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. 2012;7(2):e31492.
doi: 10.1371/journal.pone.0031492. Epub 2012 Feb 27.

Inhibition of transforming growth factor-activated kinase 1 (TAK1) blocks and reverses epithelial to mesenchymal transition of mesothelial cells

Raffaele Strippoli  1 , Ignacio Benedicto, Maria Luisa Perez Lozano, Teijo Pellinen, Pilar Sandoval, Manuel Lopez-Cabrera, Miguel Angel del Pozo
Affiliations

Inhibition of transforming growth factor-activated kinase 1 (TAK1) blocks and reverses epithelial to mesenchymal transition of mesothelial cells

Raffaele Strippoli et al. PLoS One. 2012.

Abstract

Peritoneal fibrosis is a frequent complication of peritoneal dialysis following repeated low grade inflammatory and pro-fibrotic insults. This pathological process may lead to ultrafiltration failure and eventually to the discontinuing of the therapy. Fibrosis is linked to epithelial to mesenchymal transition (EMT) of the peritoneal mesothelial cells, which acquire invasive and fibrogenic abilities. Here, we analyzed the role of the transforming growth factor-activated kinase-1 (TAK1) in the EMT of primary mesothelial cells from human peritoneum. The inhibition of TAK1 in mesenchymal-like mesothelial cells from the effluents of patients undergoing peritoneal dialysis led to the reacquisition of the apical to basolateral polarity, to increased expression of epithelial and to down-regulation of mesenchymal markers. TAK1 inhibition also resulted in decreased migratory/invasive abilities of effluent-derived mesothelial cells. Simultaneous inhibition of ERK1/2 and TAK1 pathways did not lead to an additive effect in the reacquisition of the epithelial phenotype. Inhibition of TAK1 also blocked EMT in vitro and reduced the levels of PAI-1, which is involved in fibrosis and invasion. Analysis of signalling pathways downstream of TAK1 involved in EMT induction, showed that TAK1 inhibition reduced the transcriptional activity of NF-κB and Smad3, as well as the phosphorylation of c-jun, while enhancing Smad1-5-8 activity. These results demonstrate that TAK1 is a cross-point in a network including different pro-EMT transcription factors, such as NF-κB, Snail, AP-1 and Smads. The identification of TAK1 as a main biochemical mediator of EMT and fibrosis in mesothelial cells from human peritoneum and the study of signalling pathways induced by its activity may be relevant in the design of new therapies aimed to counteract peritoneal fibrosis.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. TAK1 inhibition leads to the reversal of the EMT phenotype (MET) in MCs derived from peritoneal effluent of PD patients.
A, Confocal immunofluorescence analysis (red) of MCs from a patient undergoing PD. Cells were treated with DMSO, NP-009245, (600 nM), CI 1040 (2 µM) for 72 h. Cells were fixed and stained with rhodamine phalloidin. Nuclei were stained with Hoechst 33342 (blue). The immunofluorescence shown is representative of three independent experiments. B, Western blots showing the expression of E-cadherin in total cell lysates of MCs. Cells were treated with DMSO, NP-009245 (T) (600 nM), CI 1040 (CI) (2 µM), or a combination of the two pharmacological inhibitors for 72 h. Expression of tubulin was detected as a loading control. C, Effect of TAK1 pharmacological inhibition on E-cadherin mRNA expression in MCs from patients undergoing PD. Cells were treated as in A. Quantitative RT-PCR was performed on total RNA. Histone H3 mRNA levels were used for normalization. Bars represent means+s. e. m. of duplicate determinations in three independent experiments. D, Effect of TAK1 pharmacological inhibition on N-cadherin mRNA expression in MCs from patients undergoing PD. Cells were treated with DMSO or NP-009245, (600 nM) for 72 h. Quantitative RT-PCR was performed on total RNA. Histone H3 mRNA levels were used for normalization. Bars represent means+s. e. m. of duplicate determinations in three independent experiments. E and F, Confocal immunofluorescence analysis of MCs from patients undergoing PD. Cells were treated with DMSO or NP-009245, (600 nM) for 72 h. Cells were fixed and stained with a monoclonal antibody against cytokeratin (red) (D) or a polyclonal antibody against ZO-1 (green) (E). Nuclei were stained with Hoechst 33342 (blue). All data are representative of at least three independent experiments. * p<0.05 versus DMSO treated cells.
Figure 2
Figure 2. TAK1 inhibition limits the expression of mesenchymal markers by MCs derived from peritoneal effluent of PD patients.
A, Confocal immunofluorescence (red) of MCs from a patient undergoing PD. Cells were treated with DMSO or NP-009245 (600 nM) for 72 h. Cells were fixed and stained with a monoclonal antibody against fibronectin. Nuclei were stained with Hoechst 33342 (blue). The immunofluorescence shown is representative of three independent experiments. B, Western blots showing the expression of fibronectin, E-cadherin and vimentin in total cell lysates of MCs treated with DMSO, NP-009245 (600 nM) or CI-1040 (2 µM) for 72 h. Expression of tubulin was used as a loading control. The results shown are representative of three independent experiments. C, Effect of TAK1 pharmacological inhibition on fibronectin and E-cadherin mRNA expression in MCs from patients undergoing PD. Quantitative RT-PCR was performed on total RNA from MCs treated as in A. Histone H3 mRNA levels were used for normalization. Bars represent means+s. e. m. of duplicate determinations in three independent experiments. D, Western blots showing the expression of E-cadherin, fibronectin and TAK1 in total cell lysates of MCs transfected with either control or specific TAK1-targeting siRNAs. Scr, cells transfected with control siRNA. Data are representative of three independent experiments. E, Effect of TAK1 siRNA silencing on E-cadherin mRNA expression in MCs. Cells were treated as in D. Quantitative RT-PCR was performed; bars represent means+s. e. m. of duplicate determinations in three independent experiments. * p<0.05 versus siScrambled treated cells.
Figure 3
Figure 3. TAK1 inhibition limits the induction of EMT in HPMCs treated with TGF-β1 in combination with IL-1β.
Effect of TAK1 pharmacological inhibition on E-cadherin (A, C), Snail (B, D), fibronectin (E) and type I collagen (F) mRNA expression in HPMCs. Quantitative RT-PCR was performed on total RNA from MCs pre-treated with DMSO, or NP-009245, (600 nM) for 1 h and then left untreated or stimulated with TGF-β1 0.5 ng/ml) in combination with IL-1β 2 ng/ml) (T/I) or TGF-β1 alone at the same concentration (T) for 24 h. Histone H3 mRNA levels were used for normalization. Bars represent means+s. e. m. of duplicate determinations in three independent experiments. * p<0.05 versus DMSO treated cells.
Figure 4
Figure 4. TAK1 controls the activation of multiple transcriptional factors relevant for the induction of EMT and fibrosis.
A, Effect of TAK1 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 with NP-009245 (600 nM) for 12 h and then left untreated (NT, gray bars) or co-stimulated for 9 h with TGF-β1 (0.5 ng/ml) in combination with IL-1β (2 ng/ml) (T/I). Bars represent means+s. e. m. of triplicate determinations in three independent experiments. B, Effect of TAK1 inhibition on PAI-1 transcriptional activity. MeT-5A cells were transiently transfected with a PAI-1 reporter plasmid together with a Renilla luciferase-coding plasmid. Cells were stimulated as above. Bars represent means+s. e. m. of triplicate determinations in three independent experiments C, Western blots showing the expression of phospho-Smad3, phospho-Smad1–5, PAI-1 and phospho-c-jun in total cell lysates of HPMCs treated for different times with TGF-β1 0.5 ng/ml) in combination with IL-1β (2 ng/ml) (T/I). Expression of tubulin was used as a loading control. The results shown are representative of three independent experiments. D, Effect of TAK1 inhibition on Smad1–5–8 transcriptional activity. MeT-5A cells were transiently transfected with the BRE-luc reporter plasmid together with a Renilla luciferase-coding plasmid. Cells were pretreated with NP-009245 (600 nM) for 12 h and then left untreated (NT, gray bars) or co-stimulated for 12 h with TGF-β1 (0.5 ng/ml) in combination with IL-1β (2 ng/ml) (T/I). Bars represent means+s. e. m. of triplicate determinations in three independent experiments. E, Western blots showing the expression of phospho-Smad1–5, PAI-1 and TAK1 in whole cell lysates of HPMCs transfected with either control or specific TAK1-targeting siRNAs and then left untreated or stimulated for 4 h with TGF-β1 (0.5 ng/ml) in combination with IL-1β (2 ng/ml) (TI). Scr: cells transfected with control siRNA. Data are representative of three independent experiments F, Western blot showing the expression of phospho-Smad1–5–8 in MCs from a patient undergoing PD. Cells were treated with DMSO, CI 1040 (2 µM), NP-009245, (600 nM) for 24 h. Expression of tubulin was used as a loading control. All data are representative of at three independent experiments. * p<0.05 versus corresponding sample treated with DMSO.
Figure 5
Figure 5. TAK1 controls wound closure and invasion on type I Collagen gel in MCs from peritoneal effluent.
A, Effect of TAK1 inhibition on wound closure. Left, MCs from a patient undergoing PD were allowed to reach 100% confluency in a 6 well plate. MCs were pre-treated with DMSO or NP-009245 (600 nM) for 24 h in culture medium supplemented with 10% FCS. A scratch wound was created on the cell surface using a micropipette tip. The wound area was photographed every 30 min for 24 h by bright field microscopy (4× magnification). The width of the wound was measured and the wound closure rate was calculated. 9 different points from 3 different scratches were analyzed per condition. Only the beginning and the end of the test analysis is shown. The result described is representative of 5 independent experiments. Right, quantification of the experiment described before. P *<0.05 compared to DMSO-treated cells. B, Effect of TAK1 and MEK1 inhibition on MCs invasion on type I Collagen gel. MCs were pre-treated with DMSO, NP-009245 (600 nM) or CI-1040 (2 µM) for 24 h. Cells were allowed to invade type I Collagen gels for 24 hours. Invading cells were fixed and nuclei were counted. Nuclei were counted in ten fields per sample using a fluorescence microscope (40× magnification). Each experiment was carried out in duplicate, and at least 5 experiments were performed. P *<0.05 compared to DMSO-treated cells.
Figure 6
Figure 6. TAK1 as a checkpoint of major signalling pathways controlling EMT and fibrosis.
Rapidly induced by a wide array of pro-inflammatory and pro-fibrotic stimuli, TAK1 activation plays a central role in the induction of the EMT program. Besides controlling classical inflammatory pathways, such as NF-κB and MAPKs, TAK1 activation affects Smad3/Smad1–5 balance. TAK1 activation in MCs relies on cadherin switching, PAI-1 expression, increased ECM protein production, as well as the acquisition of invasive abilities. See text for details.
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