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，骨密度的变化是由遗传和环境因素共同决定的。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，这些基因座对人类和实验动物的骨密度变化有贡献。然而，骨密度QTL基因的识别仍然是一个巨大的挑战。在我们最近的研究中，我们已经发现Duffy抗原趋化因子受体(Darc)是位于1号染色体上的一个BMD QTL基因，它通过增加破骨细胞的分化来负向调节BMD。DARC与已知参与破骨细胞分化的趋化因子结合。关于Darc调节破骨细胞的机制，我们的初步研究表明：1)来自Darc-KO小鼠的依赖M-CSF的骨髓细胞与MCP-1和RANTES等趋化因子的结合减少；2)从Darc-KO小鼠分离的股骨显示与对照小鼠相比，TRAP阳性的骨吸收表面减少。相反，两系小鼠的骨形成参数相似；3)与空载体转染的原始细胞相比，高表达Darc的小鼠单核细胞RAW264.6细胞向趋化因子RANTES的跨内皮迁移(TEM)显著增加。这些数据表明，破骨细胞对趋化因子的透射电子显微镜部分受DARC调控。根据这些和其他的初步数据，Darc-KO小鼠表现出比野生型小鼠更高的骨密度，以及Darc在中性粒细胞/单核细胞中的已知作用，我们提出了Darc通过趋化因子调节破骨细胞前体细胞(OCP)的迁移以促进随后的融合和分化的假设。为了验证这一假说，我们将1)确定DARC在内皮细胞或OCP细胞中的过度表达并阻断其表达，是否会影响OCP细胞向单核细胞趋化蛋白-1和RANTES趋化因子的迁移；2)在Darc-KO，即仅在内皮细胞中高表达Darc的转基因小鼠和经脂多糖处理的对照小鼠中诱导趋化因子的表达，并评估趋化因子表达增加在OCP募集和体内骨吸收中的后果。我们预计，这项研究的成功完成将使人们更好地理解Darc-趋化因子相互作用在调节破骨细胞发育和炎症诱导的骨丢失中的作用。我们的假设得到证实，即DARC通过与趋化因子结合来调节破骨细胞的活动，这可能会导致诊断筛选的发展，以识别哪些患者面临更高的骨质疏松风险，以及治疗具有趋化因子诱导的高骨转换的骨质疏松症患者的治疗策略。

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.