Regulation of Osteoclast Activity by Calcium and cGMP

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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.