PTH Signaling through LRP6

 描述（由申请人提供）：骨质疏松症是美国骨折的主要原因，通常与高脂血症和相关动脉粥样硬化的人群相关。据信，促进动脉粥样硬化的相同生物活性氧化脂质（oxlipids）和低密度脂蛋白（oxLDL）也对骨产生不利影响。然而，这些生物活性脂质如何作用于骨细胞仍然是未知的。最近的人类遗传学研究表明，LRP 6突变与早发性骨质疏松症、高脂血症和早发性冠状动脉疾病有关。在我们的初步研究中，我们发现MSC质膜上的LRP 6直接结合oxlipids和oxLDL，然后通过LRP 6的内吞作用。用氧化脂质处理的MSC向成骨细胞的分化受损，对PTH、BMP和Wnt的反应降低，表明LRP 6可能介导生物活性脂质对骨的作用。我们假设骨微环境中氧化脂质的增加结合成骨细胞谱系上的LRP 6并诱导LRP 6内吞作用，导致LRP 6无法利用PTH、Wnt和BMP的信号来实现其成骨功能。该假设将通过以下具体目标进行检验。目的1将检测成骨细胞谱系细胞中响应于生物活性氧化脂质的LRP 6的内吞作用。我们将研究在野生型小鼠和ldlr-/-小鼠中，HFD与CHD相比，骨髓MSC中的细胞表面LRP 6表达是否响应于增加的生物活性脂质而降低。我们将通过监测LRP 6亚细胞定位和细胞表面LRP 6表达来研究oxLDL和单个oxlipids在培养的骨髓间充质干细胞和颅骨前成骨细胞中刺激的LRP 6内吞。目的2将检测氧化脂质-LRP 6结合的特异性和在成骨细胞系细胞中介导氧化脂质诱导的LRP 6内吞的分子途径。我们将使用已建立的蛋白质脂质覆盖测定和125 I-oxLDL-细胞结合测定来确定LRP 6是否直接结合oxlipids。还将检查介导氧化脂质诱导的MSC中LRP 6内吞作用的关键途径。目的3将确定LRP 6内吞作用的抑制是否增加MSC的成骨功能并恢复高脂血症期间PTH对骨的作用。我们将评估阻断oxlipid-LRP 6结合或抑制LRP 6内吞是否恢复受损的骨髓间充质干细胞的成骨功能和PTH对高脂血症小鼠骨的合成代谢作用。这项研究将提供骨质疏松症和高脂血症/动脉粥样硬化共同发展的机制的新认识，并产生同时治疗这两种疾病的新策略。

 DESCRIPTION (provided by applicant): Osteoporosis, the leading cause of fractures in the United States, is commonly associated with the population who have developed hyperlipidemia and associated atherosclerosis. It is believed that the same bioactive oxidized lipids (oxlipids) and low-density lipoprotein (oxLDL) that promote atherosclerosis also adversely affect bone. However, how these bioactive lipids act on bone cells remain largely unknown. Recent human genetic studies demonstrated that LRP6 mutations are associated with premature osteoporosis, hyperlipidemia, and early onset coronary artery disease. In our preliminary study, we have found that LRP6 on plasma membrane of MSCs directly binds oxlipids and oxLDL followed by LRP6 endocytosis. MSCs treated with oxlipids had impaired differentiation toward osteoblasts and reduced responses to PTH, BMPs and Wnts, indicating that LRP6 may mediate the action of bioactive lipids on bone. We hypothesize that increased oxidized lipids in bone microenvironment binds LRP6 on osteoblastic lineage of cells and induces LRP6 endocytosis, leading to the unavailability of LRP6 to transduce the signals of PTH, Wnt, and BMP for their osteogenic function. The hypothesis will be tested by the following Specific Aims. Aim 1 will examine the endocytosis of LRP6 in osteoblast lineage cells in response to bioactive oxidized lipids. We will examine whether cell surface LRP6 expression in bone marrow MSCs is reduced in response to increased bioactive lipids in wild type mice and ldlr-/- mice fed HFD vs CHD. We will examine LRP6 endocytosis stimulated by oxLDL and individual oxlipids in cultured bone marrow MSCs and calvarial preosteoblasts by monitoring LRP6 subcellular localization and cell surface LRP6 expression. Aim 2 will examine the specificity of oxlipid-LRP6 binding and the molecular pathway mediating oxidized lipid-induced LRP6 endocytosis in osteoblast lineage cells. We will define if LRP6 directly binds oxlipids using the established protein lipid overlay assay and 125I- oxLDL-cell binding assay. The key pathways mediating oxlipids-induced LRP6 endocytosis in MSCs will also be examined. Aim 3 will determine if inhibition of LRP6 endocytosis increases the osteogenic function of MSCs and restores the PTH action on bone during hyperlipidemia. We will evaluate whether blocking the oxlipid-LRP6 binding or inhibiting LRP6 endocytosis restores the impaired osteogenic function of bone marrow MSCs and the anabolic action of PTH on bone in hyperlipidemia mice. The study will provide new understanding of the mechanisms by which osteoporosis and hyperlipidemia/ atherosclerosis co-develop and yield new strategies to simultaneously treat both disorders.