Regulation of Osteoclast Activity by Calcium and cGMP

描述(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对破骨细胞的运动有调节作用。对NO的反应是由cGMP依赖的蛋白激酶I(PKG I)介导的。PKG I作用于破骨细胞附着部位的蛋白质，使细胞与骨分离。这伴随着钙释放，可能是通过IP3R受体，并伴随着涉及Mu-calain的细胞内蛋白分解。与运动相关的改变随后被逆转，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一机制。在Aim I中，我们将确定NO、cGMP和PKG I是如何调节破骨细胞附着的。这将包括对膜附着蛋白对NO反应的重排的研究，包括血管紧张素转换酶、米非菌素和α-v-β3整合素。在破骨细胞和成骨细胞中，在包括细胞伸展和雌激素在内的关键刺激存在的情况下，对NO合成的调节将具有特征。VASP将在PKG L缺陷的细胞中进行研究，在那里它也可能调节NO以外的刺激诱导的运动，如脑脊液-1。我们还将研究PKG L缺失的细胞，以评估在PKG I存在下难以检测到的NO自由基作用。在目标2中，我们将定义钙依赖机制，这些机制对于完成和逆转NO诱导的破骨细胞运动至关重要。这些研究将在正常细胞和缺乏关键途径成分的细胞中使用药物抑制剂和包括钙在内的特定介质的分析。我们将确定对NO和cGMP作出反应的钙脉冲的来源。PKG L诱导的激活肌醇-1，4，5-三磷酸受体的附着蛋白的变化将被描述。我们将确定钙激活的蛋白酶Mu-calain在运动过程中是如何发挥作用的。钙调蛋白激活的蛋白，包括磷酸二酯酶、磷酸酶和钙-三磷酸腺苷酶终止运动的机制将被确定。本课程将分析通过磷酸化和裂解对Mu-calain的调节，并将确定在运动过程中被Mu-calain修饰的蛋白质。这些研究定义的机制将突出药物干预骨质疏松症的潜在靶点。

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.