DIFFERENTIAL DOSE-DEPENDENT EFFECTS OF EPIDERMAL GROWTH-FACTOR ON GENE-EXPRESSION IN A431 CELLS - EVIDENCE FOR A SIGNAL-TRANSDUCTION PATHWAY THAT CAN BYPASS RAF-1 ACTIVATION

Epidermal growth factor (EGF), which plays an important role in normal and tumoral cell growth regulation, displays an ambivalent dose-depen dent effect on the proliferation of epithelial cells overexpressing EG F receptor. However, the underlying molecular mechanisms remain obscur e. In this study we have examined the regulation of amphiregulin (AR) gene expression by growth inhibitory (10(-9) M) and stimulatory (10(-1 2) M) EGF concentrations in A431 cells. The time course of AR messenge r RNA (mRNA) accumulation was different with 10(-12) and 10(-9) RI EGF ; AR induction by 10(-9) hz EGF peaked between I and 1.5 h, then decre ased to the basal Ies el within 2 h. Conversely, the induction by 10(- 12) M EGF was slightly delayed, but persisted for 4 h. The involvement of tyrosine phosphorylation in AR induction by EGF was suggested by t he ability of the tyrosine phosphatase inhibitor sodium orthovanadate to prolong AR expression induced by 10(-12) or 10(-9) hr EGF. In the p resence of the protein phosphatase 2A inhibitor, okadaic acid, 10(-9) M EGF induced a persistent accumulation of AR. mRNA. On the contrary, okadaic acid abrogated the stimulation of AR mRNA level induced by a l ow EGF concentration, suggesting that both EGF concentrations activate d distinct regulatory mechanisms. The signaling components involved in the differential activities of EGF in A431 cells were then examined. We previously reported a relationship between the ambivalent activity of EGF and the p42-mitogen-activated protein (MAP) kinase activity. Th us, 10(-12) M EGF induced a sustained MAP kinase activation, whereas 1 0(-9) hi EGF led to a sharp, but transitory, activation. The MAP kinas es are activated by MAP kinase kinases (MEK1 and MEK2). Whereas no sig nificant effect of 10(-12) BI EGF could be detected, 10(-9) hi EGF was shown to activate MEK1 and, to a lesser extent, MEK2. Also, both MAP kinase activation and AR induction bg 10(-9) nl, but not by 10(-12) M, EGF were inhibited by the MEK1 inhibitor PD98059. Moreover, the invol vement of c-Raf-l in the signaling pathway induced by EGF was verified . A concentration of 10(-9) M EGF induced stimulation of c-Raf-l kinas e activity, whereas 10(-12) M EGF not only failed to activate c-Raf-l, but led to a moderate decrease in its kinase activity. These results demonstrate that in EGF receptor-overexpressing cells, EGF may differe ntly affect gene expression and cell proliferation through distinct me chanisms of regulation.