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Differential dose-dependent effects of epidermal growth factor on gene expression in A431 cells: evidence for a
M Silvy1, P M Martin, N Chajry
1Laboratoire Interactions Cellulaires Intratumorales, CJF INSERM 9311, IFR Jean Roche, Faculté de Médecine Secteur Nord, Marseille, France.
Abstract:
Epidermal growth factor (EGF), which plays an important role in normal and tumoral cell growth regulation, displays an ambivalent dose-dependent effect on the proliferation of epithelial cells overexpressing EGF receptor. However, the underlying molecular mechanisms remain obscure. In this study we have examined the regulation of amphiregulin (AR) gene expression by growth inhibitory (10(-9) M) and stimulatory (10(-12) M) EGF concentrations in A431 cells. The time course of AR messenger RNA (mRNA) accumulation was different with 10(-12) and 10(-9) M EGF; AR induction by 10(-9) M EGF peaked between 1 and 1.5 h, then decreased to the basal level 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 the ability of the tyrosine phosphatase inhibitor sodium orthovanadate to prolong AR expression induced by 10(-12) or 10(-9) M EGF. In the presence 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 low EGF concentration, suggesting that both EGF concentrations activated 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. Thus, 10(-12) M EGF induced a sustained MAP kinase activation, whereas 10(-9) M EGF led to a sharp, but transitory, activation. The MAP kinases are activated by MAP kinase kinases (MEK1 and MEK2). Whereas no significant effect of 10(-12) M EGF could be detected, 10(-9) M EGF was shown to activate MEK1 and, to a lesser extent, MEK2. Also, both MAP kinase activation and AR induction by 10(-9) M, but not by 10(-12) M, EGF were inhibited by the MEK1 inhibitor PD98059. Moreover, the involvement of c-Raf-1 in the signaling pathway induced by EGF was verified. A concentration of 10(-9) M EGF induced stimulation of c-Raf-1 kinase activity, whereas 10(-12) M EGF not only failed to activate c-Raf-1, but led to a moderate decrease in its kinase activity. These results demonstrate that in EGF receptor-overexpressing cells, EGF may differently affect gene expression and cell proliferation through distinct mechanisms of regulation.
Insights
Epidermal growth factor (EGF) exhibits dual effects on epithelial cell proliferation, with distinct molecular mechanisms. Low EGF concentrations stimulate amphiregulin (AR) gene expression via sustained pathways, while high concentrations cause transient AR induction through different signaling routes.
Area of Science:
- Molecular and Cellular Biology
- Cancer Research
- Signal Transduction
Background:
- Epidermal growth factor (EGF) regulates normal and tumoral cell growth, but its dose-dependent effects on epithelial cells overexpressing the EGF receptor are complex.
- The precise molecular mechanisms underlying EGF's ambivalent, dose-dependent impact on cell proliferation and gene expression remain incompletely understood.
Purpose of the Study:
- To investigate the differential regulation of amphiregulin (AR) gene expression by low (10(-12) M) and high (10(-9) M) concentrations of EGF in A431 cells.
- To elucidate the distinct intracellular signaling pathways activated by varying EGF concentrations that lead to differential cellular responses.
Main Methods:
- Quantification of amphiregulin messenger RNA (mRNA) accumulation over time following EGF stimulation.
- Assessment of tyrosine phosphorylation and phosphatase activity using inhibitors like sodium orthovanadate and okadaic acid.
- Analysis of mitogen-activated protein (MAP) kinase pathway activation, including MEK1/2 and c-Raf-1 kinase activity, using specific inhibitors like PD98059.
Main Results:
- Low EGF (10(-12) M) induced a sustained AR mRNA expression for 4 hours, whereas high EGF (10(-9) M) caused a transient induction peaking at 1-1.5 hours.
- Distinct regulatory mechanisms were suggested by differential responses to phosphatase inhibitors; okadaic acid prolonged AR mRNA accumulation with high EGF but abrogated it with low EGF.
- Low EGF promoted sustained p42-MAP kinase activation, while high EGF induced transient activation, MEK1/2 activation, and c-Raf-1 stimulation, all inhibited by PD98059.
Conclusions:
- EGF exerts ambivalent, dose-dependent effects on AR gene expression and cell proliferation in EGF receptor-overexpressing cells.
- Distinct signaling pathways, involving differential MAP kinase and c-Raf-1 activation, mediate the contrasting cellular responses to low and high EGF concentrations.
- Understanding these distinct regulatory mechanisms is crucial for comprehending EGF's role in both normal physiology and tumoral processes.