Prostaglandin F2 alpha-induced mitogenesis in MC3T3-E1 osteoblasts: role of protein kinase-C-mediated tyrosine phosphorylation.
Prostaglandin F2 alpha (PGF2 alpha) stimulates DNA synthesis in osteoblasts through phospholipase-C-dependent increases in intracellular calcium and protein kinase-C (PKC) activity. We present evidence that stimulation of protein tyrosine phosphorylation by PKC is an additional component of the signaling pathways involved in PGF2 alpha-stimulated DNA synthesis in MC3T3-E1 osteoblast-like cells. Mitogenic doses of PGF2 alpha (42 nM) rapidly induced tyrosine phosphorylation of multiple substrates in these osteoblast-like cells. PGF2 alpha stimulated tyrosine phosphorylation of new proteins with apparent mol wt of 87, 80, 50, 47, 36, and 33 kilodaltons and up-regulated phosphorylation of preexisting tyrosine components with mol wt of 123, 112, 68, and 56 kilodaltons. Stimulation of PKC by 1.6 microM phorbol 12-myristate 13-acetate mimicked the pattern of PGF2 alpha-induced protein tyrosine phosphorylation, whereas PKC-deficient cells (induced by overnight pretreatment with 16 microM phorbol 12-myristate 13-acetate) were refractory to PGF2 alpha-stimulated protein tyrosine phosphorylation and DNA synthesis. The tyrosine kinase inhibitors tyrphostin and genistein blocked PGF2 alpha-stimulated DNA synthesis and protein tyrosine phosphorylation, and the tyrosine phosphatase inhibitor orthovanadate prolonged PGF2 alpha-stimulated tyrosine phosphorylation; these findings are consistent with activation of a putative tyrosine kinase. Calcium/calmodulin antagonists also inhibited PGF2 alpha-stimulated DNA synthesis, but the calcium-signaling pathway played no role in PGF2 alpha-induced tyrosine phosphorylation. Our findings suggest that cross-talk between receptor-mediated activation of PKC and protein tyrosine phosphorylation is an important distal signaling pathway necessary for PGF2 alpha-induced DNA synthesis in osteoblast-like cells.
Quarles
L D
LD
Department of Medicine, Duke University Medical Center, Durham, North Carolina 27710.
Haupt
D M
DM
Davidai
G
G
Middleton
J P
JP
D000818:Animals; D002118:Calcium / Q000502:physiology; D002147:Calmodulin / Q000502:physiology; D002460:Cell Line; D004247:DNA / Q000037:antagonists & inhibitors / Q000096:biosynthesis; D015237:Dinoprost / Q000494:pharmacology*; D008938:Mitosis; D010006:Osteoblasts / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism* / Q000502:physiology; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors; D015398:Signal Transduction / Q000502:physiology; D014443:Tyrosine / Q000378:metabolism*
D002147:Calmodulin; D014443:Tyrosine; D004247:DNA; D015237:Dinoprost; D011505:Protein-Tyrosine Kinases; D011493:Protein Kinase C; D002118:Calcium