Rupprecht H D, Akagi Y, Keil A, Hofer G
Medizinische Klinik IV, University Erlangen-Nürnberg, Erlangen, Germany.
Kidney Int. 2000 Jan;57(1):70-82. doi: 10.1046/j.1523-1755.2000.00828.x.
Nitric oxide inhibits growth of glomerular mesangial cells: Role of the transcription factor Egr-1.
In previous studies, we found a close link of early growth response gene-1 (Egr-1) expression to mesangial cell (MC) proliferation. Antiproliferative agents inhibited mitogen-induced Egr-1 expression. Here we investigated the effect of S-nitrosoglutathione (GSNO) on the proliferation of MCs, specifically asking how GSNO regulates the transcription factor Egr-1, which we have previously shown to be critical for the induction of MC mitogenesis.
The proliferation of MCs was measured by thymidine incorporation and cell counting. Egr-1 mRNA and protein levels were detected by Northern and Western blots. Electrophoretic mobility shift assays (EMSAs) and chloramphenicol acetyltransferase (CAT) assays were performed to test whether GSNO modulates DNA binding and transcriptional activation of Egr-1.
GSNO strongly inhibited serum-induced MC proliferation (-84% at 1 mmol/L). A mild inhibition of serum-induced Egr-1 mRNA was observed at GSNO concentrations from 50 to 200 micromol/L, whereas mRNA levels increased again at concentrations above 500 micromol/L. This increased mRNA expression, however, was not translated into Egr-1 protein. Instead, Egr-1 protein induction was inhibited (-40%). EMSAs indicated that GSNO inhibited specific binding of Egr-1 to its DNA consensus sequence. Moreover, transcriptional activation by Egr-1 in CAT assays using a reporter plasmid bearing three Egr-1 binding sites was strongly suppressed by GSNO.
Our data identify GSNO as a potent inhibitor of MC growth with potential beneficial effects in proliferative glomerular diseases. This antimitogenic property is mediated at least in part by inhibitory effects of GSNO on Egr-1 protein levels and by reducing the ability of Egr-1 to activate transcription by impairing its DNA binding activity.
一氧化氮抑制肾小球系膜细胞生长：转录因子Egr-1的作用
在先前的研究中，我们发现早期生长反应基因-1（Egr-1）的表达与系膜细胞（MC）增殖密切相关。抗增殖剂可抑制丝裂原诱导的Egr-1表达。在此，我们研究了S-亚硝基谷胱甘肽（GSNO）对MC增殖的影响，特别探讨了GSNO如何调节转录因子Egr-1，我们之前已证明该转录因子对MC有丝分裂的诱导至关重要。
通过胸腺嘧啶核苷掺入和细胞计数来测定MC的增殖。采用Northern印迹和Western印迹检测Egr-1 mRNA和蛋白水平。进行电泳迁移率变动分析（EMSA）和氯霉素乙酰转移酶（CAT）分析，以检测GSNO是否调节Egr-1的DNA结合和转录激活。
GSNO强烈抑制血清诱导的MC增殖（1 mmol/L时抑制率为-84%）。在50至200 μmol/L的GSNO浓度下，观察到血清诱导的Egr-1 mRNA有轻度抑制，而在浓度高于500 μmol/L时mRNA水平再次升高。然而，这种增加的mRNA表达并未转化为Egr-1蛋白。相反，Egr-1蛋白的诱导受到抑制（-40%）。EMSA表明GSNO抑制Egr-1与其DNA共有序列的特异性结合。此外，在使用带有三个Egr-1结合位点的报告质粒进行的CAT分析中，GSNO强烈抑制Egr-1的转录激活。
我们的数据表明GSNO是MC生长的有效抑制剂，对增殖性肾小球疾病可能具有潜在的有益作用。这种抗有丝分裂特性至少部分是由GSNO对Egr-1蛋白水平的抑制作用以及通过损害其DNA结合活性来降低Egr-1激活转录的能力介导的。
