Nitric oxide inhibits growth of glomerular mesangial cells: Role of the transcription factor EGR-1

Background. In previous studies, we found a close link of early growth resp onse gene-1 (Egr-1) expression to mesangial cell (MC) proliferation. Antipr oliferative agents inhibited mitogen-induced Egr-1 expression. Here we inve stigated 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 mito genesis. Methods. The proliferation of MCs was measured by thymidine incorporation a nd cell counting. Egr-1 mRNA and protein levels were detected by Northern a nd Western blots. Electrophoretic mobility shift assays (EMSAs) and chloram phenicol acetyltransferase (CAT) assays were performed to test whether GSNO modulates DNA binding and transcriptional activation of Egr-1. Results. GSNO strongly inhibited serum-induced MC proliferation (- 84 % at 1 mmol/L). A mild inhibition of serum-induced Egr-1 mRNA was observed at GS NO concentrations from 50 to 200 mu mol/L, whereas mRNA levels increased ag ain at concentrations above 500 mu mol/L. This increased mRNA expression, h owever, was not translated into Egr-1 protein. Instead, Egr-1 protein induc tion was inhibited (-40%). EMSAs indicated that GSNO inhibited specific bin ding of Egr-1 to its DNA consensus sequence. Moreover, transcriptional acti vation by Egr-1 in CAT assays using a reporter plasmid bearing three Egr-1 binding sites was strongly suppressed by GSNO. Conclusions. Our data identify GSNO as a potent inhibitor of MC growth with potential beneficial effects in proliferative glomerular diseases. This an timitogenic 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 activa te transcription by impairing its DNA binding activity.