Activation of p42/44 and p38 mitogen-activated protein kinases by extracellular calcium-sensing receptor agonists induces mitogenic responses in the mouse osteoblastic MC3T3-E1 cell line

Recently, substantial evidence has accumulated that the G-protein-coupled, extracellular calcium (Ca-o(2+))-sensing receptor (CaR) is expressed in bon e marrow-derived cells, including osteoblasts, stromal cells, monocytes-mac rophages, and osteoclast precursor cells. Our previous studies have shown t hat the mouse osteoblastic MC3T3-E1 cell line also expresses the CaR and ex hibits mitogenic responses when exposed to various CaR agonists. In this st udy, in order to understand the signaling pathway(s) mediating this respons e, we studied the effects of CaR agonists on the phosphorylation of p42/44 mitogen-activated protein kinase (MAPK) (Erk1/2), p38 MAPK, and c-Jun N-ter minal kinase (JNK) in MC3T3-E1 cells. Raising the level of Ca-o(2+) (4.5 mM ) or addition of the polycationic CaR agonists, gadolinium (Gd3+) (25 muM), neomycin (300 muM) or spermine (1 mM), each stimulated phosphorylation of both p42/44 and p38 MAPKs, but not JNK, as assessed using phospho-specific antibodies to the respective MAPKs. Furthermore, phosphorylation of p42/44 and p38 MAPK were markedly inhibited by their selective and potent inhibito rs, PD98059 (50 muM) and SB203580 (10 muM), respectively. Finally, the two inhibitors suppressed [H-3]thymidine incorporation into DNA in MC3T3-E1 cel ls at a normal level of Ca-o(2+) (1.8 mM) as well as when stimulated by hig h (4.5 mM) Ca-o(2+), Gd3+, or neomycin. Thus, in mouse osteoblastic MC3T3-E 1 cells, both the p42/44 and p38 MAPK cascades play pivotal roles in CaR-st imulated mitogenic responses. (C) 2000 Academic Press.