FORSKOLIN INHIBITS PROTEIN-KINASE C-INDUCED MITOGEN-ACTIVATED PROTEIN-KINASE ACTIVITY IN MC3T3-E1 OSTEOBLASTS

We recently demonstrated that stimulation of DNA synthesis in MC3T3-E1 osteoblasts involves cross-talk between protein kinase C (PKC)-depend ent pathways and activation of possible nonreceptor tyrosine kinases. In the current investigation we examined whether the Raf-1/MAP kinase kinase (MKK)/mitogen-activated protein kinase (MAPK) cascade integrate s cross-talk between G protein-coupled second messengers and protein t yrosine phosphorylation in osteoblasts. We investigated the effects on DNA synthesis, protein tyrosine phosphorylation, and Raf-1, MKK, and MAPK activities of PKC activation by phorbol 12-myristate 13-acetate ( PMA) and of cAMP elevation by forskolin (FSK) in MC3T3-E1 osteoblasts. We found that PMA-stimulated DNA synthesis was associated with increm ents in tyrosine phosphorylation of p44(mapk) (ERK1) and p42(mapk) (ER K2) and activation of Raf-1, MKK, and MAPK in these cells. FSK treatme nt of osteoblasts, which raised intracellular cAMP levels and inhibite d DNA synthesis, blocked PKC-stimulated tyrosine phosphorylation of p4 4(mapk) (ERK1) and p42(mapk) (ERK2) as well as inhibited PKC-stimulate d MAPK and Raf-1 activities. Despite this, PMA activated the intermedi ate MKK step of the Raf-1/MKK/MAPK cascade in the presence of FSK. The differential inhibition of PMA-stimulated Raf-1 and MKK activities by FSK suggests that PKC activates both Raf-1-dependent and -independent pathways in MC3T3-E1 osteoblasts. Moreover, the noncoordinate effects of FSK on PMA-stimulated MKK and MAPK activities indicates the presen ce of additional distal cAMP-dependent inhibitory mechanisms.