Zhao Xuan, Khurana Simran, Charkraborty Sharmistha, Tian Yuqian, Sedor John R, Bruggman Leslie A, Kao Hung-Ying
From the Department of Biochemistry, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106.
Rammelkamp Center for Education and Research and Department of Medicine, MetroHealth Medical Center, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106.
J Biol Chem. 2017 Feb 3;292(5):1637-1647. doi: 10.1074/jbc.M116.755546. Epub 2016 Dec 20.
Glucocorticoids are a general class of steroids that possess renoprotective activity in glomeruli through their interaction with the glucocorticoid receptor. However, the mechanisms by which glucocorticoids ameliorate proteinuria and glomerular disease are not well understood. In this study, we demonstrated that α actinin 4 (ACTN4), an actin-cross-linking protein known to coordinate cytoskeletal organization, interacts with the glucocorticoid receptor (GR) in the nucleus of human podocytes (HPCs), a key cell type in the glomerulus critical for kidney filtration function. The GR-ACTN4 complex enhances glucocorticoid response element (GRE)-driven reporter activity. Stable knockdown of ACTN4 by shRNA in HPCs significantly reduces dexamethasone-mediated induction of GR target genes and GRE-driven reporter activity without disrupting dexamethasone-induced nuclear translocation of GR. Synonymous mutations or protein expression losses in ACTN4 are associated with kidney diseases, including focal segmental glomerulosclerosis, characterized by proteinuria and podocyte injury. We found that focal segmental glomerulosclerosis-linked ACTN4 mutants lose their ability to bind liganded GR and support GRE-mediated transcriptional activity. Mechanistically, GR and ACTN4 interact in the nucleus of HPCs. Furthermore, disruption of the LXXLL nuclear receptor-interacting motif present in ACTN4 results in reduced GR interaction and dexamethasone-mediated transactivation of a GRE reporter while still maintaining its actin-binding activity. In contrast, an ACTN4 isoform, ACTN4 (Iso), that loses its actin-binding domain is still capable of potentiating a GRE reporter. Dexamethasone induces the recruitment of ACTN4 and GR to putative GREs in dexamethasone-transactivated promoters, SERPINE1, ANGPLT4, CCL20, and SAA1 as well as the NF-κB (p65) binding sites on GR-transrepressed promoters such as IL-1β, IL-6, and IL-8 Taken together, our data establish ACTN4 as a transcriptional co-regulator that modulates both dexamethasone-transactivated and -transrepressed genes in podocytes.
糖皮质激素是一类常见的类固醇，通过与糖皮质激素受体相互作用，在肾小球中具有肾脏保护活性。然而，糖皮质激素改善蛋白尿和肾小球疾病的机制尚未完全明确。在本研究中，我们证明了α辅肌动蛋白4（ACTN4），一种已知可协调细胞骨架组织的肌动蛋白交联蛋白，在人足细胞（HPCs）的细胞核中与糖皮质激素受体（GR）相互作用，人足细胞是肾小球中对肾脏滤过功能至关重要的关键细胞类型。GR-ACTN4复合物增强糖皮质激素反应元件（GRE）驱动的报告基因活性。通过shRNA在HPCs中稳定敲低ACTN4可显著降低地塞米松介导的GR靶基因诱导和GRE驱动的报告基因活性，而不会破坏地塞米松诱导的GR核转位。ACTN4中的同义突变或蛋白表达缺失与肾脏疾病相关，包括以蛋白尿和足细胞损伤为特征的局灶节段性肾小球硬化症。我们发现，与局灶节段性肾小球硬化症相关的ACTN4突变体失去了结合配体GR和支持GRE介导的转录活性的能力。从机制上讲，GR和ACTN4在HPCs的细胞核中相互作用。此外，ACTN4中存在的LXXLL核受体相互作用基序的破坏导致GR相互作用减少以及GRE报告基因的地塞米松介导的反式激活减少，同时仍保持其肌动蛋白结合活性。相反，一种失去其肌动蛋白结合结构域的ACTN4异构体ACTN4（Iso）仍然能够增强GRE报告基因。地塞米松诱导ACTN4和GR募集到地塞米松反式激活启动子（如SERPINE1、ANGPLT4、CCL20和SAA1）中的假定GRE以及GR反式抑制启动子（如IL-1β、IL-6和IL-8）上的NF-κB（p65）结合位点。综上所述，我们的数据确定ACTN4是一种转录共调节因子，可调节足细胞中地塞米松反式激活和反式抑制的基因。
