Involvement of intermediary metabolites in the pathway of extracellular Ca2+-induced mitogen-activated protein kinase activation in human fibroblasts

Human fibroblasts in culture will grow in serum-free medium containing seru m replacement factors, but without protein growth factors, as long as the C a2+ level is 1.0-2.0 mM. When the Ca2+ is reduced to 0.1 mM, the cells stop cycling, but they can he reinduced to cycle by raising the Ca2+ level to 1 .0 mM Ca2+ or ti, higher concentrations that result in activation of mitoge n-activated protein kinase (MAPK). (1,4,5) trisphosphate in the cytoplasm a nd caused a transient rise in the concentration of intracellular free Ca2+. Ca2+ induced MAPK activation was partly abolished by treatment of the cell s with pertussis toxin. It was also decreased by treatment of cells with th apsigargin, which depletes intracellular Ca2+ stores; with phorbol 12-myris tyl 13-acetate (PMA), which down-regulates protein kinase C (PKC); with the calmodulin antagonists N-(:6-aminohexyl) 5-chloro-1-naphthalenesulphonamid e HCL (W-7), and calmidazolium (24571); as well as with lanthanum, a Ca2+ c hannel inhibitor. Ca2+ stimulation did not result in phosphorylation of the c-raf-l protein. Our results suggest that extracellular Ca2+ stimulates MA PK activation through a pathway(s) involving a pertussis toxin-sensitive G protein, phospholipase C, intracellular free Ca2+, calmodulin, and PKC. (C) 1999 Elsevier Science Inc.