Glutathione suppresses TGF-beta-induced PAI-1 expression by inhibiting p38 and JNK MAPK and the binding of AP-1, SP-1, and Smad to the PAI-1 promoter.
Transforming growth factor (TGF)-beta upregulates plasminogen activator inhibitor type 1 (PAI-1) in a variety of cell types, and PAI-1 is considered to be an essential factor for the development of fibrosis. Our previous studies demonstrated that TGF-beta decreased intracellular glutathione (GSH) content in murine embryonic fibroblasts (NIH/3T3 cells), whereas treatment of the cells with GSH, which restored intracellular GSH concentration, inhibited TGF-beta-induced collagen accumulation by blocking PAI-1 expression and enhancing collagen degradation. In the present study, we demonstrate that GSH blocks TGF-beta-induced PAI-1 promoter activity in NIH/3T3 cells, which is associated with an inhibition of TGF-beta-induced JNK and p38 phosphorylation. Interestingly, although exogenous GSH does not affect phosphorylation and/or nuclear translocation of Smad2/3 and Smad4, it completely eliminates TGF-beta-induced binding of transcription factors to not only AP-1 and SP-1 but also Smad cis elements in the PAI-1 promoter. Decoy oligonucleotides (ODN) studies further demonstrate that AP-1, SP-1, and Smad ODNs abrogate the inhibitory effect of GSH on TGF-beta-induced PAI-1 promoter activity and inhibit TGF-beta-induced expression of endogenous PAI-1. Furthermore, we show that GSH reduces TGF-beta-stimulated reactive oxygen species (ROS) signal. Blocking ROS production with diphenyleneiodonium or scavenging ROS with a superoxide dismutase and catalase mimetic MnTBaP dramatically reduces TGF-beta-induced p38 and JNK phosphorylation as well as PAI-1 gene expression. In composite, these findings suggest that GSH inhibits TGF-beta-stimulated PAI-1 expression in fibroblasts by blocking the JNK/p38 pathway, probably by reducing ROS, which leads to an inhibition of the binding of transcription factors to the AP-1, SP-1, and Smad cis elements in the PAI-1 promoter.
Vayalil
Praveen K
PK
Dept. of Environmental Health Sciences, School of Public Health, Univ. of Alabama at Birmingham, Birmingham, AL 35294, USA.
Iles
Karen E
KE
Choi
Jinah
J
Yi
Ae-Kyung
AK
Postlethwait
Edward M
EM
Liu
Rui-Ming
RM
D000818:Animals; D000970:Antineoplastic Agents; D015152:Blotting, Northern; D015153:Blotting, Western; D002478:Cells, Cultured; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015870:Gene Expression / Q000187:drug effects; D005978:Glutathione / Q000494:pharmacology*; D048031:JNK Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D051379:Mice; D041681:NIH 3T3 Cells; D010766:Phosphorylation; D017395:Plasminogen Activator Inhibitor 1 / Q000096:biosynthesis / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000235:genetics*; D021381:Protein Transport; D017382:Reactive Oxygen Species; D015398:Signal Transduction; D051785:Smad Proteins / Q000378:metabolism*; D016329:Sp1 Transcription Factor / Q000378:metabolism*; D018808:Transcription Factor AP-1 / Q000378:metabolism*; D016212:Transforming Growth Factor beta / Q000037:antagonists & inhibitors / Q000494:pharmacology; D048051:p38 Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors* / Q000378:metabolism
D000970:Antineoplastic Agents; D004791:Enzyme Inhibitors; D017395:Plasminogen Activator Inhibitor 1; D017382:Reactive Oxygen Species; D051785:Smad Proteins; D016329:Sp1 Transcription Factor; D018808:Transcription Factor AP-1; D016212:Transforming Growth Factor beta; D048031:JNK Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases; D005978:Glutathione