Fig 2: Up-regulation of P-gp Expression Elevated ICl,vol and RVD of Peritoneal Fibroblast by Phosphorylating Chloride Channel-3 (ClC-3) tyrosine 342 via Src Tyrosine Kinase. (A) Comparison of volume-activated chloride currents in AFB and NFB. Iso, isotonic bath solution; Hypo, 47% hypotonic bath solution; Hyper, 47% hypertonic bath solution. (B) Suppression of MDR1 expression by sh-MDR1 transfection on ICl,vol in AFB. Cells successfully transfected with sh-MDR1 or sh-NC (green) were selected for whole-cell current recordings under the fluorescence microscope. (C and D) Differential capability of RVD in AFB and NFB and inhibition of RVD by silencing of MDR1 expression in AFB. (C) Changes in AFB and NFB cell volumes in isotonic (Iso) and 47% hypotonic (Hypo) bath solutions and RVD capacity in the hypotonic bath solution. (D) RVD capacity of AFB after treatment with Sh-NC or Sh-MDR1 (n = 20). (E) P-gp promoted phosphorylation of human ClC-3 tyrosine 342. (left panels) NIH-3T3 cells were co-transfected with human MDR1-GFP vectors and ClC-3-Flag or ClC-3-Y432F. Immunoblots of IP with anti-phosphotyrosine and anti-ClC-3 antibodies from extracts of NIH-3T3 cells were probed with an antibody against Flag. (right panels) Effects of down-regulation of P-gp expression or inhibition of Src kinase by SU6656 on phosphorylation of human ClC-3 tyrosine induced by TGF ß1 and hypotension. pTyr: phosphotyrosine. **: P < 0.01. (F) P-gp-mediated Src phosphorylation induced by hypotonicity and TGF ß1. Representative western blot (upper) and densitometric analysis (lower) showed that phospho-Src (pY416) induced by hypotonicity and TGF ß1 was abolished by down-regulation of P-gp expression. **: P < 0.01. (G) P-gp binds to Src kinase. (left two panels) P-gp does not bind ClC-3. Immunoblots of co-IP with anti-Flag antibody from extracts of NIH-3T3 cells co-transfected with MDR1/GFP and ClC-3/Flag, probed with antibodies against P-gp or ClC-3 (upper panel). Control experiment with anti-Flag antibody from extracts of NIH-3T3 cells co-transfected with K18/GFP and ClC-3/Flag probed with anti-K18 (centre panel). (Lower panel) Co-IP with anti-GFP antibody from NIH-3T3 cells extract was probed with anti-Src and anti-P-gp. Cells were transfected with human MDR1-GFP vectors.

Fig 3: Levels of MRP-1 (A) and P-gp (B) measured by whole cell ELISA after exposure of cells to hydralazine, with or without YC-1, during three cycles. Results are represented as mean value ± s.e.m (n = 6). * p < 0.05 versus normoxia, # p < 0.05 versus without YC-1 treatment.

Fig 4: Altering P-gp Expression or Blocking ICl,vol affects Cell Migration and Proliferation. (A and B) Migratory ability of human and rat AFB overexpressing P-gp and NFB. Confluent monolayers were scratched and then cultured in medium containing 0.5% FCS for 48 h (A). Data shown in (B) are mean ± SEM. (C) Representative photographs of wound-healing motility assays for rat AFB cells transiently transfected with siRNA against MDR1. (D) Average migratory width of AFB cells treated with MDR1 siRNA or inhibitors. Data are mean ± SEM. (E and F) Comparison of proliferative capability between rat AFB and NFB cells (E) and quantification of multiple visual fields (n = 4, F). Bright-field photographs of primary cultured live cells were taken at different times. Dark areas indicate peritoneal adhesion tissue or adjacent normal peritoneum tissue. (G) Growth curve for rat AFB cells transfected with si-MDR1 or treated with different inhibitors. Cells were cultured in 96-well plates in medium with 10% serum. Relative cell numbers were determined by CCK-8 colorimetric assay. Data shown are the results of three independent experiments (mean ± SEM). si-MDR1 transfection and all inhibitors except for tariquidar significantly inhibited the proliferation of AFB cells at day 5 and 7 (P < 0.01 vs si-NC).

Fig 5: TGF-ß1 Up-regulated MDR1 Expression by TGF-ß1/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-ß1 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-ß1 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-ß1 promoted peritoneal adhesion formation and P-gp expression of adhesion tissue. (D) Representative photographic images of rat peritoneal adhesions. Intraperitoneal injection of exogenous TGF-ß1 (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-ß1 upregulated P-gp expression in adhesion tissues. ** P < 0.01 vs Control. (F and G) Inhibition of the TGF-ß1/Smad pathway prevented induction of P-gp expression by TGF-ß1. (F) TGF-ß type I receptor (TßRI) inhibitor SB431542 inhibited P-gp expression induced by TGF-ß1. (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-ß1. (H - K) TGF-ß1 enhances activity of the MDR1 promoter via the TGF-ß1/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-ß1 (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-ß1 or the histone deacetylase (HDAC) inhibitor panobinostat (LBH589). TGF-ß type I receptor (TßRI) inhibitor SB431542 and histone acetyltransferase CBP/p300 inhibitor C646 abolished the increased activity induced by TGF-ß1. * P < 0.05; ** P < 0.01. (L) Schematic model depicting the proposed mechanism of up-regulation of MDR1 expression induced by TGF-ß1 via the TGF-ß-Smad signaling pathway and histone H3 acetylation.