Hormones – Cytokines – Signaling

Kidney International (2005) 68, 972–984; doi:10.1111/j.1523-1755.2005.00491.x

MAPK/AP-1-dependent regulation of PAI-1 gene expression by TGF-bold beta in rat mesangial cells

BAOLIANG GUO1, KEN INOKI1, MOTOHIDE ISONO, HIROYUKI MORI, KEIZO KANASAKI, TOSHIRO SUGIMOTO, SATOSHI AKIBA, TAKASHI SATO, BAOFENG YANG, RYUICHI KIKKAWA, ATSUNORI KASHIWAGI, MASAKAZU HANEDA and DAISUKE KOYA

Department of Medicine, Shiga University of Medical Science, Otsu, Shiga, Japan Department of Pathological Biochemistry, Kyoto Pharmaceutical University, Kyoto, Japan Harbin Medical University, Harbin, China and Second Department of Medicine, Asahikawa Medical College, Asahikawa, Japan

Correspondence: Daisuke Koya, M.D., Ph.D, Department of Medicine, Shiga University of Medical Science, Seta, Otsu, Shiga 520–2192, Japan E-mail: koya@belle.shiga-med.ac.jp

1Dr. Guo and Dr. Inoki contributed equally to this work.

Received 4 January 2005; Revised 21 March 2005; Accepted 30 March 2005.

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Abstract

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.

Conclusion

 

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.

Keywords:

PAI-1, TGF-beta, ERK, AP-1, INF-gamma

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