Nitric oxide down-regulates connective tissue growth factor in rat mesangial cells.

Abstract

Nitric oxide (NO) exerts complex regulatory actions on mesangial cell (MC) biology, such as inhibition of proliferation, adhesion or contractility and induction of apoptosis. In our previous studies the NO-donor S-nitroso-glutathione (GSNO) was found to be a potent inhibitor of MC growth. This effect was mediated at least in part by inhibitory effects of GSNO on the transcription factor early growth response gene-1 (Egr-1) [10]. We therefore were interested in the regulation of gene expression in MC after treatment with NO. To identify the genes that are regulated by NO in MC, gene expression was analyzed by representational difference analysis. Expression of connective tissue growth factor (CTGF) was studied by Northern and Western blot analyses. Cultured rat MCs treated with GSNO for 8 hours were compared with unstimulated MCs and the CTGF mRNA was found to be down-regulated. The down-regulation was dose-dependent and transient, with a maximum inhibition seen after 6 hours. In parallel, down-regulation of CTGF protein by GSNO was observed by Western blot analysis. Other NO-donors such as S-nitroso-N-acetyl-D,L-penicillamine and spermine-NO showed similar effects. The induction of the inducible NO-synthase by TNF-alpha, IL-1beta and LPS provoked a transient down-regulation of CTGF mRNA, an effect that could be partially overcome by pretreatment with the NOS-inhibitor Nomega-nitro-l-arginine methyl ester. The observed NO-effect could be simulated by treatment with the stable cGMP analog 8br-cGMP, and was abolished by blocking the guanylyl cyclase with the inhibitor NS2028. NO acts as a strong repressor of CTGF expression in cultured rat MC. Thus, in addition to its antiproliferative effects, NO potentially exerts antifibrotic activity by down-regulation of CTGF.