Regulation of Osteoclast Activity by Calcium and cGMP

描述（由申请人提供）：调节骨量的刺激，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的应答由cgmp依赖性蛋白激酶I （PKG I）介导。PKG - 1作用于破骨细胞附着部位的蛋白质，使细胞与骨分离。这伴随着Ca2+的释放，可能通过IP3R受体，以及涉及mu-calpain的细胞内蛋白水解。然后，与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。我们将以人类骨细胞为主要测试系统进一步研究这一机制。在目的1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α -v- β 3整合素。在包括细胞拉伸和雌激素在内的关键刺激存在下，NO合成的调节将在破骨细胞和成骨细胞中被表征。VASP将在PKG - l缺陷细胞中进行研究，其中VASP也可能调节由NO以外的刺激（如CSF-1）诱导的运动。PKG l-缺陷细胞也将被研究以评估no自由基的作用，这在PKG i存在时很难检测到。在Aim 2中，我们将定义Ca2+依赖机制，这对于完成和逆转no诱导的破骨细胞运动至关重要。这些研究将在正常细胞和缺乏关键通路成分的细胞中使用药理学抑制剂和特定介质（包括Ca2+）的测定。我们将确定响应NO和cGMP而产生的Ca脉冲的来源。PKG诱导的肌醇-1,4,5-三磷酸受体的附着蛋白的变化将被表征。我们将确定Ca2+活化的蛋白酶mu-calpain在运动过程中的功能。钙调素激活蛋白（包括磷酸二酯酶、磷酸酶和Ca2+- atp酶）终止运动的机制将被确定。将分析通过磷酸化和裂解对mu-calpain的调节，并确定在运动过程中被mu-calpain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.