, bone formation parameters were similar in both lines of mice; and 3) mouse monocyte RAW264.6 cells overexpressing Darc exhibited significant increase in transendothelial migration (TEM) towards the chemokine RANTES compared to RAW cells transfected with empty vector. These data demonstrate that osteoclast TEM towards chemokines is in part regulated by Darc. Based on these and other preliminary data that Darc-KO mice exhibit higher BMD compared to wild type mice and the known action of Darc in neutrophils/monocyteTEM, we propose the hypothesis that Darc regulates the migration of osteoclast precursor (OCP) cells through chemokines for subsequent fusion and differentiation. To test this hypothesis, we will 1) determine if the overexpression and the blockage of Darc expression in endothelial cells or in OCP cells will affect the transmigration of OCP cells towards MCP-1 and RANTES chemokines; 2) induce chemokine expression in Darc-KO, transgenic mice that overexpress Darc only in endothelial cells and control mice by treatment with lipopolysaccharide and evaluate the consequence of increased chemokine expression on OCP recruitment and bone resorption in vivo. We anticipate that successful completion of this study would lead to increased understanding of the role of Darc-chemokine interaction in regulating osteoclast development and inflammation-induced bone loss. The confirmation of our hypothesis that Darc regulates osteoclast activity via binding to chemokines could lead to development of diagnostic screens to identify patients who are at increased risk for osteoporosis and therapeutic strategies to treat osteoporotic patients with chemokine-induced high bone turnover. PUBLIC HEALTH RELEVANCE: Low peak bone mineral density (BMD) has been established as an important risk factor for osteoporosis. Because >70% variation in peak BMD is genetically determined, it is of great importance to identify the genes and their mechanisms that contribute to variation in peak BMD. In this regard, we have recently identified that genetic variation in duffy antigen receptor for chemokines (Darc) regulates BMD negatively by influencing osteoclastic bone resorption. This study will test a novel hypothesis that DARC regulates osteoclast mobility by modulating the activity of chemokines. Our confirmation of the hypothesis that Darc regulates osteoclast function by binding to chemokines could lead to future development of diagnostic screens to identify patients who are at risk for osteoporosis and therapeutic strategies to treat osteoporotic patients with chemokine- induced high bone turnover.