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. 2016 Jan 1;6(2):204–218. doi: 10.7150/thno.13907

Figure 3.

Figure 3

TGF-β1 Up-regulated MDR1 Expression by TGF-β1/Smad Signaling Pathway and Histone H3 Acetylation. (A) The effect of physical factors, cytokines and growth factors associated with peritoneal injury on MDR1 mRNA expression in NFB. (B and C) Reinfusion of peritoneal fluid obtained from rats with peritoneal adhesions resulted in more serious peritoneal adhesions. (B) Peritoneal injury increased the concentration of TGF-β1 in peritoneal fluid, which reached the highest level at day 7. (C) Representative photographic images of rat peritoneal adhesions. Peritoneal fluid with high endogenous TGF-β1 was reinfused into peritoneal cavities of rats with mild peritoneal injuries, resulting in more serious peritoneal adhesions of higher grade and a greater rate of adhesions (For detailed statistical results, see Supplemental Table 1). (D and E) TGF-β1 promoted peritoneal adhesion formation and P-gp expression of adhesion tissue. (D) Representative photographic images of rat peritoneal adhesions. Intraperitoneal injection of exogenous TGF-β1 (150 ng/kg) induced more serious peritoneal adhesions of higher grade and a greater rate of adhesions (For detailed statistical results, see Supplemental Table 2). (E) Intraperitoneal injection of TGF-β1 upregulated P-gp expression in adhesion tissues. ** P < 0.01 vs Control. (F and G) Inhibition of the TGF-β1/Smad pathway prevented induction of P-gp expression by TGF-β1. (F) TGF-β type I receptor (TβRI) inhibitor SB431542 inhibited P-gp expression induced by TGF-β1. (G) Silencing of Smad 2 or 3 expression by transfection with Si-Smad 2 or -Smad 3, respectively, prevented induction of P-gp expression by TGF-β1. (H - K) TGF-β1 enhances activity of the MDR1 promoter via the TGF-β1/Smad signaling pathway and promotion of histone H3 acetylation. Chromatin immunoprecipitation (ChIP) analysis of the binding of Smad2/3 and histone H3 to the MDR1 promoter in AFB (H) and acetylated H3 at the MDR1 promoter in rat NFB and NIH3T3 cells treated with TGF-β1 (I). RPL30 was used as a positive control. (J) Detection of global acetylated histone H3 in rat NFB treated with different factors. (K) Induction of MDR1 promoter activity by co-transfection with either histone H3, Smad 2, or Smad 3 expression plasmids plus pMDR1(-1202) reporter plasmid and TGF-β1 or the histone deacetylase (HDAC) inhibitor panobinostat (LBH589). TGF-β type I receptor (TβRI) inhibitor SB431542 and histone acetyltransferase CBP/p300 inhibitor C646 abolished the increased activity induced by TGF-β1. * P < 0.05; ** P < 0.01. (L) Schematic model depicting the proposed mechanism of up-regulation of MDR1 expression induced by TGF-β1 via the TGF-β-Smad signaling pathway and histone H3 acetylation.