MAPK/AP-1-dependent regulation of PAI-1 gene expression by TGF-beta in rat mesangial cells.
BACKGROUND
Receptor-regulated Smads and/or mitogen-activated protein kinases (MAPKs) are involved in transforming growth factor-beta (TGF-beta)-induced expression of various genes, including plasminogen activator inhibitor-1 (PAI-1). Because the sequence of the promoter region in rat PAI-1 gene differs from that in the human gene, we examined the mechanisms of TGF-beta-induced rat PAI-1 expression in rat mesangial cells.


METHODS
TGF-beta1-induced PAI-1 and c-fos mRNA expressions were determined by Northern blot analysis. Activation of MAPKs and Smad proteins was evaluated by an immunoblot analysis. DNA binding activities of nuclear protein were examined by using an electrophoretic mobility shift assay (EMSA). The activities of PAI-1 promoter were measured by a luciferase reporter assay.


RESULTS
Extracellular-regulated kinase (ERK) and c-Jun NH-terminal kinase (JNK) phosphorylation, c-fos mRNA expression, and activator protein-1 (AP-1) DNA binding activity stimulated by TGF-beta1 were completely suppressed by the ERK kinase (MEK) inhibitors. EMSA and reporter analysis revealed that an AP-1-like sequence located in the proximal region of the rat PAI-1 promoter was the target for TGF-beta1, and the disruption of this AP-1-like sequence suppressed basal and TGF-beta1-induced promoter activation. TGF-beta1 also stimulated nuclear translocation of Smads and binding to palindromic Smad binding element (SBE) located in the rat PAI-1 promoter, without being affected by MEK inhibitor. Point mutation and deletion of palindromic SBE did not affect TGF-beta1-induced rat PAI-1 promoter activity. Moreover, interferon-gamma (IFN-gamma) inhibited TGF-beta1-induced PAI-1 expression through selectively suppressing the ERK-AP-1 pathway.


CONCLUSIONS
These results suggest that the essential requirement of MAPK/AP-1 activation for TGF-beta1-induced PAI-1 expression is unique to rat mesangial cells.
Guo
Baoliang
B
Department of Medicine, Shiga University of Medical Science, Otsu, Shiga, Japan.
Inoki
Ken
K
Isono
Motohide
M
Mori
Hiroyuki
H
Kanasaki
Keizo
K
Sugimoto
Toshiro
T
Akiba
Satoshi
S
Sato
Takashi
T
Yang
Baofeng
B
Kikkawa
Ryuichi
R
Kashiwagi
Atsunori
A
Haneda
Masakazu
M
Koya
Daisuke
D
D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology; D002478:Cells, Cultured; D004791:Enzyme Inhibitors / Q000494:pharmacology; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D015870:Gene Expression / Q000187:drug effects / Q000502:physiology; D007371:Interferon-gamma / Q000494:pharmacology; D048031:JNK Mitogen-Activated Protein Kinases / Q000378:metabolism; D007678:Kidney Glomerulus / Q000166:cytology*; D020935:MAP Kinase Signaling System / Q000187:drug effects / Q000502:physiology*; D050527:Mesangial Cells / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D010766:Phosphorylation / Q000187:drug effects; D017395:Plasminogen Activator Inhibitor 1 / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000502:physiology; D016760:Proto-Oncogene Proteins c-fos / Q000235:genetics; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D051785:Smad Proteins / Q000378:metabolism; D018808:Transcription Factor AP-1 / Q000378:metabolism*; D016212:Transforming Growth Factor beta / Q000378:metabolism* / Q000494:pharmacology; D053773:Transforming Growth Factor beta1
D000970:Antineoplastic Agents; D004791:Enzyme Inhibitors; D017395:Plasminogen Activator Inhibitor 1; D016760:Proto-Oncogene Proteins c-fos; D012333:RNA, Messenger; D051785:Smad Proteins; C508960:TGFB1 protein, human; C508962:Tgfb1 protein, rat; D018808:Transcription Factor AP-1; D016212:Transforming Growth Factor beta; D053773:Transforming Growth Factor beta1; D007371:Interferon-gamma; D048049:Extracellular Signal-Regulated MAP Kinases; D048031:JNK Mitogen-Activated Protein Kinases