Regulation of mitogen-activated protein kinase phosphatase-1 expression by extracellular signal-related kinase-dependent and Ca2+-dependent signal pathways in rat-1 cells

Stimulation of Rat-1 cells with lysophosphatidic acid (LPA) or epidermal growth factor (EGF) results in a biphasic, sustained activation of extracellular signal-regulated kinase 1 (ERK1). Pretreatment of Rat-1 cells with either cycloheximide or sodium orthovanadate had Little effect on the early peak of ERK1 activity but potentiated the sustained phase, Cycloheximide also potentiated ERK1 activation in Rat-1 cells expressing Delta Raf-1:ER, an estradiol-regulated form of the oncogenic, human Raf-l, Since cycloheximide did not potentiate MEK activity but abrogated the expression of mitogen-activated protein kinase phosphatase (MKP-1) normally seen in response to EGF and LPA, we speculated that the level of MKP-1 expression may be an important regulator of ERK1 activity in Rat-1 cells, Inhibition of LPA-stimulated MEK and ERR activation with PD98059 and pertussis toxin, a selective inhibitor of G(i)-protein-coupled signaling pathways, reduced LPA-stimulated MKP-1 expression by only 50%, suggesting the presence of additional MEK- and ERR-independent pathways for MKP-1 expression, Specific activation of the MEK/ERK pathway by Delta Raf-1:ER had little or no effect on MKP-1 expression, suggesting that activation of the Raf/MEK/ERK pathway is necessary but not sufficient for MKP-1 expression in Rat-1 cells, Activation of PKC played little part in growth factor-stimulated MKP-1 expression, but LPA- and EGF-induced MKP-1 expression was blocked by buffering [Ca2+](i), leading to a potentiation of the sustained phase of ERK1 activation without potentiating MEK activity, in Rat-1 Delta Raf-1:ER cells, we observed a strong synergy of MKP-1 expression when cells were stimulated with estradiol, in the presence of ionomycin, phorbol la-myristate 13-acetate, or okadaic acid under conditions where these agents did not synergize for ERR activation, These results suggest that activation of the Raf/MEK/ERK pathway is insufficient to induce expression of MKP-1 but instead requires other signals, such as Ca2+, to fully reconstitute the response seen with growth factors. In this way, ERR-dependent and -independent signals may regulate MKP-1 expression, the magnitude of sustained ERK1 activity, and therefore gene expression.