Hormones – Cytokines – Signaling
Kidney International (2002) 62, 401–411; doi:10.1046/j.1523-1755.2002.00462.x
Nitric oxide down-regulates connective tissue growth factor in rat mesangial cells
Annette Keil, Ingrid E Blom, Roel Goldschmeding and Harald D Rupprecht
Medizinische Klinik IV, University Erlangen-Nürnberg, Erlangen, Germany; Department of Pathology, University Medical Center, Utrecht, The Netherlands; and Medizinische Poliklinik, Ludwig Maximilians University Munich, Munich, Germany
Correspondence: Prof. Dr med. Harald D. Rupprecht, Ludwig Maximilians Universität München, Medizinische Klinik – Innenstadt, Ziemssenstra
e 1, 80336 München, Germany. E-mail: Harald.Rupprecht@medinn.med.uni-muenchen.de
Received 29 May 2001; Revised 15 November 2001; Accepted 8 March 2002.
Abstract
Nitric oxide down-regulates connective tissue growth factor in rat mesangial cells.
Background
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.
Methods
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.
Results
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-
, IL-1
and LPS provoked a transient down-regulation of CTGF mRNA, an effect that could be partially overcome by pretreatment with the NOS-inhibitor N
-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.
Conclusion
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.
Keywords:
representational difference analysis, cell proliferation, adhesion, apoptosis, fibrosis and NO, S-nitroso-glutathione
Abbreviations:
8br-cGMP, 8-bromo-cyclic guanosine-3', S-monophosphate; CTGF, connective tissue growth factor; ECL, enhanced chemiluminescence; DMEM, Dulbecco's modified Eagle's medium; EDTA, ethylenediaminetetraacetic acid; Egr-1, early growth response gene-1; GBM, glomerular basement membrane; GSH, glutathione sulfhydryl; GSNO, S-nitrosoglutathione; HSP, horseradish peroxidase; HUVECs, human umbilical vein endothelial cells; ICAM, intracellular adhesion molecule; IL-1, interleukin-1; iNOS, inducible nitric oxide synthase; L-NAME, N-nitro-L-arginine methyl ester; L-NMMA, NG-monomethyl-L-arginine; LPS, lipopolysaccharide; MC, mesangial cells; nNOS, neuronal nitric oxide synthase; NO, nitric oxide; NOS, nitric oxide synthase; PCR, polymerase chain reaction; PDGF, platelet-derived growth factor; PMSF, phenylmethylsulfonyl fluoride; RDA, representational difference analysis; SNAP, S-nitroso-N-acetyl-D,L-penicillamine; SNP, sodium nitroprusside; SPARC, secreted protein acidic and rich in cysteine; TGF-, transforming growth factor-; TNF-, tumor necrosis factor-; VEGF, vascular endothelial growth factor; VSMC, vascular smooth muscle cells
