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

Abstract

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-E1 osteoblasts. We found that elevated Ca e stimulated PLC-γ1 tyrosine phosphorylation in a time-dependent and Ca e-concentration-dependent manner. The maximal increase in PLC-γ1 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] i). 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-β. U73122 blocked the Ca e-promoted, but not PDGF-promoted, PLC-γ1 tyrosine phosphorylation, suggesting that the activation of PLC-β is upstream of PLC-γ1 activation. Inhibition of protein kinase C (PKC) disrupted Ca e-stimulated tyrosine phosphorylation of PLC-γ1. In addition, exposure to pertussis toxin or exogenous activation of protein kinase A (PKA) inhibited PLC-γ1 tyrosine phosphorylation in response to Ca e. The results indicate that: (a) the osteoblast CasR activates PLC-γ1 downstream of PLC-β in a PKC-dependent manner; (b) PKA is a negative regulator of Ca e-promoted PLC-γ1 phosphorylation; and (c) Gq and Gi are both involved in the CasR-mediated phosphorylation of PLC-γ1.