Huang Songming, Zhang Aihua, Ding Guixia, Chen Ronghua
Department of Nephrology, Nanjing Children's Hospital, Nanjing Medical Univ., Nanjing, China.
Am J Physiol Renal Physiol. 2009 Jun;296(6):F1323-33. doi: 10.1152/ajprenal.90428.2008. Epub 2009 Apr 1.
Aldosterone (Aldo) stimulates glomerular mesangial cell (MC) proliferation, in part, through an ERK1/2-dependent pathway. In this study, we examined whether Aldo activation of ERK1/2 in MC is mediated through redox-dependent EGF receptor (EGFR) transactivation, as well as the involvement of other signaling mechanisms in Aldo-induced MC proliferation. Aldo increased human MC proliferation, as determined by [(3)H]thymidine incorporation and cell counts. This increase in proliferation was blocked by inhibition of the mineralocorticoid receptor (MR). Continuing our observations downstream in the signaling pathway, we examined the ability of Aldo to activate both the Ras/MAPK and the PI3K signaling pathways. Aldo increased Ki-RasA and Ki-RasA:GTP levels, and sequentially phosphorylated c-Raf, MAPK kinase (MEK1/2), and ERK1/2. Ki-RasA small interfering RNA (siRNA), the c-Raf inhibitor GW5074, and the MEK1/2 inhibitor PD98059 reduced Aldo-induced cell proliferation by approximately 65%. Aldo also increased phosphorylation of PI3K, Akt, the mammalian target of rapamycin (mTOR), and the 70-kDa ribosomal S6 kinase (p70S6K1). Inhibition of the PI3K pathways by the selective PI3K inhibitor LY 294002, an Akt inhibitor, or the mTOR inhibitor rapamycin reduced cell proliferation by 51%. Combining LY 294002 and PD98059 completely blocked Aldo-induced MC proliferation. Next, we confirmed that Aldo exerts its effect on MAPK and PI3K activation, as well as on cell proliferation, by activating the EGFR. Pretreatment with the EGFR antagonist AG1478 inhibited MC proliferation, as well as the activation of Ras/MAPK and PI3K/Akt, suggesting that Ras/MAPK and PI3K/Akt activation occur downstream of EGFR activation. Finally, we examined the role of reactive oxygen species (ROS) in Aldo-induced transactivation of the EGFR. Aldo-induced ROS were predominantly generated by mitochondria. Pretreatment with the antioxidant N-acetyl-l-cysteine, catalase, SOD, mitochondrial respiratory chain complex I inhibitor rotenone (Rot), NADPH oxidase inhibitor apocynin, and DPI significantly inhibited Aldo-stimulated MC proliferation as well as EGFR transactivation. However, Rot reduced MC proliferation more potently than apocynin and DPI. In conclusion, Aldo stimulated cell proliferation through MR-mediated, redox-sensitive EGFR transactivation, which was dependent on the Ki-RasA/c-Raf/MEK/ERK and PI3K/Akt/mTOR/p70S6K1 signaling pathways in human MCs.
醛固酮（Aldo）部分通过细胞外信号调节激酶1/2（ERK1/2）依赖的途径刺激肾小球系膜细胞（MC）增殖。在本研究中，我们检测了醛固酮在MC中对ERK1/2的激活是否通过氧化还原依赖的表皮生长因子受体（EGFR）转活化介导，以及其他信号机制在醛固酮诱导的MC增殖中的作用。通过[³H]胸腺嘧啶核苷掺入和细胞计数测定，醛固酮增加了人MC增殖。这种增殖的增加被盐皮质激素受体（MR）的抑制所阻断。在信号通路中继续我们的观察，我们检测了醛固酮激活Ras/丝裂原活化蛋白激酶（MAPK）和磷脂酰肌醇-3激酶（PI3K）信号通路的能力。醛固酮增加了Ki-RasA和Ki-RasA：GTP水平，并依次磷酸化c-Raf、丝裂原活化蛋白激酶激酶（MEK1/2）和ERK1/2。Ki-RasA小干扰RNA（siRNA）、c-Raf抑制剂GW5074和MEK1/2抑制剂PD98059使醛固酮诱导的细胞增殖减少了约65%。醛固酮还增加了PI3K、蛋白激酶B（Akt）、雷帕霉素靶蛋白（mTOR）和70 kDa核糖体S6激酶（p70S6K1）的磷酸化。选择性PI3K抑制剂LY 294002、Akt抑制剂或mTOR抑制剂雷帕霉素对PI3K通路的抑制使细胞增殖减少了51%。联合使用LY 294002和PD98059完全阻断了醛固酮诱导的MC增殖。接下来，我们证实醛固酮通过激活EGFR对MAPK和PI3K激活以及细胞增殖发挥作用。用EGFR拮抗剂AG1478预处理可抑制MC增殖以及Ras/MAPK和PI3K/Akt的激活，这表明Ras/MAPK和PI3K/Akt激活发生在EGFR激活的下游。最后，我们检测了活性氧（ROS）在醛固酮诱导的EGFR转活化中的作用。醛固酮诱导的ROS主要由线粒体产生。用抗氧化剂N-乙酰-L-半胱氨酸、过氧化氢酶、超氧化物歧化酶（SOD）、线粒体呼吸链复合体I抑制剂鱼藤酮（Rot）、烟酰胺腺嘌呤二核苷酸磷酸（NADPH）氧化酶抑制剂阿朴吗啡和二苯基碘（DPI）预处理可显著抑制醛固酮刺激的MC增殖以及EGFR转活化。然而，Rot比阿朴吗啡和DPI更有效地降低了MC增殖。总之，醛固酮通过MR介导的、氧化还原敏感的EGFR转活化刺激细胞增殖，这依赖于人MC中的Ki-RasA/c-Raf/MEK/ERK和PI3K/Akt/mTOR/p70S6K1信号通路。
