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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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Differential, site-specific effects of TGFβ on EMyT-related protein expression. (A) Cell cultures were prepared and treated without or with TGFβ as in Figure 1. Samples obtained from the intact and wound regions were processed for Western blotting using antibodies against the indicated proteins. After densitometry, GAPDH-normalized values were renormalized to reflect fold changes compared with the intact, untreated control. Data are expressed as mean ± SEM; n ≥ 3. (B) Protein expression in the TGFβ-treated (72 h) monolayer as a function of the distance from the wound edge. Samples (2-mm strips) were collected from several rows adjacent to the wound edge at 5-mm increments and probed for the indicated proteins (top). After densitometry, GAPDH-normalized values were renormalized to the intact, untreated controls and plotted against the distance from the wound. (C) Site-specific protein expression was visualized by immunostaining for SMA and Smad3 in wound-adjacent areas of TGFβ-treated (72 h) monolayers. The solid white line denotes the wound edge. Right, representative intensity profiles for SMA and Smad3 staining obtained along the dotted lines (au, arbitrary units).
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Figure 2: Differential, site-specific effects of TGFβ on EMyT-related protein expression. (A) Cell cultures were prepared and treated without or with TGFβ as in Figure 1. Samples obtained from the intact and wound regions were processed for Western blotting using antibodies against the indicated proteins. After densitometry, GAPDH-normalized values were renormalized to reflect fold changes compared with the intact, untreated control. Data are expressed as mean ± SEM; n ≥ 3. (B) Protein expression in the TGFβ-treated (72 h) monolayer as a function of the distance from the wound edge. Samples (2-mm strips) were collected from several rows adjacent to the wound edge at 5-mm increments and probed for the indicated proteins (top). After densitometry, GAPDH-normalized values were renormalized to the intact, untreated controls and plotted against the distance from the wound. (C) Site-specific protein expression was visualized by immunostaining for SMA and Smad3 in wound-adjacent areas of TGFβ-treated (72 h) monolayers. The solid white line denotes the wound edge. Right, representative intensity profiles for SMA and Smad3 staining obtained along the dotted lines (au, arbitrary units).

Mentions: Our previous results suggest that alterations in protein stability also contribute to the steady-state expression of myogenic regulators in EMyT (Charbonney et al., 2011). Therefore we sought to determine how the observed mRNA changes correlate with the expression of the corresponding proteins. To this end we performed Western blot analysis of samples obtained at both loci either from untreated layers or after short- or long-term TGFβ treatment corresponding to the early or peak phases of EMyT. No SMA protein was expressed at either location in the absence of or after 6-h TGFβ exposure, nor was any site-specific difference observed in Smad3, β-catenin, or MRTF protein levels (Supplemental Figure S1B). After 72 h (Figure 2A), we detected robust SMA expression exclusively in the TGFβ-exposed wound. Of importance, Smad3 protein expression was strongly reduced in the TGFβ-treated wound region compared with the untreated control, whereas no major change was observed in the wound without TGFβ treatment or in the intact area exposed to TGFβ. β-Catenin protein expression was markedly increased in the TGFβ-treated wound sample and appeared modestly elevated after the individual stimuli. MRTF protein levels did not seem to significantly differ under any condition.


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)

Differential, site-specific effects of TGFβ on EMyT-related protein expression. (A) Cell cultures were prepared and treated without or with TGFβ as in Figure 1. Samples obtained from the intact and wound regions were processed for Western blotting using antibodies against the indicated proteins. After densitometry, GAPDH-normalized values were renormalized to reflect fold changes compared with the intact, untreated control. Data are expressed as mean ± SEM; n ≥ 3. (B) Protein expression in the TGFβ-treated (72 h) monolayer as a function of the distance from the wound edge. Samples (2-mm strips) were collected from several rows adjacent to the wound edge at 5-mm increments and probed for the indicated proteins (top). After densitometry, GAPDH-normalized values were renormalized to the intact, untreated controls and plotted against the distance from the wound. (C) Site-specific protein expression was visualized by immunostaining for SMA and Smad3 in wound-adjacent areas of TGFβ-treated (72 h) monolayers. The solid white line denotes the wound edge. Right, representative intensity profiles for SMA and Smad3 staining obtained along the dotted lines (au, arbitrary units).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3814143&req=5

Figure 2: Differential, site-specific effects of TGFβ on EMyT-related protein expression. (A) Cell cultures were prepared and treated without or with TGFβ as in Figure 1. Samples obtained from the intact and wound regions were processed for Western blotting using antibodies against the indicated proteins. After densitometry, GAPDH-normalized values were renormalized to reflect fold changes compared with the intact, untreated control. Data are expressed as mean ± SEM; n ≥ 3. (B) Protein expression in the TGFβ-treated (72 h) monolayer as a function of the distance from the wound edge. Samples (2-mm strips) were collected from several rows adjacent to the wound edge at 5-mm increments and probed for the indicated proteins (top). After densitometry, GAPDH-normalized values were renormalized to the intact, untreated controls and plotted against the distance from the wound. (C) Site-specific protein expression was visualized by immunostaining for SMA and Smad3 in wound-adjacent areas of TGFβ-treated (72 h) monolayers. The solid white line denotes the wound edge. Right, representative intensity profiles for SMA and Smad3 staining obtained along the dotted lines (au, arbitrary units).
Mentions: Our previous results suggest that alterations in protein stability also contribute to the steady-state expression of myogenic regulators in EMyT (Charbonney et al., 2011). Therefore we sought to determine how the observed mRNA changes correlate with the expression of the corresponding proteins. To this end we performed Western blot analysis of samples obtained at both loci either from untreated layers or after short- or long-term TGFβ treatment corresponding to the early or peak phases of EMyT. No SMA protein was expressed at either location in the absence of or after 6-h TGFβ exposure, nor was any site-specific difference observed in Smad3, β-catenin, or MRTF protein levels (Supplemental Figure S1B). After 72 h (Figure 2A), we detected robust SMA expression exclusively in the TGFβ-exposed wound. Of importance, Smad3 protein expression was strongly reduced in the TGFβ-treated wound region compared with the untreated control, whereas no major change was observed in the wound without TGFβ treatment or in the intact area exposed to TGFβ. β-Catenin protein expression was markedly increased in the TGFβ-treated wound sample and appeared modestly elevated after the individual stimuli. MRTF protein levels did not seem to significantly differ under any condition.

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