Nitric oxide induces TIMP-1 expression by activating the transforming growth factor beta-Smad signaling pathway.
Excessive accumulation of the extracellular matrix is a hallmark of many inflammatory and fibrotic diseases, including those of the kidney. This study addresses the question whether NO, in addition to inhibiting the expression of MMP-9, a prominent metalloprotease expressed by mesangial cells, additionally modulates expression of its endogenous inhibitor TIMP-1. We demonstrate that exogenous NO has no modulatory effect on the extracellular TIMP-1 content but strongly amplifies the early increase in cytokine-induced TIMP-1 mRNA and protein levels. We examined whether transforming growth factor beta (TGFbeta), a potent profibrotic cytokine, is involved in the regulation of NO-dependent TIMP-1 expression. Experiments utilizing a pan-specific neutralizing TGFbeta antibody demonstrate that the NO-induced amplification of TIMP-1 is mediated by extracellular TGFbeta. Mechanistically, NO causes a rapid increase in Smad-2 phosphorylation, which is abrogated by the addition of neutralizing TGFbeta antisera. Similarly, the NO-dependent increase in Smad-2 phosphorylation is prevented in the presence of an inhibitor of TGFbeta-RI kinase, indicating that the NO-dependent activation of Smad-2 occurs via the TGFbeta-type I receptor. Furthermore, activation of the Smad signaling cascade by NO is corroborated by the NO-dependent increase in nuclear Smad-4 level and is paralleled by increased DNA binding of Smad-2/3 containing complexes to a TIMP-1-specific Smad-binding element (SBE). Reporter gene assays revealed that NO activates a 0.6-kb TIMP-1 gene promoter fragment as well as a TGFbeta-inducible and SBE-driven control promoter. Chromatin immunoprecipitation analysis also demonstrated DNA binding activity of Smad-3 and Smad-4 proteins to the TIMP-1-specific SBE. Finally, by enzyme-linked immunosorbent assay, we demonstrated that NO causes a rapid increase in TGFbeta(1) levels in cell supernatants. Together, these experiments demonstrate that NO by induction of the Smad signaling pathway modulates TIMP-1 expression.
Akool
El-Sayed
el-S
Pharmazentrum Frankfurt/ZAFES, Klinikum der Johann Wolfgang Goethe-Universität, D-60590 Frankfurt am Main, Germany.
Doller
Anke
A
Müller
Roswitha
R
Gutwein
Paul
P
Xin
Cuiyan
C
Huwiler
Andrea
A
Pfeilschifter
Josef
J
Eberhardt
Wolfgang
W
D030201:Activin Receptors, Type I / Q000378:metabolism; D000818:Animals; D001483:Base Sequence; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D019175:DNA Methylation; D005786:Gene Expression Regulation / Q000187:drug effects; D007375:Interleukin-1 / Q000494:pharmacology; D050527:Mesangial Cells / Q000187:drug effects / Q000378:metabolism; D009500:Neutralization Tests; D009569:Nitric Oxide / Q000378:metabolism*; D020030:Nitric Oxide Donors / Q000494:pharmacology; D010766:Phosphorylation; D011401:Promoter Regions, Genetic; D017346:Protein Serine-Threonine Kinases; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats; D000077293:Receptor, Transforming Growth Factor-beta Type I; D018125:Receptors, Transforming Growth Factor beta / Q000378:metabolism; D026423:S-Nitroso-N-Acetylpenicillamine / Q000494:pharmacology; D015398:Signal Transduction / Q000187:drug effects; D051896:Smad Proteins, Receptor-Regulated / Q000378:metabolism*; D051899:Smad2 Protein / Q000378:metabolism; D051900:Smad3 Protein / Q000378:metabolism; D051901:Smad4 Protein / Q000378:metabolism; D019715:Tissue Inhibitor of Metalloproteinase-1 / Q000235:genetics* / Q000378:metabolism*; D016212:Transforming Growth Factor beta / Q000037:antagonists & inhibitors / Q000378:metabolism*; D053773:Transforming Growth Factor beta1
D007375:Interleukin-1; D020030:Nitric Oxide Donors; D012333:RNA, Messenger; D018125:Receptors, Transforming Growth Factor beta; D051896:Smad Proteins, Receptor-Regulated; D051899:Smad2 Protein; C497784:Smad2 protein, rat; D051900:Smad3 Protein; C497787:Smad3 protein, rat; D051901:Smad4 Protein; C490811:Smad4 protein, rat; C508962:Tgfb1 protein, rat; D019715:Tissue Inhibitor of Metalloproteinase-1; D016212:Transforming Growth Factor beta; D053773:Transforming Growth Factor beta1; D009569:Nitric Oxide; D026423:S-Nitroso-N-Acetylpenicillamine; D017346:Protein Serine-Threonine Kinases; D030201:Activin Receptors, Type I; D000077293:Receptor, Transforming Growth Factor-beta Type I; C000625843:Tgfbr1 protein, rat