cAMP-mediated growth inhibition in fibroblasts is not mediated via mitogen-activated protein (MAP) kinase (ERK) inhibition - cAMP-dependent protein kinase induces a temporal shift in growth factor-stimulated MAP kinases

Growth factors stimulate fibroblast cell division by activating the recently identified mitogen-activated protein kinase (MAP kinase) signaling cascade. In contrast to our previous work (Kahan, K., Seuwen, K., 13369-13375), several reports have suggested that an elevation in intracellular cAMP blocks cell proliferation by attenuating MAP kinase activation. Hence we re-examined the effect of a long term increase in intracellular cAMP and therefore cAMP-dependent protein kinase (PKA) activation on the MAP kinase cascade in CCL39 fibroblasts. The concomitant addition of cAMP-elevating agents prostaglandin E, (PGE(1)) and IBMX did not inhibit the mitogen-mediated activation of p44 MAP kinase. However, a 5-min PGE(1)/IBMX pretreatment abolished the MAP kinase response, in a manner correlating with the extent of PKA activity. This inhibition was temporal in nature, and while modifying the time course of growth factor-mediated p44 MAP kinase, activation did not diminish the magnitude of the response. Thus the major peak of MAP kinase activity normally present 5 min after alpha-thrombin addition was now evident at 10 min in the presence of PGE(1)/IBMX. CCL39 cell proliferation could reflect either a reduction in the number of cells entering the cell cycle or a delay in the time required to go through the cycle. Bromodeoxyuridine labeling experiments revealed that the cAMP-mediated inhibition of DNA synthesis in CCL39 cell was not due to a delay in S phase entry, but was due to a reduction in the number of cells entering S phase. Thus we conclude that although PKA activation may slightly modify the time course of MAP kinase activation in response to mitogens in CCL39 cells, the PKA-mediated inhibition of cell division occurs through modulation of an intracellular target, distinct from the p42/p44 MAP kinase cascade.