Silvy M, Martin P M, Chajry N, Berthois Y
Laboratoire Interactions Cellulaires Intratumorales, CJF INSERM 9311, IFR Jean Roche, Faculté de Médecine Secteur Nord, Marseille, France.
Endocrinology. 1998 May;139(5):2382-91. doi: 10.1210/endo.139.5.5981.
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.
表皮生长因子（EGF）在正常和肿瘤细胞生长调节中发挥重要作用，对过表达EGF受体的上皮细胞增殖呈现矛盾的剂量依赖性效应。然而，其潜在的分子机制仍不清楚。在本研究中，我们检测了生长抑制浓度（10⁻⁹ M）和刺激浓度（10⁻¹² M）的EGF对A431细胞中双调蛋白（AR）基因表达的调节。AR信使核糖核酸（mRNA）积累的时间进程在10⁻¹² M和10⁻⁹ M EGF作用下有所不同；10⁻⁹ M EGF诱导的AR在1至1.5小时达到峰值，然后在2小时内降至基础水平。相反，10⁻¹² M EGF诱导的AR延迟出现，但持续4小时。酪氨酸磷酸酶抑制剂原钒酸钠能够延长10⁻¹²或10⁻⁹ M EGF诱导的AR表达，提示酪氨酸磷酸化参与了EGF诱导的AR表达。在蛋白磷酸酶2A抑制剂冈田酸存在的情况下，10⁻⁹ M EGF诱导AR mRNA持续积累。相反，冈田酸消除了低浓度EGF诱导的AR mRNA水平的升高，表明两种EGF浓度激活了不同的调节机制。随后检测了A431细胞中EGF不同活性所涉及的信号成分。我们之前报道了EGF的矛盾活性与p42-丝裂原活化蛋白（MAP）激酶活性之间的关系。因此，10⁻¹² M EGF诱导MAP激酶持续激活，而10⁻⁹ M EGF导致MAP激酶迅速但短暂的激活。MAP激酶由MAP激酶激酶（MEK1和MEK2）激活。虽然未检测到10⁻¹² M EGF有显著作用，但10⁻⁹ M EGF可激活MEK1，并在较小程度上激活MEK2。此外，MEK1抑制剂PD98059可抑制10⁻⁹ M EGF诱导的MAP激酶激活和AR诱导，但不影响10⁻¹² M EGF。此外，还验证了c-Raf-1参与EGF诱导的信号通路。10⁻⁹ M EGF浓度可诱导c-Raf-1激酶活性增强，而10⁻¹² M EGF不仅未能激活c-Raf-1，反而导致其激酶活性适度降低。这些结果表明，在过表达EGF受体的细胞中，EGF可能通过不同的调节机制对基因表达和细胞增殖产生不同影响。
