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 对破骨细胞附着位点蛋白质的作用使细胞从骨上分离。这伴随着 Ca2 的释放，可能是通过 IP3R 受体，以及涉及 mu-钙蛋白酶的细胞内蛋白水解。然后，与运动相关的变化被逆转，使破骨细胞能够在新位置恢复骨质降解。我们将使用人类骨细胞作为我们的主要测试系统进一步研究这一机制。在目标 I 中，我们将确定 NO、cGMP 和 PKG I 如何调节破骨细胞附着。这将包括响应 NO 的膜附着蛋白重排的研究，包括 VASP、migfilin 和 alpha-v-beta3 整合素。在细胞拉伸和雌激素等关键刺激存在下，NO 合成的调节将在破骨细胞和成骨细胞中进行表征。 VASP 将在 PKG l 缺陷的细胞中进行研究，其中它还可以调节由 NO 以外的刺激（例如 CSF-1）诱导的运动。还将研究 PKG I 缺陷细胞，以评估 NO 自由基的作用，这在 PKG I 存在的情况下很难检测到。在目标 2 中，我们将定义 Ca2+ 依赖性机制，该机制对于完成和逆转 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对正常细胞和缺乏关键途径成分的细胞中的特定介质（包括 Ca2+）进行测定。我们将确定响应 NO 和 cGMP 而发生的 Ca 脉冲的来源。 PKG1诱导的激活肌醇-1,4,5-三磷酸受体的附着蛋白的变化将得到表征。我们将确定 Ca2 激活蛋白酶 mu-calpain 在运动过程中如何发挥作用。将确定钙调蛋白激活蛋白（包括磷酸二酯酶、磷酸酶和Ca2-ATP酶）终止运动的机制。将分析 mu-钙蛋白酶通过磷酸化和切割的调节，并鉴定在运动过程中被 mu-钙蛋白酶修饰的蛋白质。这些研究定义的机制将突出骨质疏松症药物干预的潜在目标。

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.