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 serum replacement factors, but without protein growth factors, as long as the Ca2+ level is 1.0-2.0 mM. When the Ca2+ is reduced to 0.1 mM, the cells stop cycling, but they can be reinduced to cycle by raising the Ca2+ level to 1.0 mM Ca2+ or to higher concentrations that result in activation of mitogen-activated protein kinase (MAPK). We now report that exposure of human fibroblasts to extracellular Ca2+ increased the level of inositol (1,4,5)-trisphosphate in the cytoplasm and caused a transient rise in the concentration of intracellular free Ca2+. Ca2+-induced MAPK activation was partly abolished by treatment of the cells with pertussis toxin. It was also decreased by treatment of cells with thapsigargin, which depletes intracellular Ca2+ stores; with phorbol 12-myristyl 13-acetate (PMA), which down-regulates protein kinase C (PKC); with the calmodulin antagonists N-(6-aminohexyl)-5-chloro-1-naphthalenesulphonamide HCl (W-7), and calmidazolium (24571); as well as with lanthanum, a Ca2+ channel inhibitor. Ca2+ stimulation did not result in phosphorylation of the c-raf-1 protein. Our results suggest that extracellular Ca2+ stimulates MAPK activation through a pathway(s) involving a pertussis toxin-sensitive G protein, phospholipase C, intracellular free Ca2+, calmodulin, and PKC.
Huang
S
S
Department of Microbiology, The Cancer Center, Michigan State University, East Lansing 48824-1302, USA.
Maher
V M
VM
McCormick
J
J
D000818:Animals; D002118:Calcium / Q000378:metabolism* / Q000494:pharmacology; D002121:Calcium Channel Blockers / Q000494:pharmacology; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D002147:Calmodulin / Q000037:antagonists & inhibitors; D002455:Cell Division; D002460:Cell Line; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005110:Extracellular Space; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D007424:Intracellular Fluid / Q000378:metabolism; D007527:Isoenzymes / Q000502:physiology; D007811:Lanthanum / Q000494:pharmacology; D051379:Mice; D009543:Nifedipine / Q000494:pharmacology; D037342:Pertussis Toxin; D051966:Phospholipase C gamma; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism; D019908:Proto-Oncogene Proteins c-raf / Q000378:metabolism; D015398:Signal Transduction; D012867:Skin / Q000166:cytology; D013449:Sulfonamides / Q000494:pharmacology; D019284:Thapsigargin / Q000494:pharmacology; D010738:Type C Phospholipases / Q000502:physiology; D014700:Verapamil / Q000494:pharmacology; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D002121:Calcium Channel Blockers; D002147:Calmodulin; D004791:Enzyme Inhibitors; D007527:Isoenzymes; D013449:Sulfonamides; D010566:Virulence Factors, Bordetella; C040184:W 12; C017967:W 7; D019284:Thapsigargin; D007811:Lanthanum; D014700:Verapamil; D037342:Pertussis Toxin; D019908:Proto-Oncogene Proteins c-raf; D011493:Protein Kinase C; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D010738:Type C Phospholipases; D051966:Phospholipase C gamma; D009543:Nifedipine; D002118:Calcium