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Differential topical susceptibility to TGFβ in intact and injured regions of the epithelium: key role in myofibroblast transition.

Speight P, Nakano H, Kelley TJ, Hinz B, Kapus A - Mol. Biol. Cell (2013)

Bottom Line: We show that TGFβ elicits dramatically different responses at these two loci.Mechanistically, three transcriptional coactivators whose localization is regulated by cell contact integrity are critical for these local effects.Remarkably, active TAZ stimulates the SMA and suppresses the Smad3 promoter, whereas TAZ silencing prevents wound-restricted expression of SMA and loss of Smad3.

View Article: PubMed Central - PubMed

Affiliation: Keenan Research Centre, Li Ka Shing Knowledge Institute, St. Michael's Hospital, and Department of Surgery, University of Toronto, Toronto, ON M5B 1W8, Canada Department of Immunology, Juntendo University School of Medicine, Tokyo 113-8421, Japan Division of Pediatric Pulmonology, Case Western Reserve University, Cleveland, OH 44106 Laboratory of Tissue Repair and Regeneration, Matrix Dynamics Group, Faculty of Dentistry, University of Toronto, Toronto, ON M5S 3E2, Canada.

ABSTRACT
Induction of epithelial-myofibroblast transition (EMyT), a robust fibrogenic phenotype change hallmarked by α-smooth muscle actin (SMA) expression, requires transforming growth factor-β1 (TGFβ) and the absence/uncoupling of intracellular contacts. This suggests that an "injured" epithelium may be topically susceptible to TGFβ. To explore this concept, we use an epithelial wound model in which intact and contact-deprived regions of the same monolayer can be analyzed simultaneously. We show that TGFβ elicits dramatically different responses at these two loci. SMA expression and initially enhanced nuclear Smad3 accumulation followed by Smad3 mRNA and protein down-regulation occur exclusively at the wound. Mechanistically, three transcriptional coactivators whose localization is regulated by cell contact integrity are critical for these local effects. These are myocardin-related transcription factor (MRTF), the driver of the SMA promoter; β-catenin, which counteracts the known inhibitory effect of Smad3 on MRTF and maintains MRTF protein stability and mRNA expression in the wound; and TAZ, a Hippo effector and Smad3 retention factor. Remarkably, active TAZ stimulates the SMA and suppresses the Smad3 promoter, whereas TAZ silencing prevents wound-restricted expression of SMA and loss of Smad3. Such locus-specific reprogramming might play key roles in wound healing and the susceptibility of the injured epithelium to fibrogenesis.

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Related in: MedlinePlus

β-Catenin is essential for maintaining MRTF stability and mRNA expression in the wound. (A) Cells were transfected with NR or β-catenin–specific siRNA and treated with or without TGFβ for 72 h. Samples collected from the wound edge or the intact area were probed for the indicated proteins. (B) SMA mRNA analysis performed in samples prepared as in A. (C) To assess the time dependence of MRTF down-regulation in β-catenin–depleted cells, cultures were transfected as in A and treated with TGFβ for 24 or 72 h. MRTF expression, normalized to GAPDH, was quantified by densitometry. (D) After transfection with NR or β-catenin siRNA as in A, cultures were exposed to TGFβ for 24 h. Cells collected from indicated areas were subjected to immunoprecipitation using an anti-MRTF antibody. The precipitates were probed for MRTF and coprecipitating GSK-3β and Smad3. The graph shows densitometric quantification of the cosedimented GSK-3β normalized to the precipitated MRTF (mean ± SEM; n = 3). (E) MRTF mRNA analysis performed in samples prepared as in A.
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Figure 3: β-Catenin is essential for maintaining MRTF stability and mRNA expression in the wound. (A) Cells were transfected with NR or β-catenin–specific siRNA and treated with or without TGFβ for 72 h. Samples collected from the wound edge or the intact area were probed for the indicated proteins. (B) SMA mRNA analysis performed in samples prepared as in A. (C) To assess the time dependence of MRTF down-regulation in β-catenin–depleted cells, cultures were transfected as in A and treated with TGFβ for 24 or 72 h. MRTF expression, normalized to GAPDH, was quantified by densitometry. (D) After transfection with NR or β-catenin siRNA as in A, cultures were exposed to TGFβ for 24 h. Cells collected from indicated areas were subjected to immunoprecipitation using an anti-MRTF antibody. The precipitates were probed for MRTF and coprecipitating GSK-3β and Smad3. The graph shows densitometric quantification of the cosedimented GSK-3β normalized to the precipitated MRTF (mean ± SEM; n = 3). (E) MRTF mRNA analysis performed in samples prepared as in A.

Mentions: Because MRTF is indispensable for SMA expression (Masszi et al., 2010) and our recent studies implicated β-catenin as a regulator of MRTF stability (Charbonney et al., 2011), we sought to determine whether β-catenin affects MRTF expression in our model and, if so, whether this occurs in a topically differential manner. To this end, we transfected confluent monolayers harboring a central wound region with nonrelated (NR) or β-catenin–specific siRNA for 24 h and then left them untreated or exposed to TGFβ for 72 h. The specific siRNA completely eliminated β-catenin protein expression both in the intact area and in the wound (Figure 3A). Concomitantly, it prevented TGFβ-induced SMA mRNA and protein expression and reduced MRTF protein expression in the wound (Figure 3, A and B), indicating that β-catenin is an absolute requisite for the myogenic response. To assess the kinetics of the loss of MRTF, we compared the changes in MRTF protein expression in the intact and wound areas after 24 and 72 h of TGFβ exposure (Figure 3C). In the intact region there was no substantive difference in MRTF expression after short- or long-term TGFβ treatment, irrespective of whether β-catenin was down-regulated. Whereas β-catenin silencing did not alter MRTF protein levels in the wound at 24 h, it did cause a significant reduction by 72 h, pointing to a wound-specific, gradually developing, β-catenin–counteracted loss.


Differential topical susceptibility to TGFβ in intact and injured regions of the epithelium: key role in myofibroblast transition.

Speight P, Nakano H, Kelley TJ, Hinz B, Kapus A - Mol. Biol. Cell (2013)

β-Catenin is essential for maintaining MRTF stability and mRNA expression in the wound. (A) Cells were transfected with NR or β-catenin–specific siRNA and treated with or without TGFβ for 72 h. Samples collected from the wound edge or the intact area were probed for the indicated proteins. (B) SMA mRNA analysis performed in samples prepared as in A. (C) To assess the time dependence of MRTF down-regulation in β-catenin–depleted cells, cultures were transfected as in A and treated with TGFβ for 24 or 72 h. MRTF expression, normalized to GAPDH, was quantified by densitometry. (D) After transfection with NR or β-catenin siRNA as in A, cultures were exposed to TGFβ for 24 h. Cells collected from indicated areas were subjected to immunoprecipitation using an anti-MRTF antibody. The precipitates were probed for MRTF and coprecipitating GSK-3β and Smad3. The graph shows densitometric quantification of the cosedimented GSK-3β normalized to the precipitated MRTF (mean ± SEM; n = 3). (E) MRTF mRNA analysis performed in samples prepared as in A.
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Related In: Results  -  Collection

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Figure 3: β-Catenin is essential for maintaining MRTF stability and mRNA expression in the wound. (A) Cells were transfected with NR or β-catenin–specific siRNA and treated with or without TGFβ for 72 h. Samples collected from the wound edge or the intact area were probed for the indicated proteins. (B) SMA mRNA analysis performed in samples prepared as in A. (C) To assess the time dependence of MRTF down-regulation in β-catenin–depleted cells, cultures were transfected as in A and treated with TGFβ for 24 or 72 h. MRTF expression, normalized to GAPDH, was quantified by densitometry. (D) After transfection with NR or β-catenin siRNA as in A, cultures were exposed to TGFβ for 24 h. Cells collected from indicated areas were subjected to immunoprecipitation using an anti-MRTF antibody. The precipitates were probed for MRTF and coprecipitating GSK-3β and Smad3. The graph shows densitometric quantification of the cosedimented GSK-3β normalized to the precipitated MRTF (mean ± SEM; n = 3). (E) MRTF mRNA analysis performed in samples prepared as in A.
Mentions: Because MRTF is indispensable for SMA expression (Masszi et al., 2010) and our recent studies implicated β-catenin as a regulator of MRTF stability (Charbonney et al., 2011), we sought to determine whether β-catenin affects MRTF expression in our model and, if so, whether this occurs in a topically differential manner. To this end, we transfected confluent monolayers harboring a central wound region with nonrelated (NR) or β-catenin–specific siRNA for 24 h and then left them untreated or exposed to TGFβ for 72 h. The specific siRNA completely eliminated β-catenin protein expression both in the intact area and in the wound (Figure 3A). Concomitantly, it prevented TGFβ-induced SMA mRNA and protein expression and reduced MRTF protein expression in the wound (Figure 3, A and B), indicating that β-catenin is an absolute requisite for the myogenic response. To assess the kinetics of the loss of MRTF, we compared the changes in MRTF protein expression in the intact and wound areas after 24 and 72 h of TGFβ exposure (Figure 3C). In the intact region there was no substantive difference in MRTF expression after short- or long-term TGFβ treatment, irrespective of whether β-catenin was down-regulated. Whereas β-catenin silencing did not alter MRTF protein levels in the wound at 24 h, it did cause a significant reduction by 72 h, pointing to a wound-specific, gradually developing, β-catenin–counteracted loss.

Bottom Line: We show that TGFβ elicits dramatically different responses at these two loci.Mechanistically, three transcriptional coactivators whose localization is regulated by cell contact integrity are critical for these local effects.Remarkably, active TAZ stimulates the SMA and suppresses the Smad3 promoter, whereas TAZ silencing prevents wound-restricted expression of SMA and loss of Smad3.

View Article: PubMed Central - PubMed

Affiliation: Keenan Research Centre, Li Ka Shing Knowledge Institute, St. Michael's Hospital, and Department of Surgery, University of Toronto, Toronto, ON M5B 1W8, Canada Department of Immunology, Juntendo University School of Medicine, Tokyo 113-8421, Japan Division of Pediatric Pulmonology, Case Western Reserve University, Cleveland, OH 44106 Laboratory of Tissue Repair and Regeneration, Matrix Dynamics Group, Faculty of Dentistry, University of Toronto, Toronto, ON M5S 3E2, Canada.

ABSTRACT
Induction of epithelial-myofibroblast transition (EMyT), a robust fibrogenic phenotype change hallmarked by α-smooth muscle actin (SMA) expression, requires transforming growth factor-β1 (TGFβ) and the absence/uncoupling of intracellular contacts. This suggests that an "injured" epithelium may be topically susceptible to TGFβ. To explore this concept, we use an epithelial wound model in which intact and contact-deprived regions of the same monolayer can be analyzed simultaneously. We show that TGFβ elicits dramatically different responses at these two loci. SMA expression and initially enhanced nuclear Smad3 accumulation followed by Smad3 mRNA and protein down-regulation occur exclusively at the wound. Mechanistically, three transcriptional coactivators whose localization is regulated by cell contact integrity are critical for these local effects. These are myocardin-related transcription factor (MRTF), the driver of the SMA promoter; β-catenin, which counteracts the known inhibitory effect of Smad3 on MRTF and maintains MRTF protein stability and mRNA expression in the wound; and TAZ, a Hippo effector and Smad3 retention factor. Remarkably, active TAZ stimulates the SMA and suppresses the Smad3 promoter, whereas TAZ silencing prevents wound-restricted expression of SMA and loss of Smad3. Such locus-specific reprogramming might play key roles in wound healing and the susceptibility of the injured epithelium to fibrogenesis.

Show MeSH
Related in: MedlinePlus