Omentum-derived MCs undergo EMT upon exposure to peritonitis effluent. (A) Photomicrographs of confluent monolayers of human primary omental MCs, non-treated (NT) or treated for 72 hours with peritoneal effluent (diluted 1:1 with culture medium) from a PD patient suffering acute peritonitis. (B) Confocal immunofluorescence (red) of non-treated and treated MCs stained with monoclonal antibodies against pan-cytokeratin (72-hour stimulation). Nuclei were stained with Hoechst 33342 (blue). (C) Western blots showing the expression of E-cadherin and fibronectin in total cell lysates of MCs treated as indicated for 1, 24 or 72 hours. Expression of α-tubulin was detected as a loading control. Data are representative of three independent experiments.

NF-κB nuclear translocation is inhibited by IκBα super-repressor expression. (A) Confocal immunofluorescence of NF-κB expression and localization. Human primary peritoneal MCs were co-stimulated with TGF-β1 and IL-1β for the indicated times; NT, non-treated controls. Fixed and permeabilized cells were stained with a polyclonal antibody against p65 NF-κB. The histogram shows mean fluorescence intensities of nuclear NF-κB staining quantified using the software LAS-AS from Leica. Bars represent s.e.m. A total of 50 cells were analyzed per condition; AU, arbitrary units. (B) Human primary peritoneal MCs stimulated with peritonitis effluent or control medium were analyzed as in (A). ***P<0.0001 compared with medium-treated cells. (C) Primary omental MCs were infected with either a Cop Green-tagged retrovirus encoding an IκBα super-repressor (IκB, lower panels) or with empty Cop Green-tagged virus (empty vector, upper panels). Cells were then co-stimulated for 30 minutes with TGF-β1 and IL-1β. Fixed and permeabilized cells were stained for p65 NF-κB (yellow in overlay image). Nuclei were stained with Hoechst 33342 (blue in overlay). Green fluorescence shows infected cells. The spectral confocal technology allowed discrimination between yellow and green emitted fluorescence. Overlay images show overlapping of Hoechst 33342, anti-NF-κB and Cop Green staining. Data shown are representative of three independent experiments.

ERK controls E-cadherin and cytokeratin downregulation during EMT of cytokine-stimulated MCs. (A) Western blot showing expression of phosphorylated (active) ERK (pERK, top) in MC total lysates. Primary omental MCs were left untreated (NT) or co-stimulated with TGF-β1 and IL-1β (T/I) for the times indicated. As a loading control, samples were probed for total ERK expression (bottom). (B) Primary omental MCs incubated for 24 hours with dialysis fluid (D), non-peritonitis effluent (E) or peritonitis effluent (P) were analyzed as in (A). (C) Western blot showing expression of E-cadherin and phosphorylated (active) ERK in MC total lysates. Omental MCs were pretreated with DMSO or U0126 (20 μM), and treated for 24 hours with TGF-β1 and IL-1β as indicated. Total ERK expression was detected as a loading control. (D) Confocal immunofluorescence analysis of cytokeratin expression. Omental MCs were pretreated with DMSO or U0126 (20 μM) and then co-stimulated with TGF-β1 and IL-1β for 56 hours. Cells were fixed, permeabilized, and stained with a monoclonal antibody against pan-cytokeratin. (E) Effect of ERK inhibition on E-cadherin mRNA expression in MCs. Cells were pretreated with DMSO (gray bars) or U0126 (black bars) and co-stimulated for the times indicated with TGF-β1 and IL-1β (T/I); NT, non-stimulated controls. Quantitative RT-PCR was performed on total RNA. Histone H3 mRNA levels were used for normalization. Bars represent the means ± s.e.m. of duplicate determinations from three independent experiments.

ERK and NF-κB control Snail1 expression induced by TGF-β1 plus IL-1β co-stimulation in MCs. (A) Effect of ERK inhibition on Snail1 mRNA expression in MCs. Omental MCs were pretreated with DMSO (gray bars) or 20 μM U0126 (black bars) and co-stimulated as indicated with TGF-β1 and IL-1β (T/I); NT, non-treated. Quantitative RT-PCR was performed on total RNA. Histone H3 mRNA expression was used for normalization. Bars represent means ± s.e.m. of duplicate determinations from three independent experiments. (B) Effect of ERK inhibition on Snail1 protein immunofluorescence. Omental MCs were pretreated with DMSO or U0126 (20 μM) and treated as indicated for 24 hours. LiCl (40 mM) and MG132 (10 μM) were added to cells 4 hours before the end of the stimulation. Cells were fixed, permeabilized, and stained with a polyclonal antibody against Snail1 before being subjected to confocal microscopy analysis. Panels show Hoechst 33342 staining of cell nuclei (nuclei) and immunofluorescence staining of Snail1 expression. The results shown are from a single experiment that was representative of three independently performed experiments. (C) Effect of NF-κB inhibition on Snail1 mRNA expression in MCs. Human primary omental mesothelial cultures (omentum, left panel) or the MeT-5A MC line (right panel) were infected with a retrovirus encoding an IκBα super-repressor (IκB) or empty control plasmid (CopG). After infection, MeT-5A cultures were sorted for green fluorescence to obtain near pure cultures of infected cells. Cultures were left untreated (NT, gray) or co-stimulated for 24 hours with TGF-β1 and IL-1β (black), and quantitative RT-PCR was performed on total RNA. Histone H3 mRNA expression was used for normalization. Bars represent means ± s.e.m. of duplicate determinations from six independent experiments.

Omentum-derived MCs undergo EMT upon TGF-β1 and IL-1β stimulation. (A) Photomicrographs of confluent monolayers of human primary omental MCs, non-treated (NT) or treated with TGF-β1 (0.5 ng/ml) in combination with IL-1β (2 ng/ml) for 24 hours (T/I). (B) Confocal immunofluorescence (red) of non-treated and treated MCs stained with monoclonal antibodies against E-cadherin (24-hour stimulation) and with pan-cytokeratin (56-hour stimulation). Nuclei were stained with Hoechst 33342 (blue). (C) Western blots showing the expression of E-cadherin, N-cadherin and fibronectin in total cell lysates of MCs treated as indicated with TGF-β1 and IL-1β for 24 or 48 hours. Expression of α-tubulin was detected as a loading control. Data are representative of more than ten independent experiments.

Downregulation of epithelial markers in cytokine-stimulated MCs requires NF-κB signaling activity. (A) Western blots showing the expression of E-cadherin and IκB in total MC lysates. Primary omental MCs were infected with either Cop Green-tagged retrovirus encoding an IκBα super-repressor (IκB) or with empty Cop Green-tagged virus and stimulated for 24 hours with TGF-β1 and IL-1β (T/I) as indicated; NT, non-treated cells. Expression of α-tubulin was detected as a loading control. The histogram shows E-cadherin/α-tubulin band intensity ratios from a representative experiment. (B) Effect of NF-κB inhibition on E-cadherin mRNA expression in MCs. MeT-5A MCs were infected as above with retrovirus encoding an IκBα super-repressor or empty control plasmid (CopG). After infection, MeT-5A cultures were sorted for green fluorescence to obtain near pure cultures of infected cells. Cultures were left untreated (gray bars) or co-stimulated for 24 hours with TGF-β1 and IL-1β (black bars) and quantitative RT-PCR was performed on total RNA. Histone H3 mRNA levels were used for normalization. Bars represent the means ± s.e.m. from six independent experiments. (C) Confocal immunofluorescence analysis of cytokeratin expression. Cells infected and stimulated as in (A) were fixed, permeabilized, and stained with a monoclonal antibody against pan-cytokeratin. Green, Cop Green fluorescence.

ERK controls NF-κB nuclear translocation and transcriptional activity induced by TGF-β1 and IL-1β stimulation in MCs. (A) Confocal immunofluorescence analysis of NF-κB expression and localization. Omental MCs were pretreated with DMSO or U0126 (20 μM) and then co-stimulated for the times indicated with TGF-β1 and IL-1β (T/I); NT, non-treated. Fixed and permeabilized cells were stained with a polyclonal antibody against p65 NF-κB. The results shown are from a single experiment that was representative of three independently performed experiments. (B) Effect of ERK inhibition on NF-κB transcriptional activity. MeT-5A cells were transiently transfected with the KBF-luc reporter plasmid together with Renilla luciferase. Cells were pretreated as indicated with U0126 (20 μM) and then left untreated (NT, gray bars) or co-stimulated for the times indicated with TGF-β1 and IL-1β (T/I, black bars). Bars represent means ± s.e.m. determinations from three independent experiments carried out in duplicate. *P<0.05 compared with DMSO-treated cells. (C) Omental MCs were pretreated with DMSO or U0126 (20 μM) and treated as indicated. Qualitative RT-PCR was performed with specific primers for COX-2 and Histone 3.

Inhibition of ERK and NF-κB can reverse the EMT in transdifferentiated MCs from the peritoneal effluent of patients undergoing continuous ambulatory peritoneal dialysis (CAPD). (A) Western blot showing expression of pERK in monolayer cultures of MCs derived from either the peritoneal effluent of patients undergoing CAPD or from normal omentum. Confluent monolayers of omental MCs from control donors or from CAPD patients were lysed, and lysates were blotted with monoclonal antibodies against pERK. Total ERK expression was detected as a loading control. The histogram shows pERK/total ERK band intensity ratios. *P<0.05 control donors compared with CAPD patients. (B) Photomicrographs of confluent monolayers of non-epithelioid effluent-derived MCs treated for 48 hours with DMSO (NT) or U0126 (20 μM). (C) Top, western blot showing expression of E-cadherin in monolayer cultures of effluent-derived MCs or control MCs from normal omentum (O). Confluent monolayers of MCs were treated with DMSO or U0126. Alternatively, cells were infected with either a retrovirus encoding an IκBα super-repressor (IκB) or with empty virus (CopGreen). After 48 hours, cells were lysed and the lysates subjected to western blotting with monoclonal anti-E-cadherin antibody. Expression of α-tubulin was detected as a loading control. Bottom, densitometry of western blots showing E-cadherin expression upon treatment with DMSO or U0126 (patients 1-6), or infection with IκBα super-repressor (IκB) or empty virus (CopG) (patients 1, 2, 5, 6). *P<0.05 compared with DMSO-treated or empty-vector-infected cells. (D) Top, confocal immunofluorescence analysis of the effect of ERK inhibition on NF-κB and cytokeratin expression in transitional peritoneal mesothelium. Confluent monolayers of effluent-derived MCs (patient 10), showing non-epithelioid morphology, were treated with DMSO or U0126 for 56 hours. Cells were stained with polyclonal anti-p65 NF-κB and monoclonal anti-cytokeratin antibodies. Nuclei were stained with Hoechst 33342 (blue). Bottom-left, the histogram shows mean fluorescence intensities of nuclear NF-κB staining from cells treated as in the top panel (patients 1–3, 9, 10), quantified using the software LAS-AS from Leica. Bars represent s.e.m. A total of 250 cells were analyzed per condition; AU, arbitrary units. Bottom-center, western blots showing cytokeratin and E-cadherin expression in non-epithelioid effluent-derived MCs treated with DMSO (D) or U0126 (U0) (patients 11–13). Bottom-right, quantification of the western blot shown in the middle panel. (E) Snail1 and E-cadherin mRNA expression in effluent-derived MCs (patients 7–10) treated with DMSO (gray) or U0126 (black) for 24 hours. Quantitative RT-PCR was performed on total RNA. Histone H3 mRNA expression was used for normalization. Bars represent means ± s.e.m. of duplicate determinations from four independent experiments using cells from four different patients. *P<0.05 compared with DMSO-treated cells.