Calcium-sensing receptor-mediated activation of phospholipase C-gamma1 is downstream of phospholipase C-beta 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-E1 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)P3], 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)P3 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.
Godwin
S L
SL
Department of Orthodontics, University of Connecticut Health Center, Farmington, CT 06030, USA. godwin@nso2.uchc.edu
Soltoff
S P
SP
D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002478:Cells, Cultured; D005110:Extracellular Space / Q000378:metabolism; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000378:metabolism; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D020962:Heterotrimeric GTP-Binding Proteins / Q000378:metabolism; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism; D007527:Isoenzymes / Q000378:metabolism*; D051379:Mice; D010006:Osteoblasts / Q000166:cytology / Q000187:drug effects / Q000201:enzymology*; D037342:Pertussis Toxin; D054799:Phospholipase C beta; D051966:Phospholipase C gamma; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism*; D044169:Receptors, Calcium-Sensing; D011956:Receptors, Cell Surface / Q000378:metabolism*; D015290:Second Messenger Systems / Q000502:physiology; D010738:Type C Phospholipases / Q000378:metabolism*; D014443:Tyrosine / Q000378:metabolism; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D007527:Isoenzymes; D044169:Receptors, Calcium-Sensing; D011956:Receptors, Cell Surface; D010566:Virulence Factors, Bordetella; D014443:Tyrosine; D015544:Inositol 1,4,5-Trisphosphate; D037342:Pertussis Toxin; D011493:Protein Kinase C; D010738:Type C Phospholipases; D054799:Phospholipase C beta; D051966:Phospholipase C gamma; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D020962:Heterotrimeric GTP-Binding Proteins; D002118:Calcium