Calcium-sensing receptor-mediated activation of phospholipase C-γ1 is downstream of phospholipase C-β and protein kinase C in MC3T3-E1 osteoblasts
Calcium-sensing receptor-mediated activation of phospholipase C-γ1 is downstream of phospholipase C-β and protein kinase C in MC3T3-E1 osteoblasts
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DOI：
10.1016/s8756-3282(01)00700-1
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发表时间：
2002-04-01
期刊：
影响因子：
4.1
通讯作者：
Soltoff, SP
中科院分区：
文献类型：
--
作者：
Godwin, SL;Soltoff, SP
细胞外钙(Ca-e)升高通过钙敏感受体(CaSR)刺激MC3T3-E1成骨细胞的趋化和有丝分裂。这些成骨细胞的CA-e介导的趋化作用依赖于磷脂酶C(PLC)，并被胃肠道蛋白抑制剂百日咳毒素阻断。在这项研究中，我们研究了CaSR刺激MC3T3-E I成骨细胞PLC活性的信号机制。我们发现，升高的Ca-e以时间依赖和Ca-e浓度依赖的方式刺激PLC-Gamma1酪氨酸磷酸化。钙离子浓度从1.8 mmol/L增加3.2 mmol/L后3~5min，PLC-Gamma1酪氨酸磷酸化水平最高，升高的钙离子还促进磷脂酰肌醇-4，5-二磷酸的第二信使--三磷酸肌醇[INS(1，4，5)P-3]和胞内游离钙[[Ca+2](1)]迅速升高。CaSR介导的INS(1，4，5)P-3和[Ca+2](I)增加的动力学以及钙-e刺激的[Ca+2](I)对U73122(PLC抑制剂)的敏感性共同表明成骨细胞CaSR通过GQ与PLC-β偶联。U73122阻断Ca-e促进的PLC-Gamma1酪氨酸磷酸化，但不阻断PDGF促进的PLC-Gamma1酪氨酸磷酸化，提示PLC-β的激活是PLC-Gamma1激活的上游。蛋白激酶C(PKC)的抑制阻断了钙-e刺激的PLC-、Gamma1酪氨酸磷酸化。此外，暴露于百日咳毒素或外源性蛋白激酶A(PKA)激活抑制了PLC-Gamma1酪氨酸磷酸化对Ca-e的反应。结果表明：(A)成骨细胞CaSR以PKC依赖的方式激活PLC-Gamma1下游的PLC-Gamma1；(B)PKA是Ca-e促进的PLC-Gamma1磷酸化的负调节因子；(C)GQ和GI均参与CaSR介导的PLC-Gamma1的磷酸化。(C)2002年，爱思唯尔科学公司。保留所有权利。
Elevated extracellular calcium (Ca-e) stimulates both chemotaxis and mitogenesis of MC3T3-E1 osteoblasts via a calcium-sensing receptor (CasR). Ca-e-mediated chemotaxis of these bone-forming cells is dependent on phospholipase C (PLC) and blocked by the Gi-protein inhibitor pertussis toxin. In this study, we examine the signaling mechanisms by which the CasR stimulates PLC activity in MC3T3-E I osteoblasts. We found that elevated Ca-e stimulated PLC-gamma1 tyrosine phosphorylation in a time-dependent and Ca-e-concentration-dependent manner. The maximal increase in PLC-gamma1 tyrosine phosphorylation was observed 3-5 min after increasing Ca-e by 3.2 mmol/L from 1.8 mmol/L. Elevated Ca-e also promoted a rapid increase in both inositol 1,4,5-trisphosphate [Ins(1,4,5)P-3], a second messenger formed by PLC-mediated hydrolysis of phosphatidylinositol 4,5-bisphosphate, and cytosolic free calcium ([Ca+2](1)). The kinetics of the CasR-mediated increases in Ins(1,4,5)P-3 and [Ca+2](i) and the sensitivity of the Ca-e-stimulated elevation in [Ca+2](i) to U73122 (a PLC inhibitor) together suggest that the osteoblast CasR is coupled via Gq to PLC-beta. U73122 blocked the Ca-e-promoted, but not PDGF-promoted, PLC-gamma1 tyrosine phosphorylation, suggesting that the activation of PLC-beta is upstream of PLC-gamma1 activation. Inhibition of protein kinase C (PKC) disrupted Ca-e-stimulated tyrosine phosphorylation of PLC-,gamma1. In addition, exposure to pertussis toxin or exogenous activation of protein kinase A (PKA) inhibited PLC-gamma1 tyrosine phosphorylation in response to Ca-e. The results indicate that: (a) the osteoblast CasR activates PLC-gamma1 downstream of PLC-beta in a PKC-dependent manner; (b) PKA is a negative regulator of Ca-e-promoted PLC-gamma1 phosphorylation; and (c) Gq and Gi are both involved in the CasR-mediated phosphorylation of PLC-gamma1. (C) 2002 by Elsevier Science Inc. All rights reserved.