| Summary: | 碩士 === 國防醫學院 === 生物及解剖學研究所 === 88 === Osteoclastic bone resorption is initiated by integrin (avb3) adhesion to the bone surface RGD-containing peptide, followed by cytoskeletal rearrangement and formation of the sealing zone that polarizes the cell. Calcitonin (CT) inhibits bone resorption by causing detachment of osteoclasts. In HEK-293 cells, CT induced G protein-coupled signaling cascades, which involve adenylyl cyclase, phospholipase C, protein kinase A, protein kinase C, Erk1/2, and HEF1. However, the CT-induced signaling effectors in osteoclasts are not well characterized. Proline-rich tyrosine kinase 2 (PYK2) is a member of the focal adhesion kinase family. PYK2 localized in the sealing zone of osteoclasts, activated by ligation of integrin, and then activated src, is an important signaling molecule for bone resorption. Role of PYK2 in the signaling pathways that link G protein-coupled receptors with mitogen-activated protein kinase has been reported. In the present study, we address the role played by PYK2 as a potential effector downstream of CT-induced signaling pathways. In isolated authentic rabbit osteoclasts cultured on collagen-I coated plate, CT-induced detachment of the cells, effects on PYK2 tyrosine phosphorylation and distribution were examined. CT induced a dose-dependent decrease of PYK2 tyrosine phosphorylation which is blocked by pre-treatment of the protein kinase C inhibitor calphostin C and the phosphatase inhibitors pervanadate and okadaic acid, but not the protein kinase A inhibitor H89 or the Erk1/2 inhibitor PD98059. Similar results were obtained in the CT-induced detachment assay. Transient increase of intracellular calcium concentration following CT treatments also appeared to modulate both osteoclast retraction/detachment and PYK2 phosphorylation. Furthermore, confocal microscopy analysis indicated that CT treatments caused redistribution of PYK2 from cell periphery to central portion of the cytosol and decrease of tyrosine phosphorylation levels of PYK2. SHPTP1 (Src homology 2 domain-containing phosphatase) coimmuno-precipitated with PYK2 and its tyrosine phosphorylation state was increased by CT. Taken together, our findings suggest that PYK2 may play an important role in mediating CT-induced cytoskeletal rearrangement in osteoclasts by modulating protein kinase C, SHPTP1, and intracellular calcium concentration.
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