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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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Intracellular distribution of cell contact– and/or TGFβ-sensitive transcriptional coactivators Smad3, MRTF, and TAZ in cells residing in the intact or wound-adjacent areas of the monolayer. Cells were plated on coverslips affixed with a tape (wound) and treated with or without TGFβ for 6 h. Cells were then immunostained for (A) Smad3, (B) TAZ, or (C) MRTF and counterstained with the nuclear dye DAPI. The nuclear vs. cytosolic distribution of the corresponding proteins in cells at the wound edge or in the intact region without or after TGFβ treatment was visualized by immunofluorescence microscopy.
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Figure 4: Intracellular distribution of cell contact– and/or TGFβ-sensitive transcriptional coactivators Smad3, MRTF, and TAZ in cells residing in the intact or wound-adjacent areas of the monolayer. Cells were plated on coverslips affixed with a tape (wound) and treated with or without TGFβ for 6 h. Cells were then immunostained for (A) Smad3, (B) TAZ, or (C) MRTF and counterstained with the nuclear dye DAPI. The nuclear vs. cytosolic distribution of the corresponding proteins in cells at the wound edge or in the intact region without or after TGFβ treatment was visualized by immunofluorescence microscopy.

Mentions: Although the decrease in Smad3 expression in the TGFβ-treated wound was in good agreement with our previous studies suggesting that Smad3 is a negative regulator of MRTF, it was also somewhat surprising since low cell density or low calcium-mediated contact disruption have been reported to facilitate Smad2/3 signaling (Varelas et al., 2010). The proposed mechanism is that these stimuli promote the nuclear translocation of the Hippo pathway transcriptional regulators TAZ and YAP, which in turn act as nuclear retention factors for Smad2/3, thereby facilitating their accumulation and effects (Varelas et al., 2008, 2010). To test whether a similar mechanism is present in the near-wound areas and whether nuclear Smad3 levels follow a biphasic pattern, we compared TGFβ-induced Smad3 translocation at the wound edge and in the intact area after 6 h of stimulation (Figure 4A). In the absence of TGFβ, Smad3 was cytosolic in both the intact and wound regions. We often observed slight perinuclear accumulation with clear nuclear exclusion. After 6 h of TGFβ stimulation, there was a dramatic difference in the distribution of Smad3 in the wound versus the intact region. Cells along the wound showed pronounced nuclear Smad3 accumulation, whereas cells residing in the intact area exhibited only somewhat enhanced perinuclear labeling (Figure 4A). Pronounced nuclear accumulation was not restricted to a single cell row but was present in several rows behind the edge, reminiscent of the belt from which Smad3 disappeared upon long-term stimulation (Figure 2C). It is worth noting that intact epithelial monolayers are responsive to Smad3 signaling in terms of Smad3 phosphorylation, translocation, and transcriptional responses (Varelas et al., 2008, 2010; Masszi et al., 2010; Supplemental Figure S2). However, Smad3 retention is strongly potentiated by the absence of cell contacts (Varelas et al., 2008, 2010). Moreover, early, enhanced Smad3 responsiveness may underlie subsequent Smad3 down-regulation as well. Indeed, nuclear accumulation and posttranslational modification of Smad3 were recently reported to be prerequisite for Smad3 degradation (see Discussion).


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)

Intracellular distribution of cell contact– and/or TGFβ-sensitive transcriptional coactivators Smad3, MRTF, and TAZ in cells residing in the intact or wound-adjacent areas of the monolayer. Cells were plated on coverslips affixed with a tape (wound) and treated with or without TGFβ for 6 h. Cells were then immunostained for (A) Smad3, (B) TAZ, or (C) MRTF and counterstained with the nuclear dye DAPI. The nuclear vs. cytosolic distribution of the corresponding proteins in cells at the wound edge or in the intact region without or after TGFβ treatment was visualized by immunofluorescence microscopy.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 4: Intracellular distribution of cell contact– and/or TGFβ-sensitive transcriptional coactivators Smad3, MRTF, and TAZ in cells residing in the intact or wound-adjacent areas of the monolayer. Cells were plated on coverslips affixed with a tape (wound) and treated with or without TGFβ for 6 h. Cells were then immunostained for (A) Smad3, (B) TAZ, or (C) MRTF and counterstained with the nuclear dye DAPI. The nuclear vs. cytosolic distribution of the corresponding proteins in cells at the wound edge or in the intact region without or after TGFβ treatment was visualized by immunofluorescence microscopy.
Mentions: Although the decrease in Smad3 expression in the TGFβ-treated wound was in good agreement with our previous studies suggesting that Smad3 is a negative regulator of MRTF, it was also somewhat surprising since low cell density or low calcium-mediated contact disruption have been reported to facilitate Smad2/3 signaling (Varelas et al., 2010). The proposed mechanism is that these stimuli promote the nuclear translocation of the Hippo pathway transcriptional regulators TAZ and YAP, which in turn act as nuclear retention factors for Smad2/3, thereby facilitating their accumulation and effects (Varelas et al., 2008, 2010). To test whether a similar mechanism is present in the near-wound areas and whether nuclear Smad3 levels follow a biphasic pattern, we compared TGFβ-induced Smad3 translocation at the wound edge and in the intact area after 6 h of stimulation (Figure 4A). In the absence of TGFβ, Smad3 was cytosolic in both the intact and wound regions. We often observed slight perinuclear accumulation with clear nuclear exclusion. After 6 h of TGFβ stimulation, there was a dramatic difference in the distribution of Smad3 in the wound versus the intact region. Cells along the wound showed pronounced nuclear Smad3 accumulation, whereas cells residing in the intact area exhibited only somewhat enhanced perinuclear labeling (Figure 4A). Pronounced nuclear accumulation was not restricted to a single cell row but was present in several rows behind the edge, reminiscent of the belt from which Smad3 disappeared upon long-term stimulation (Figure 2C). It is worth noting that intact epithelial monolayers are responsive to Smad3 signaling in terms of Smad3 phosphorylation, translocation, and transcriptional responses (Varelas et al., 2008, 2010; Masszi et al., 2010; Supplemental Figure S2). However, Smad3 retention is strongly potentiated by the absence of cell contacts (Varelas et al., 2008, 2010). Moreover, early, enhanced Smad3 responsiveness may underlie subsequent Smad3 down-regulation as well. Indeed, nuclear accumulation and posttranslational modification of Smad3 were recently reported to be prerequisite for Smad3 degradation (see Discussion).

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