DISSOCIATION OF CAMP-STIMULATED MITOGENESIS FROM ACTIVATION OF THE MITOGEN-ACTIVATED PROTEIN-KINASE CASCADE IN SWISS 3T3 CELLS

Elevation of intracellular cAMP by forskolin, 8-bromoadenosine 3':5'-cyclic monophosphate, and prostaglandin E(1), in synergy with insulin, stimulated DNA synthesis in quiescent Swiss 3T3 cells to the same level achieved by platelet-derived growth factor (PDGF) or bombesin. Both forskolin and 8-bromoadenosine 3':5'-cyclic monophosphate stimulated a significant increase in cell number which, in the presence of insulin, reached the same level achieved with PDGF. Treatment with either PDGF or bombesin caused a marked and persistent stimulation of p42(MAPK) and p44(MAPK). In striking contrast, no activation was seen with mitogenic combinations of cAMP as shown by three different assays. Swiss 3T3 cells stably transfected with a constitutively activated Gs alpha subunit were 100-fold more sensitive to the mitogenic effects of forskolin but in this distinct cellular model forskolin did not activate p42(MAPK). Swiss 3T3 cells stably transfected with interfering mutants of MEK-1 showed a 60% decrease in PDGF-stimulated p42 MAPK activation, but there was no inhibition of the mitogenic effect of forskolin in these cells. Furthermore, the upstream kinases MEK-1/MEK-2 and p74(raf-1) were not activated by mitogenic combinations of cAMP while PDGF caused marked stimulation of their activity. Treatment of 3T3 cells with forskolin attenuated PDGF-stimulated p74(raf-1) and p42(MAPK) activation but enhanced the mitogenic effects of this agent. Mitogenic combinations of cAMP strongly stimulated the phosphorylation and activation of p70(s6k) an effect that was inhibited by rapamycin. This agent markedly inhibited cAMP-stimulated DNA synthesis suggesting a critical role for p70(s6k) in cAMP mitogenic signaling. These results demonstrate that cAMP-induced mitogenesis can be dissociated from activation of the mitogen-activated protein kinase cascade and that this is not an obligatory point of convergence in mitogenic signaling in Swiss 3T3 cells.