Calcium-sensing receptor-mediated activation of phospholipase C-γ1 is downstream of phospholipase C-β and protein kinase C in MC3T3-E1 osteoblasts

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.