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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

Interplay between contact-regulated and TGFβ-induced signaling promotes wound-restricted EMyT. The proposed mechanisms are the following. Contact disruption promotes the nuclear translocation of three junction-regulated transcriptional coactivators: β-catenin, MRTF, and TAZ. TGFβ concomitantly induces nuclear translocation of Smad3. β-Catenin activates mesenchymal genes and maintains the stability and expression of MRTF, which in turn drives the SMA promoter. TAZ further facilitates the SMA promoter and also acts as a Smad3 retention factor. The latter effect is likely responsible for the enhanced, wound-specific Smad3 accumulation in the initial phase of EMyT. Augmented early Smad3 signaling temporarily inhibits the action of MRTF, but it also primes Smad3 for subsequent degradation. Moreover, TAZ suppresses the Smad3 promoter. Ultimately these effects lead to a strong reduction in Smad3 expression. Reduced Smad3 levels allow the disinhibition of MRTF, whereas they may be sufficient to promote other (e.g., TAZ-dependent) inputs, thereby both liberating and supporting the myogenic phase of the transition (the ± sign indicates both effects of Smad3). Together these events ensure locus-specific phenotypic reprogramming and temporally coordinate the mesenchymal and myogenic phases of EMyT.
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Figure 7: Interplay between contact-regulated and TGFβ-induced signaling promotes wound-restricted EMyT. The proposed mechanisms are the following. Contact disruption promotes the nuclear translocation of three junction-regulated transcriptional coactivators: β-catenin, MRTF, and TAZ. TGFβ concomitantly induces nuclear translocation of Smad3. β-Catenin activates mesenchymal genes and maintains the stability and expression of MRTF, which in turn drives the SMA promoter. TAZ further facilitates the SMA promoter and also acts as a Smad3 retention factor. The latter effect is likely responsible for the enhanced, wound-specific Smad3 accumulation in the initial phase of EMyT. Augmented early Smad3 signaling temporarily inhibits the action of MRTF, but it also primes Smad3 for subsequent degradation. Moreover, TAZ suppresses the Smad3 promoter. Ultimately these effects lead to a strong reduction in Smad3 expression. Reduced Smad3 levels allow the disinhibition of MRTF, whereas they may be sufficient to promote other (e.g., TAZ-dependent) inputs, thereby both liberating and supporting the myogenic phase of the transition (the ± sign indicates both effects of Smad3). Together these events ensure locus-specific phenotypic reprogramming and temporally coordinate the mesenchymal and myogenic phases of EMyT.

Mentions: What are the molecular mechanisms by which the absence or disassembly of cell contacts synergizes with TGFβ-triggered myofibroblast formation? Our results suggest interplay among three contact- and cytoskeleton-dependent transcriptional coactivators and Smad3 (Figure 7). Previous studies showed that contact disruption stimulates RhoA and Rac, which in turn induce (transient) nuclear translocation of MRTF (Fan et al., 2007; Busche et al., 2008). TGFβ prolongs the nuclear accumulation of MRTF by a yet-unknown mechanism. However, the activity of MRTF on the SMA promoter is negatively regulated by Smad3, and thus additional factors are necessary to relieve this inhibition and augment SMA expression. One such factor is β-catenin, which, when released from the injured contacts (or present in the cytosol in high enough concentration), binds to Smad3 and mitigates its negative action on MRTF (Charbonney et al., 2011). In addition, β-catenin counteracts the adaptor function of Smad3, preventing it from recruiting GSK-3β, which phosphorylates and primes MRTF for degradation (Charbonney et al., 2011). Our present work shows that β-catenin, through the above-described mechanism, plays an essential permissive role in the stabilization of the MRTF protein specifically in the wound. In addition, β-catenin is also needed for sustained MRTF mRNA expression at this locus. Such β-catenin–dependent transcriptional regulation of MRTF might explain the recent finding that Wnt2-knockout embryos show reduced MRTF expression (Goss et al., 2011). Overall β-catenin may support MRTF function via both mechanisms, explaining at least in part why β-catenin is required for SMA expression during EMyT (Charbonney et al., 2011) and fibrogenesis (Zhou et al., 2012).


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)

Interplay between contact-regulated and TGFβ-induced signaling promotes wound-restricted EMyT. The proposed mechanisms are the following. Contact disruption promotes the nuclear translocation of three junction-regulated transcriptional coactivators: β-catenin, MRTF, and TAZ. TGFβ concomitantly induces nuclear translocation of Smad3. β-Catenin activates mesenchymal genes and maintains the stability and expression of MRTF, which in turn drives the SMA promoter. TAZ further facilitates the SMA promoter and also acts as a Smad3 retention factor. The latter effect is likely responsible for the enhanced, wound-specific Smad3 accumulation in the initial phase of EMyT. Augmented early Smad3 signaling temporarily inhibits the action of MRTF, but it also primes Smad3 for subsequent degradation. Moreover, TAZ suppresses the Smad3 promoter. Ultimately these effects lead to a strong reduction in Smad3 expression. Reduced Smad3 levels allow the disinhibition of MRTF, whereas they may be sufficient to promote other (e.g., TAZ-dependent) inputs, thereby both liberating and supporting the myogenic phase of the transition (the ± sign indicates both effects of Smad3). Together these events ensure locus-specific phenotypic reprogramming and temporally coordinate the mesenchymal and myogenic phases of EMyT.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

Show All Figures
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Figure 7: Interplay between contact-regulated and TGFβ-induced signaling promotes wound-restricted EMyT. The proposed mechanisms are the following. Contact disruption promotes the nuclear translocation of three junction-regulated transcriptional coactivators: β-catenin, MRTF, and TAZ. TGFβ concomitantly induces nuclear translocation of Smad3. β-Catenin activates mesenchymal genes and maintains the stability and expression of MRTF, which in turn drives the SMA promoter. TAZ further facilitates the SMA promoter and also acts as a Smad3 retention factor. The latter effect is likely responsible for the enhanced, wound-specific Smad3 accumulation in the initial phase of EMyT. Augmented early Smad3 signaling temporarily inhibits the action of MRTF, but it also primes Smad3 for subsequent degradation. Moreover, TAZ suppresses the Smad3 promoter. Ultimately these effects lead to a strong reduction in Smad3 expression. Reduced Smad3 levels allow the disinhibition of MRTF, whereas they may be sufficient to promote other (e.g., TAZ-dependent) inputs, thereby both liberating and supporting the myogenic phase of the transition (the ± sign indicates both effects of Smad3). Together these events ensure locus-specific phenotypic reprogramming and temporally coordinate the mesenchymal and myogenic phases of EMyT.
Mentions: What are the molecular mechanisms by which the absence or disassembly of cell contacts synergizes with TGFβ-triggered myofibroblast formation? Our results suggest interplay among three contact- and cytoskeleton-dependent transcriptional coactivators and Smad3 (Figure 7). Previous studies showed that contact disruption stimulates RhoA and Rac, which in turn induce (transient) nuclear translocation of MRTF (Fan et al., 2007; Busche et al., 2008). TGFβ prolongs the nuclear accumulation of MRTF by a yet-unknown mechanism. However, the activity of MRTF on the SMA promoter is negatively regulated by Smad3, and thus additional factors are necessary to relieve this inhibition and augment SMA expression. One such factor is β-catenin, which, when released from the injured contacts (or present in the cytosol in high enough concentration), binds to Smad3 and mitigates its negative action on MRTF (Charbonney et al., 2011). In addition, β-catenin counteracts the adaptor function of Smad3, preventing it from recruiting GSK-3β, which phosphorylates and primes MRTF for degradation (Charbonney et al., 2011). Our present work shows that β-catenin, through the above-described mechanism, plays an essential permissive role in the stabilization of the MRTF protein specifically in the wound. In addition, β-catenin is also needed for sustained MRTF mRNA expression at this locus. Such β-catenin–dependent transcriptional regulation of MRTF might explain the recent finding that Wnt2-knockout embryos show reduced MRTF expression (Goss et al., 2011). Overall β-catenin may support MRTF function via both mechanisms, explaining at least in part why β-catenin is required for SMA expression during EMyT (Charbonney et al., 2011) and fibrogenesis (Zhou et al., 2012).

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