Abstract
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., Meloche, S. and Pouysségur, J. (1992) J. Biol. Chem. 267, 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, (PGE1) and IBMX did not inhibit the mitogen-mediated activation of p44 MAP kinase. However, a 5-min PGE1/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 PGE1/IBMX. CCL39 cell proliferation is inhibited by elevated cAMP levels. Such an inhibition 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 cells 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.
Highlights
In normal untransformed fibroblast cell lines, it has long been appreciated [2] that a significant elevation of intracellular cAMP levels may potently inhibit cell growth and division
As elevated cAMP levels inhibit cell proliferation mediated by either Gprotein-coupled receptors or receptors with intrinsic tyrosine kinase activity, it was assumed that the target for protein kinase (PKA) was downstream of the initial signaling events and likely to be a central player in the mitogenic response [2]
As we have noted previously, the concomitant addition of an agent such as PGE1, which together with the phosphodiesterase inhibitor, IBMX, provokes a robust increase in the intracellular cAMP levels [1, 30], does not modify the stimulation of p44 MAP kinase elicited by three different mitogens: ␣-thrombin, FGF, or serum in M1-81 cells
Summary
In normal untransformed fibroblast cell lines, it has long been appreciated [2] that a significant elevation of intracellular cAMP levels may potently inhibit cell growth and division. As elevated cAMP levels inhibit cell proliferation mediated by either Gprotein-coupled receptors or receptors with intrinsic tyrosine kinase activity, it was assumed that the target for PKA was downstream of the initial signaling events and likely to be a central player in the mitogenic response [2]. In a widely studied model of growth factor signaling, the CCL39 fibroblast cell line, a 42- and 44-kDa MAP kinase have been identified [7] Both proteins form part of a signaling complex involving the sequential activation of Ras, a MAP kinase kinase kinase (MAPKKK or MEKK), a MAP kinase kinase (MAPKK or MEK), with MAP kinase being the final member of this cascade [6, 8].
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