Regulation of Osteoclast Activity by Calcium and cGMP

描述（由申请人提供）：调节骨量的刺激物，包括雌激素，调节骨细胞中一氧化氮（NO）的产生。NO是骨降解的重要调节因子。我们的研究表明，NO调节破骨细胞的运动。对NO的反应由cGMP依赖性蛋白激酶I（PKG I）介导。PKG I作用于破骨细胞附着部位的蛋白质，使细胞从骨上脱离。这是伴随着Ca 2+释放，可能通过IP 3R受体，并通过细胞内蛋白水解涉及μ-钙蛋白酶。然后，运动相关的变化被逆转，允许破骨细胞在新的位置恢复骨降解。我们将使用人骨细胞作为我们的主要测试系统来进一步研究这种机制。在目的我，我们将确定如何NO，cGMP和PKG我调节破骨细胞附着。这将包括对响应于NO的膜附着蛋白的重排的研究，包括VASP、migfilin和α-v-β 3整联蛋白。在关键刺激物（包括细胞拉伸和雌激素）的存在下，NO合成的调节将在破骨细胞和成骨细胞中表征。将在PKGl缺陷细胞中研究VASP，其中它还可以调节由NO以外的刺激物（例如CSF-1）诱导的运动性。还将研究PKG I缺陷细胞以评价NO自由基作用，这在PKG I存在下难以检测。在目标2中，我们将定义钙离子依赖性机制，这对完成和逆转NO诱导的破骨细胞运动至关重要。这些研究将使用药理学抑制剂和特定介质的测定，包括正常细胞和缺乏关键途径成分的细胞中的Ca 2+。我们将确定响应于NO和cGMP而发生的Ca脉冲的来源。将表征PKG 1诱导的激活肌醇-1，4，5-三磷酸受体的附着蛋白的变化。我们将确定钙激活蛋白酶μ-钙蛋白酶在运动过程中的功能。将确定钙调素激活蛋白（包括磷酸二酯酶、磷酸酶和Ca 2 +-ATP酶）终止运动性的机制。将分析磷酸化和切割对mu-钙蛋白酶的调节，并鉴定在运动过程中被mu-钙蛋白酶修饰的蛋白质。这些研究确定的机制将突出骨质疏松症药物干预的潜在靶点。

DESCRIPTION (provided by applicant): Stimuli that regulate bone mass, including estrogen, modulate nitric oxide (NO) production in bone cells. NO is an important regulator of bone degradation. Our studies showed that NO regulates osteoclast motility. Response to NO is mediated by the cGMP-dependent protein kinase I (PKG I). PKG I action on proteins at the osteoclast's attachment site allow the cell to detach from bone. This is accompanied by Ca2+ release, probably via the IP3R receptor, and by intracellular proteolysis involving mu-calpain. Motility-related changes are then reversed, allowing the osteoclast to resume bone degradation in a new location. We will study this mechanism further using human bone cells as our principal test system. In Aim I we will determine how NO, cGMP, and PKG I regulate osteoclast attachment. This will include studies of rearrangement of membrane- attachment proteins in response to NO, including VASP, migfilin and the alph-v-beta3 integrin. The regulation of NO synthesis in the presence of key stimuli including cell stretch and estrogen will be characterized in osteoclasts and in osteoblasts. VASP will be studied in PKG l-deficient cells, where it may also regulate motility induced by stimuli other than NO, such as CSF-1. PKG l-deficient cells will also be studied to evaluate NO-free radical actions, which are difficult to detect in the presence of PKG I. In Aim 2, we will define Ca2+-dependent mechanisms that are critical to completing and reversing NO-induced osteoclast motility. These studies will use pharmacological inhibitors and assays for specific mediators, including Ca2+, in normal cells and in cells deficient in key pathway constituents. We will determine the source of Ca pulses that occur in response to NO and cGMP. PKG l-induced changes in attachment proteins that activate the inositol-1,4,5-trisphosphate receptor will be characterized. We will determine how the Ca2+ activated proteinase mu-calpain functions during motility. The mechanisms by which calmodulin-activated proteins including phosphodiesterase, phosphatase, and Ca2+-ATPase terminate motility will be determined. Regulation of mu-calpain by phosphorylation and cleavage will be analyzed, and proteins that are modified by mu-calpain during motility will be identified. The mechanisms defined by these studies will highlight potential targets for pharmacological intervention in osteoporosis.