Sarközi Rita, Miller Bradley, Pollack Verena, Feifel Elisabeth, Mayer Gert, Sorokin Andrey, Schramek Herbert
Clinical Division of Nephrology, Department of Internal Medicine, Medical University of Innsbruck, Innsbruck, Austria.
J Cell Physiol. 2007 Apr;211(1):88-100. doi: 10.1002/jcp.20909.
The MEK1-ERK1/2 signaling pathway has been implicated in the regulation of renal epithelial cell proliferation, epithelial-to-mesenchymal transition and the induction of an invasive cell phenotype. Much less information is available about the MEK5-ERK5 module and its role in renal epithelial cell proliferation and differentiation. In the present study we have investigated the regulation of these two families of extracellular signal-regulated kinases in epidermal growth factor (EGF)-stimulated human kidney-2 (HK-2) cells and a possible interaction between ERK1/2 and ERK5. Here we report that 5 ng/ml EGF led to a strong stimulation of HK-2 cell proliferation, which was largely U0126-sensitive. Both synthetic MEK1/2 inhibitors U0126 and Cl-1040, when used at 10 and 1 microM, respectively, inhibited basal and EGF-induced ERK1/2 phosphorylation but not ERK5 phosphorylation. Long-term inhibition of MEK1/2-ERK1/2 signaling and/or vanadate-sensitive protein phosphatases enhanced and prolonged EGF-induced ERK5 phosphorylation, while transient expression of an adenoviral constitutively active MEK1 (Ad-caMEK1) construct completely blocked EGF-induced ERK5 phosphorylation. Expression of Ad-caMEK1 in HK-2 cells resulted in the upregulation of the dual-specificity phosphatases MKP-3/DUSP6, MKP-1/DUSP1, and DUSP5. The EGF-mediated time-dependent induction of MKP-3, MKP-1 and DUSP5 mRNA levels was U0126-sensitive at a concentration, which blocked EGF-mediated ERK1/2 phosphorylation but not ERK5 phosphorylation. Furthermore, U0126 inhibited EGF-induced MKP-3 and MKP-1 protein expression. Both MKP-3 and MKP-1 co-immunoprecipitated with ERK5 in unstimulated as well as in EGF-stimulated HK-2 cells. These results suggest the existence of an ERK1/2-driven negative feed-back regulation of ERK5 signaling in EGF-stimulated HK-2 cells, which is mediated by MKP-3, DUSP5 and/or MKP-1.
MEK1-ERK1/2信号通路与肾上皮细胞增殖、上皮-间质转化及侵袭性细胞表型的诱导调控有关。关于MEK5-ERK5模块及其在肾上皮细胞增殖和分化中的作用,目前所知甚少。在本研究中,我们调查了这两个细胞外信号调节激酶家族在表皮生长因子(EGF)刺激的人肾-2(HK-2)细胞中的调控情况以及ERK1/2与ERK5之间可能存在的相互作用。在此我们报告,5 ng/ml EGF可强烈刺激HK-2细胞增殖,这在很大程度上对U0126敏感。两种合成的MEK1/2抑制剂U0126和Cl-1040,分别以10和1 μM的浓度使用时,可抑制基础状态及EGF诱导的ERK1/2磷酸化,但不抑制ERK5磷酸化。长期抑制MEK1/2-ERK1/2信号通路和/或钒酸盐敏感蛋白磷酸酶可增强并延长EGF诱导的ERK5磷酸化,而腺病毒组成型活性MEK1(Ad-caMEK1)构建体的瞬时表达则完全阻断EGF诱导的ERK5磷酸化。在HK-2细胞中表达Ad-caMEK1会导致双特异性磷酸酶MKP-3/DUSP6、MKP-1/DUSP1和DUSP5上调。EGF介导的MKP-3、MKP-1和DUSP5 mRNA水平的时间依赖性诱导在一定浓度下对U0126敏感,该浓度可阻断EGF介导的ERK1/2磷酸化,但不阻断ERK5磷酸化。此外,U0126抑制EGF诱导的MKP-3和MKP-1蛋白表达。在未刺激及EGF刺激的HK-2细胞中,MKP-3和MKP-1均与ERK5共免疫沉淀。这些结果表明,在EGF刺激的HK-2细胞中存在由ERK1/2驱动的ERK5信号负反馈调节,其由MKP-3、DUSP5和/或MKP-1介导。