Prostaglandin-dependent activation of ERK mediates cell proliferation induced by transforming growth factor beta in mouse osteoblastic cells.

Transforming growth factor beta (TGF(beta)) is a major coupling factor for bone turnover and is known to stimulate osteoblastic proliferation. Recent information indicates that, in addition to the Smad pathway, TGF(beta) also activates MAP kinases in osteoblastic cells. The role of these signaling cascades in cell proliferation induced by TGF(beta) as well as the cellular and molecular mechanisms of their activation by TGF(beta) has been investigated in this study. In MC3T3-E1 cells, TGF(beta) enhanced cell proliferation by about 2-fold and induced activation of the three MAP kinases, extracellular regulated kinase (ERK), p38, and c-Jun N-terminal kinase (JNK). Surprisingly, however, whereas activation of Smad2 was rapid and maximal after 15-min incubation, activation of MAP kinases was delayed with p38 stimulation detected after 1-h exposure and activation of ERK and JNK after 3 h, suggesting indirect activation of MAP kinases by TGF(beta). Among factors known to be released in response to TGF(beta) in osteoblastic cells and influence their growth, prostaglandins (PGs) were good candidates that were further investigated for mediating TGF(beta)-induced activation of MAP kinases and cell proliferation. Indomethacin, a selective inhibitor of PG synthesis, completely blunted cell proliferation induced by TGF(beta) and markedly reduced activation of MAP kinases without influencing Smad2 phosphorylation. EP4A, a specific PGE2 receptor antagonist, also blunted TGF(beta)-induced osteoblastic proliferation. In addition to these effects, PGE2 rapidly activated MAP kinases in MC3T3-E1 cells and increased cell proliferation by about 2-fold. The role of each MAP kinases in mediating TGF(beta)- and PGE2-induced cell proliferation was investigated using selective inhibitors. U0126, a specific inhibitor of the ERK pathway, completely blocked both TGF(beta)- and PGE2-induced cell proliferation whereas SB203580 and SP600125, which are selective inhibitors of, respectively, p38 and JNK pathways, had no effect. Finally, the effect of PGE2 on activation of ERK was mimicked by phorbol esters and not by forskolin, and was associated with activation of protein kinase C. This latter effect and the stimulation of ERK induced by PGE2 were completely blocked by a specific inhibitor of PKC. In conclusion, data presented in this study strongly suggest that the local release of PGE2 is involved in cell proliferation induced by TGF(beta) in osteoblastic cells. This effect is mediated by the ERK pathway activated by a PKC-dependent mechanism.