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P300-dependent STAT3 acetylation is necessary for angiotensin II-induced pro-fibrotic responses in renal tubular epithelial cells.

Ni J, Shen Y, Wang Z, Shao DC, Liu J, Kong YL, Fu LJ, Zhou L, Xue H, Huang Y, Zhang W, Yu C, Lu LM - Acta Pharmacol. Sin. (2014)

Bottom Line: Treatment of the cells with Ang II (1 μmol/L) significantly increased STAT3 phosphorylation on Tyr705 through JAK2 activation, and dose-dependently increased the expression of fibronectin, collagen IV and TGF-β1.Pretreatment with curcumin, an inhibitor of JAK2 and p300, blocked Ang II-induced effects.Knockdown of p300 significantly decreased STAT3 acetylation on Lys685, and abolished Ang II-stimulated STAT3 phosphorylation on Tyr705, whereas pretreatment of the cells with C646, a selective inhibitor of p300, inhibited Ang II-induced STAT3 nuclear translocation and the expression of TGF-β1, collagen IV and fibronectin.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Pathophysiology, Shanghai Medical College, Fudan University, Shanghai 200032, China.

ABSTRACT

Aim: To explore the signal transducer and activator of transcription 3 (STAT3) signaling pathway, especially STAT3 acetylation, in angiotensin II (Ang II)-induced pro-fibrotic responses in renal tubular epithelial cells.

Methods: Rat renal tubular epithelial cell line (NRK-52E) was used. STAT3 acetylation and phosphorylation, as well as the expression of fibronectin, collagen IV and transforming growth factor-β1 (TGF-β1) were examined using Western blotting. The level and localization of STAT3 phosphorylation on Tyr705 were detected with fluorescence immunocytochemistry. The cells were transfected with a plasmid vector carrying p300 gene or siRNA targeting p300 to regulate p300 expression.

Results: Overexpression of p300 significantly increased STAT3 acetylation on Lys685, STAT3 phosphorylation on Tyr705, and the expression of TGF-β1, collagen IV and fibronectin in the cells. Treatment of the cells with Ang II (1 μmol/L) significantly increased STAT3 phosphorylation on Tyr705 through JAK2 activation, and dose-dependently increased the expression of fibronectin, collagen IV and TGF-β1. Pretreatment with curcumin, an inhibitor of JAK2 and p300, blocked Ang II-induced effects. Knockdown of p300 significantly decreased STAT3 acetylation on Lys685, and abolished Ang II-stimulated STAT3 phosphorylation on Tyr705, whereas pretreatment of the cells with C646, a selective inhibitor of p300, inhibited Ang II-induced STAT3 nuclear translocation and the expression of TGF-β1, collagen IV and fibronectin. Pretreatment of the cells with AG490, a JAK2 inhibitor, markedly inhibited Ang II-induced STAT3 phosphorylation on Tyr705 and fibronectin expression.

Conclusion: p300-dependent STAT3 acetylation is necessary for Ang II-induced STAT3 phosphorylation and the consequent pro-fibrotic responses in renal tubular epithelial cells in vitro.

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Effect of C646 on Ang II-induced pro-fibrotic responses in NRK-52E cells. Cells were treated with Ang II (1 μmol/L) for 48 h with or without a 1-h pretreatment with C646 (10 μmol/L). Fibronectin, collagen IV and TGF-β1 were analyzed by Western blot analysis. Media containing Ang II or C646 were changed every 24 h. Data are presented as the mean±SEM of 6 experiments. bP<0.05, cP<0.01 compared with control [Ang II (−) and C646 (−)]. fP<0.01 compared with Ang II only.
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fig9: Effect of C646 on Ang II-induced pro-fibrotic responses in NRK-52E cells. Cells were treated with Ang II (1 μmol/L) for 48 h with or without a 1-h pretreatment with C646 (10 μmol/L). Fibronectin, collagen IV and TGF-β1 were analyzed by Western blot analysis. Media containing Ang II or C646 were changed every 24 h. Data are presented as the mean±SEM of 6 experiments. bP<0.05, cP<0.01 compared with control [Ang II (−) and C646 (−)]. fP<0.01 compared with Ang II only.

Mentions: siRNA targeted against p300 efficiently decreased p300 protein levels, but control siRNA did not have the same effect (Figure 7A). The knockdown of p300 decreased STAT3 Lys685 acetylation (Figure 7B) and abolished the Ang II-stimulated up-regulation of STAT3 Tyr705 phosphorylation (Figure 7C), but not Ser727 phosphorylation (Figure 7D). The pretreatment of cells with C646, a selective inhibitor of p30050,51,52, rapidly inhibited Ang II-induced STAT3 nuclear translocation, which was proportional to STAT3 Tyr705 phosphorylation (Figure 8). C646 also attenuated fibronectin, collagen IV and TGF-β1 expression induced by Ang II (Figure 9).


P300-dependent STAT3 acetylation is necessary for angiotensin II-induced pro-fibrotic responses in renal tubular epithelial cells.

Ni J, Shen Y, Wang Z, Shao DC, Liu J, Kong YL, Fu LJ, Zhou L, Xue H, Huang Y, Zhang W, Yu C, Lu LM - Acta Pharmacol. Sin. (2014)

Effect of C646 on Ang II-induced pro-fibrotic responses in NRK-52E cells. Cells were treated with Ang II (1 μmol/L) for 48 h with or without a 1-h pretreatment with C646 (10 μmol/L). Fibronectin, collagen IV and TGF-β1 were analyzed by Western blot analysis. Media containing Ang II or C646 were changed every 24 h. Data are presented as the mean±SEM of 6 experiments. bP<0.05, cP<0.01 compared with control [Ang II (−) and C646 (−)]. fP<0.01 compared with Ang II only.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4155527&req=5

fig9: Effect of C646 on Ang II-induced pro-fibrotic responses in NRK-52E cells. Cells were treated with Ang II (1 μmol/L) for 48 h with or without a 1-h pretreatment with C646 (10 μmol/L). Fibronectin, collagen IV and TGF-β1 were analyzed by Western blot analysis. Media containing Ang II or C646 were changed every 24 h. Data are presented as the mean±SEM of 6 experiments. bP<0.05, cP<0.01 compared with control [Ang II (−) and C646 (−)]. fP<0.01 compared with Ang II only.
Mentions: siRNA targeted against p300 efficiently decreased p300 protein levels, but control siRNA did not have the same effect (Figure 7A). The knockdown of p300 decreased STAT3 Lys685 acetylation (Figure 7B) and abolished the Ang II-stimulated up-regulation of STAT3 Tyr705 phosphorylation (Figure 7C), but not Ser727 phosphorylation (Figure 7D). The pretreatment of cells with C646, a selective inhibitor of p30050,51,52, rapidly inhibited Ang II-induced STAT3 nuclear translocation, which was proportional to STAT3 Tyr705 phosphorylation (Figure 8). C646 also attenuated fibronectin, collagen IV and TGF-β1 expression induced by Ang II (Figure 9).

Bottom Line: Treatment of the cells with Ang II (1 μmol/L) significantly increased STAT3 phosphorylation on Tyr705 through JAK2 activation, and dose-dependently increased the expression of fibronectin, collagen IV and TGF-β1.Pretreatment with curcumin, an inhibitor of JAK2 and p300, blocked Ang II-induced effects.Knockdown of p300 significantly decreased STAT3 acetylation on Lys685, and abolished Ang II-stimulated STAT3 phosphorylation on Tyr705, whereas pretreatment of the cells with C646, a selective inhibitor of p300, inhibited Ang II-induced STAT3 nuclear translocation and the expression of TGF-β1, collagen IV and fibronectin.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Pathophysiology, Shanghai Medical College, Fudan University, Shanghai 200032, China.

ABSTRACT

Aim: To explore the signal transducer and activator of transcription 3 (STAT3) signaling pathway, especially STAT3 acetylation, in angiotensin II (Ang II)-induced pro-fibrotic responses in renal tubular epithelial cells.

Methods: Rat renal tubular epithelial cell line (NRK-52E) was used. STAT3 acetylation and phosphorylation, as well as the expression of fibronectin, collagen IV and transforming growth factor-β1 (TGF-β1) were examined using Western blotting. The level and localization of STAT3 phosphorylation on Tyr705 were detected with fluorescence immunocytochemistry. The cells were transfected with a plasmid vector carrying p300 gene or siRNA targeting p300 to regulate p300 expression.

Results: Overexpression of p300 significantly increased STAT3 acetylation on Lys685, STAT3 phosphorylation on Tyr705, and the expression of TGF-β1, collagen IV and fibronectin in the cells. Treatment of the cells with Ang II (1 μmol/L) significantly increased STAT3 phosphorylation on Tyr705 through JAK2 activation, and dose-dependently increased the expression of fibronectin, collagen IV and TGF-β1. Pretreatment with curcumin, an inhibitor of JAK2 and p300, blocked Ang II-induced effects. Knockdown of p300 significantly decreased STAT3 acetylation on Lys685, and abolished Ang II-stimulated STAT3 phosphorylation on Tyr705, whereas pretreatment of the cells with C646, a selective inhibitor of p300, inhibited Ang II-induced STAT3 nuclear translocation and the expression of TGF-β1, collagen IV and fibronectin. Pretreatment of the cells with AG490, a JAK2 inhibitor, markedly inhibited Ang II-induced STAT3 phosphorylation on Tyr705 and fibronectin expression.

Conclusion: p300-dependent STAT3 acetylation is necessary for Ang II-induced STAT3 phosphorylation and the consequent pro-fibrotic responses in renal tubular epithelial cells in vitro.

Show MeSH
Related in: MedlinePlus