J Korean Med Sci.  2009 Jan;24(Suppl 1):S195-S203. 10.3346/jkms.2009.24.S1.S195.

Aldosterone-induced TGF-beta1 Expression is Regulated by Mitogen-Activated Protein Kinases and Activator Protein-1 in Mesangial Cells

• Han JS
• Cho BS
• Yang CW
• Kim YS

• KMID: 1778162
• DOI: http://doi.org/10.3346/jkms.2009.24.S1.S195

Abstract

Aldosterone has been shown to stimulate renal TGF-beta1 expression. However, the mechanisms for aldosterone-induced TGF-beta1 expression have not been clearly determined in mesangial cells. We examined the role of extracellular-signal regulated kinase 1 and 2 (ERK1/2), c-Jun N-terminal kinase (JNK) and activator protein- 1 (AP-1) in the aldosterone-induced TGF-beta1 expression in rat mesangial cells. TGF-beta1 protein in the conditioned medium released from rat mesangial cells was measured by sandwich ELISA, TGF-beta1 mRNA expression was analyzed by Northern blotting, AP-1 DNA binding activity was measured by EMSA and the ERK1/2, JNK activity was analyzed by western blotting. Aldosterone significantly stimulated TGF-beta1 protein production and TGF-beta1 mRNA expression in mesangial cells in a dose-dependent manner. Aldosterone significantly increased AP-1 DNA binding activity in mesangial cells. Pre-treatment of cells with AP-1 inhibitor, curcumin, blocked aldosterone-induced AP-1 DNA binding activity as well as aldosterone-induced TGF-beta1 production. Aldosterone increased phosphorylation of ERK1/2 and JNK in mesangial cells. Pre-treatment of cells with ERK1/2 inhibitor, PD98059, or JNK inhibitor, SP600125 significantly inhibited aldosterone-induced ERK1/2 and JNK activity and subsequently TGF-beta1 production, respectively. We conclude that aldosteroneinduced TGF-beta1 expression in mesangial cells is regulated by the ERK1/ 2, JNK and AP-1 intracellular signaling pathways.

Keyword

Aldosterone; Transforming Growth Factor beta1; Extracellular Signal-Regulated MAP Kinases; JNK Mitogen-Activated Protein Kinases; Transcription Factor AP-1

MeSH Terms

Aldosterone/*pharmacology
Animals
Culture Media, Conditioned/pharmacology
DNA/metabolism
Extracellular Signal-Regulated MAP Kinases/metabolism
*Gene Expression Regulation, Enzymologic
Humans
*MAP Kinase Signaling System
Mesangial Cells/*metabolism
Models, Biological
Phosphorylation
Protein Binding
Rats
Transcription Factor AP-1/*metabolism
Transforming Growth Factor beta1/*biosynthesis

Figure

• 

  Fig. 1 Aldosterone stimulates TGF-β1 production in mesangial cells. (A) Mesangial cells were incubated with or without aldosterone (5 ng/mL) for the indicated time points (○, untreated: •, treated with aldosterone). *p<0.05 vs. untreated control. (B) Cells were incubated with various dose of aldosterone or angiotensin II (10-7 M) or a combination of aldosterone and angiotensin II for 24 hr. *p<0.05 vs. untreated control. †p<0.05 vs. angiotensin II-treated cells. (C) Cells were pre-incubated with or without spironolactone (10-9 M) for 60 min and then incubated with aldosterone (5 ng/mL) for 16 hr. *p<0.05 vs. untreated control. †p<0.05 vs. aldosterone-treated cells. In all of (A), (B) and (C) TGF-β1 protein was measured by ELISA. Results from 5 independent experiments are shown as mean±SEM. (D) TGF-β1 protein expression was measured by western blot analysis after cells were pre-incubated with or without spironolactone (10-9 M) for 60 min, and then incubated with aldosterone. *p<0.01 vs. untreated control. †p<0.05 vs. aldosterone-treated cells.

• 

  Fig. 2 Aldosterone stimulates TGF-β1 mRNA expression in mesangial cells. (A) Cells were incubated with various dose of aldosterone (5 ng/mL) or angiotensin II (10-7 M) or a combination of aldosterone and angiotensin II for 16 hr. *p<0.05 vs. untreated control. †p<0.05 vs. angiotensin II-treated cells. (B) Cells were pre-incubated with or without spironolactone (10-9 M) for 60 min and then incubated with aldosterone (5 ng/mL) for 16 hr. *p<0.05 vs. untreated control. †p<0.05 vs. aldosterone-treated cells. In both (A) and (B), Upper panel, the autoradiograph is a representative of five independent experiments with similar results. Lower panel, the optical density of autoradiographic signals was quantified and calculated as the ratio of TGF-β1 to GAPDH mRNA. Results are expressed as fold increase over untreated (represented as 1) in densitometric arbitrary units. Each value represents the mean±SEM.

• 

  Fig. 3 Role for AP-1 activation in the aldosterone-induced TGF-β1 expression. (A) Time course for AP-1 activation by aldosterone. Cells were incubated with aldosterone (5 ng/mL) for the indicated time points. AP-1 DNA binding activity in the nuclear extracts was measured by EMSA using AP-1 consensus oligonucleotides. *p<0.05 vs. untreated control. (B) Inhibition of aldosterone-induced AP-1 activity by curcumin. Cells were pre-incubated with or without AP-1 inhibitor, curcumin (12.5 µM), for 15 min, and then stimulated with aldosterone (5 ng/mL) for 15 min, and EMSA was performed. *p<0.05 vs. untreated control. †p<0.05 vs. aldosterone-treated cells. In some experiments, the nuclear extracts from aldosterone-stimulated mesangial cells were pre-incubated with or without an antibody to c-jun or c-fos for 1 hr at room temperature and were then analyzed for AP-1 binding activity. Pre-incubation of nuclear extracts with c-jun or c-fos antibody induced supershift of aldosterone-induced AP-1 activity (upper panel). In both (A) and (B) Left panel, the autoradiograph is a representative of five independent experiments with similar results. Right panel, the optical density of autoradiographic signals was quantified and calculated as the ratio of aldosterone-treated cells to untreated control. Results are expressed as fold increase over untreated control (represented as 1) in densitometric arbitrary units. Each value represents the mean±SEM. (C) Inhibition of aldosterone-induced TGF-β1 production by curcumin. Cells were pre-incubated with or without curcumin (12.5 µM) for 15 min, and then incubated with aldosterone (5 ng/mL) for 24 hr and TGF-β1 protein was measured by ELISA. *p<0.05 vs. untreated control. †p<0.05 vs. aldosterone-treated cells.

• 

  Fig. 4 Role for JNK phosphorylation in the aldosterone-induced TGF-β1 expression. (A) Cells were incubated with aldosterone (5 ng/mL) for various time points, and phosphorylated JNK was detected by Western blot analysis. *p<0.05 vs. untreated control. (B) Inhibition of aldosterone-induced JNK phosphorylation by spironolactone. Cells were pre-incubated with or without spironolactone (10-9 M) for 60 min, and then incubated with aldosterone (5 ng/mL) for 10 min, and western blot was performed. *p<0.05 vs. untreated control. †p<0.05 vs. aldosterone-treated cells. In both (A) and (B) Upper panel, the autoradiograph is a representative of five independent experiments with similar results. Lower panel, the optical density of autoradiographic signals was quantified and calculated as the ratio of aldosterone-treated cells to untreated control. Results are expressed as fold increase over untreated control (represented as 1) in densitometric arbitrary units. Each value represents the mean±SEM. (C) Inhibition of aldosterone-induced JNK phosphorylation by SP600125. Cells were pre-incubated with or without SP600125 (20 µM) for 15 min, and then incubated with aldosterone (5 ng/mL) for 24 hr, and TGF-β1 protein was measured by ELISA. *p<0.05 vs. untreated control. †p<0.05 vs. aldosterone-treated cells.

• 

  Fig. 5 Role for ERK1/2 phosphorylation in aldosterone-induced TGF-β1 expression. (A) Cells were incubated with aldosterone (5 ng/mL) for various time points, and phosphorylated ERK1/2 was detected by Western blot analysis. *p<0.05 vs. untreated control. (B) Inhibition of aldosterone-induced ERK1/2 phosphorylation by spironolactone. Cells were pre-incubated with or without spironolactone (10-9 M) for 60 min, and then incubated with aldosterone (5 ng/mL) for 10 min, and western blot was performed. *p<0.05 vs. untreated control. †p<0.05 vs. aldosterone-treated cells. In both a and b Upper panel, the autoradiograph is a representative of five independent experiments with similar results. Lower panel, the optical density of autoradiographic signals was quantified and calculated as the ratio of aldosterone-treated cells to untreated control. Results are expressed as fold increase over untreated control (represented as 1) in densitometric arbitrary units. Each value represents the mean±SEM. (C) Inhibition of aldosterone-induced ERK1/2 phosphorylation by PD98059. Cells were pre-incubated with or without PD98059 (50 µM) for 15 min, and then incubated with aldosterone (5 ng/mL for 24 hr, and TGF-β1 protein was measured by ELISA. *p<0.05 vs. untreated control. †p<0.05 vs. aldosterone-treated cells.

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