From the Department of Medicine, McGill University Health Centre, Montreal, Quebec H3A 2B4, Canada and.
J Biol Chem. 2014 Feb 14;289(7):4206-18. doi: 10.1074/jbc.M113.506816. Epub 2013 Dec 19.
Visceral glomerular epithelial cells (GEC), also known as podocytes, are vital for the structural and functional integrity of the glomerulus. The actin cytoskeleton plays a central role in maintaining GEC morphology. In a rat model of experimental membranous nephropathy (passive Heymann nephritis (PHN)), complement C5b-9-induced proteinuria was associated with the activation of the actin regulator small GTPase, RhoA. The mechanisms of RhoA activation, however, remained unknown. In this study, we explored the role of the epithelial guanine nucleotide exchange factor, GEF-H1, in complement-induced RhoA activation. Using affinity precipitation to monitor GEF activity, we found that GEF-H1 was activated in glomeruli isolated from rats with PHN. Complement C5b-9 also induced parallel activation of GEF-H1 and RhoA in cultured GEC. In GEC in which GEF-H1 was knocked down, both basal and complement-induced RhoA activity was reduced. On the other hand, GEF-H1 knockdown augmented complement-mediated cytolysis, suggesting a role for GEF-H1 and RhoA in protecting GEC from cell death. The MEK1/2 inhibitor, U0126, and mutation of the ERK-dependent phosphorylation site (T678A) prevented complement-induced GEF-H1 activation, indicating a role for the ERK pathway. Further, complement induced GEF-H1 and microtubule accumulation in the perinuclear region. However, both the perinuclear accumulation and the activation of GEF-H1 were independent of microtubules and myosin-mediated contractility, as shown using drugs that interfere with microtubule dynamics and myosin II activity. In summary, we have identified complement-induced ERK-dependent GEF-H1 activation as the upstream mechanism of RhoA stimulation, and this pathway has a protective role against cell death.
内脏肾小球上皮细胞（GEC），也称为足细胞，对肾小球的结构和功能完整性至关重要。肌动蛋白细胞骨架在维持 GEC 形态中起着核心作用。在实验性膜性肾病（被动 Heymann 肾炎（PHN））的大鼠模型中，补体 C5b-9 诱导的蛋白尿与肌动蛋白调节剂小 GTP 酶 RhoA 的激活有关。然而，RhoA 激活的机制尚不清楚。在这项研究中，我们探讨了上皮细胞鸟嘌呤核苷酸交换因子 GEF-H1 在补体诱导的 RhoA 激活中的作用。使用亲和沉淀监测 GEF 活性，我们发现 PHN 大鼠肾小球中 GEF-H1 被激活。补体 C5b-9 还诱导培养的 GEC 中 GEF-H1 和 RhoA 的平行激活。在 GEF-H1 被敲低的 GEC 中，基础和补体诱导的 RhoA 活性均降低。另一方面，GEF-H1 敲低增强了补体介导的细胞溶解，表明 GEF-H1 和 RhoA 在保护 GEC 免受细胞死亡方面发挥作用。MEK1/2 抑制剂 U0126 和 ERK 依赖性磷酸化位点（T678A）的突变阻止了补体诱导的 GEF-H1 激活，表明 ERK 途径的作用。此外，补体诱导 GEF-H1 和微管在核周区的积累。然而，GEF-H1 的核周积累和激活都与微管和肌球蛋白介导的收缩性无关，如使用干扰微管动力学和肌球蛋白 II 活性的药物所示。总之，我们已经确定补体诱导的 ERK 依赖性 GEF-H1 激活是 RhoA 刺激的上游机制，该途径在细胞死亡中具有保护作用。
