Liu Fei, Ma Xiao-Jun, Wang Qing-Zhu, Zhao Yan-Yan, Wu Li-Na, Qin Gui-Jun
Department of Endocrinology and Metabolism, The First Affiliated Hospital of Zhengzhou University, Zhengzhou 450052, China.
Nephrol Dial Transplant. 2014 Oct;29(10):1879-87. doi: 10.1093/ndt/gfu202. Epub 2014 Jun 8.
Hyperproliferation of glomerular mesangial cells (MCs) is a major pathological characteristic in the early stage of diabetic nephropathy (DN). We have previously confirmed that forkhead transcription factor O1 (FoxO1) was significantly downregulated in both the renal cortex of DN rats and MCs cultured under high-glucose (HG) conditions, but the effects and mechanisms of FoxO1 involved in the hyperproliferation of MCs are still unclear. This study aims to investigate whether FoxO1 regulates the hyperproliferation of MCs induced under high-glucose conditions, through modulating the cyclin-dependent kinase inhibitor (CKI), p27.
Lentiviral vectors of LV-constitutively active FoxO1 (CA-FoxO1) and LV-small interfering RNA (siRNA)-FoxO1 were constructed to up- and downregulate FoxO1. Similarly, LV-NC-FoxO1 was used as negative control (NC). Rat MCs were cultured in normal glucose (5.6 mM) medium, HG (30 mM) medium, HG with LV-NC-FoxO1, HG with LV-CA-FoxO1 and HG with LV-siRNA-FoxO1 for 72 h. Cell proliferation, cell cycle progression, messenger RNA and protein expression of FoxO1, p27, cyclin D1 and CDK4 were detected by methyl thiazolyl tetrazolium assay, flow cytometry, quantitative real-time polymerase chain reaction and western blotting, respectively.
MCs exposed to HG medium triggered hyperproliferation of MCs. Nevertheless, overexpression of FoxO1 caused by LV-CA-FoxO1 promoted cell cycle arrest at the G0/G1 phase and attenuated proliferation, which was associated with upregulation of p27 and downregulation of cyclin D1 and CDK4. Moreover, specific degradation of FoxO1 by LV-siRNA-FoxO1 caused a decrease of p27, increase of cyclin D1 and CDK4, overrode the limited cell cycle and stimulated proliferation of MCs.
Overexpression of FoxO1 caused upregulation of p27, which promoted cell cycle arrest and inhibited hyperproliferation of MCs induced by HG. Degradation of FoxO1 caused an increase in p27 and stimulated MC proliferation. These findings unveil part of the molecular mechanism of FoxO1 regulation of MC hyperproliferation induced by HG.
肾小球系膜细胞（MCs）过度增殖是糖尿病肾病（DN）早期的主要病理特征。我们之前已经证实，在DN大鼠的肾皮质以及在高糖（HG）条件下培养的MCs中，叉头转录因子O1（FoxO1）均显著下调，但FoxO1参与MCs过度增殖的作用及机制仍不清楚。本研究旨在探讨FoxO1是否通过调节细胞周期蛋白依赖性激酶抑制剂（CKI）p27来调控高糖条件下诱导的MCs过度增殖。
构建LV-组成型激活FoxO1（CA-FoxO1）和LV-小干扰RNA（siRNA）-FoxO1慢病毒载体，分别上调和下调FoxO1。同样，LV-NC-FoxO1用作阴性对照（NC）。将大鼠MCs在正常葡萄糖（5.6 mM）培养基、HG（30 mM）培养基、含LV-NC-FoxO1的HG培养基、含LV-CA-FoxO1的HG培养基和含LV-siRNA-FoxO1的HG培养基中培养72小时。分别通过甲基噻唑基四氮唑法、流式细胞术、定量实时聚合酶链反应和蛋白质印迹法检测细胞增殖、细胞周期进程、FoxO1、p27、细胞周期蛋白D1和细胞周期蛋白依赖性激酶4（CDK4）的信使核糖核酸和蛋白质表达。
暴露于HG培养基的MCs引发了MCs的过度增殖。然而，LV-CA-FoxO1导致的FoxO1过表达促进细胞周期停滞在G0/G1期并减弱增殖，这与p27上调以及细胞周期蛋白D1和CDK4下调有关。此外，LV-siRNA-FoxO1导致的FoxO1特异性降解导致p27减少、细胞周期蛋白D1和CDK4增加，克服了有限的细胞周期并刺激了MCs的增殖。
FoxO1过表达导致p27上调，从而促进细胞周期停滞并抑制HG诱导的MCs过度增殖。FoxO1降解导致p27增加并刺激MCs增殖。这些发现揭示了FoxO1调控HG诱导的MCs过度增殖的部分分子机制。
