The Role of PYK2/Src in Calcitonin-induced Podosome Reassembly and Sealing Zone Detachment in Osteoclasts

• Main Authors: Wen-Zhi Lin, 林文智
• Other Authors: Jia-Fwu Shyu
• Format: Others
• Language: zh-TW
• Published: 2005
• Online Access: http://ndltd.ncl.edu.tw/handle/96009504852448586015

碩士 === 國防醫學院 === 生物及解剖學研究所 === 93 === Osteoclasts (OCs) are multinucleated, terminally differentiated cells which play an essential role in bone resorption. To resorb bone, OCs attach to extracellular matrix or the bone surface via specialized attachment structures called podosomes, which form a prominent F-actin-rich ring that is thought to correspond to the sealing zone of resorbing OCs. It had been showed that Pyk2/Src/Cbl complex is critical to the αvβ3 integrin-mediated signaling in podosome. The concept of outside-in and inside-out of integrin-mediated signaling has been elucidated in depth through the study of Pyk2/Src/Cbl complex. Calcitonin (CT) is a 32-amino acid polypeptide which inhibits OC motility, induces OC retraction, and disrupts the actin-ring structure of OCs. Thus it is reasonable to assume that the Pyk2/Src/Cbl complex in podosome could be the potential target for the CT-induced signaling. In isolated authentic rabbit OCs cultured on extracellular matrix-coated Petri dish, we previously showed that CT induced dephosphorylation and redistribution of Pyk2. The dephosphorylation of Pyk2 would be expected to prevent the formation of Pyk2/Src/Cbl complex and therefore inhibit OC motility and attachment. However, the effects of CT on Pyk2/Src/Cbl complex function and their distribution in OCs are not clear. We therefore investigated whether CT affects Pyk2/Src association and Src phosphorylation in OCs by using the immunoprecipitation and Western blot methods. The result showed that CT induced an increase of Src tyrosine phosphorylation and Pyk2/Src association. Using immunofluorescent confocal analysis, we showed that CT induces dephosphorylation of Pyk2 in the sealing zone and increase Src tyrosine phosphorylation and Pyk2/Src association in the central region of OCs. Further examination using specific Y416 and Y527 phosphorylation antibodies of Src showed that CT induced increase of Y416 but not Y527 phosphorylation. The increase of Y416 and decrease of Y527 phosphorylation were found in the central region of OCs after CT stimulation. In conclusions, CT may induce podosome reassembly and sealing zone detachment by decrease Pyk2 phosphorylation in the sealing zone and increase Y416 phosphorylation of Src and Pyk2/Src association in the central region of OCs.