Limits...
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.

Show MeSH

Related in: MedlinePlus

TAZ activates and potentiates the SMA promoter and suppresses the Smad3 promoter. (A, B) MRTF potently drives the SMA promoter but fails to inhibit the Smad3 promoter. Cells were transfected with MRTF along with either the SMA promoter luciferase or the Smad3 promoter luciferase reporter system. (C) Cells were cotransfected with the SMA reporter and either active (S89A) TAZ or control plasmid. Cells were then left untreated or exposed to the individual or combined stimuli of the two-hit scheme (TGFβ or/and LCM), and 24 h later luciferase activities were determined. Note that TAZ increases the activity of the SMA promoter and potentiates the stimulating effect of the other stimuli. (D) Confluent cells were cotransfected with active TAZ and the Smad3 promoter construct, and 24 h later luciferase activities were determined as in C.
© Copyright Policy - creative-commons
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3814143&req=5

Figure 5: TAZ activates and potentiates the SMA promoter and suppresses the Smad3 promoter. (A, B) MRTF potently drives the SMA promoter but fails to inhibit the Smad3 promoter. Cells were transfected with MRTF along with either the SMA promoter luciferase or the Smad3 promoter luciferase reporter system. (C) Cells were cotransfected with the SMA reporter and either active (S89A) TAZ or control plasmid. Cells were then left untreated or exposed to the individual or combined stimuli of the two-hit scheme (TGFβ or/and LCM), and 24 h later luciferase activities were determined. Note that TAZ increases the activity of the SMA promoter and potentiates the stimulating effect of the other stimuli. (D) Confluent cells were cotransfected with active TAZ and the Smad3 promoter construct, and 24 h later luciferase activities were determined as in C.

Mentions: In search for the mechanism underlying the concomitant SMA mRNA expression and Smad3 mRNA suppression, first we considered that MRTF might inhibit Smad3 transcription. To assess this possibility, we overexpressed MRTF along with either an SMA or a Smad3 promoter luciferase construct. Although MRTF overexpression drove the SMA promoter 25-fold (testifying to its activity; Figure 5A), it failed to affect the Smad3 promoter (Figure 5B). As an alternative, we tested the effect of active and constitutively nuclear TAZ (S89A) on these promoters. For these cotransfection experiments, where the wound edge cannot be targeted specifically, we used our two-hit scheme (LCM and TGFβ; Masszi et al., 2004, 2010). Surprisingly, expression of active TAZ induced a four- to fivefold activation of the SMA promoter and potentiated its activation to the same extent by the individual and combined effect of TGFβ and LCM, attaining an overall >40-fold stimulation under the two-hit conditions (Figure 5C). Moreover, overexpression of active TAZ inhibited the Smad3 promoter by 80% (Figure 5D). To test whether TAZ indeed regulates SMA and Smad3 mRNA expression in the context of the wound model, we transfected the cells with NR or TAZ-specific siRNA and measured the message for these proteins at both loci with or without TGFβ treatment. As expected, the specific siRNA efficiently suppressed the message for TAZ under all conditions (Figure 6A). Remarkably, down-regulation of TAZ completely eliminated the induction of SMA mRNA (Figure 6B) and prevented the loss of Smad3 mRNA (Figure 6C) in the TGFβ-exposed wound. To verify that these changes are reflected on the corresponding protein levels, we compared the expression of SMA and Smad3 protein in samples obtained from the intact untreated region with those collected from the TGFβ-treated wound area. Elimination of TAZ prevented both the expression of SMA and the down-regulation of Smad3 in the TGFβ-treated wound region (Figure 6D). Thus TAZ is an essential factor for the topical sensitivity of the contact-deprived epithelium in terms of both SMA expression and Smad3 suppression. However, it should be mentioned that TAZ alone is not sufficient to drive SMA expression, as indicated by the fact that neither wounding (which induces nuclear accumulation of TAZ) nor the expression of constitutively active nuclear TAZ induced detectable SMA protein expression. Nonetheless, consistent with the potentiation of the promoter (Figure 5C), expression of active TAZ rendered TGFβ capable of inducing SMA expression. Although TGFβ alone never provoked SMA expression in a confluent monolayer, it induced SMA expression in a significant fraction of active TAZ-transfected cells (Supplemental Figure S3, A and B).


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)

TAZ activates and potentiates the SMA promoter and suppresses the Smad3 promoter. (A, B) MRTF potently drives the SMA promoter but fails to inhibit the Smad3 promoter. Cells were transfected with MRTF along with either the SMA promoter luciferase or the Smad3 promoter luciferase reporter system. (C) Cells were cotransfected with the SMA reporter and either active (S89A) TAZ or control plasmid. Cells were then left untreated or exposed to the individual or combined stimuli of the two-hit scheme (TGFβ or/and LCM), and 24 h later luciferase activities were determined. Note that TAZ increases the activity of the SMA promoter and potentiates the stimulating effect of the other stimuli. (D) Confluent cells were cotransfected with active TAZ and the Smad3 promoter construct, and 24 h later luciferase activities were determined as in C.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 5: TAZ activates and potentiates the SMA promoter and suppresses the Smad3 promoter. (A, B) MRTF potently drives the SMA promoter but fails to inhibit the Smad3 promoter. Cells were transfected with MRTF along with either the SMA promoter luciferase or the Smad3 promoter luciferase reporter system. (C) Cells were cotransfected with the SMA reporter and either active (S89A) TAZ or control plasmid. Cells were then left untreated or exposed to the individual or combined stimuli of the two-hit scheme (TGFβ or/and LCM), and 24 h later luciferase activities were determined. Note that TAZ increases the activity of the SMA promoter and potentiates the stimulating effect of the other stimuli. (D) Confluent cells were cotransfected with active TAZ and the Smad3 promoter construct, and 24 h later luciferase activities were determined as in C.
Mentions: In search for the mechanism underlying the concomitant SMA mRNA expression and Smad3 mRNA suppression, first we considered that MRTF might inhibit Smad3 transcription. To assess this possibility, we overexpressed MRTF along with either an SMA or a Smad3 promoter luciferase construct. Although MRTF overexpression drove the SMA promoter 25-fold (testifying to its activity; Figure 5A), it failed to affect the Smad3 promoter (Figure 5B). As an alternative, we tested the effect of active and constitutively nuclear TAZ (S89A) on these promoters. For these cotransfection experiments, where the wound edge cannot be targeted specifically, we used our two-hit scheme (LCM and TGFβ; Masszi et al., 2004, 2010). Surprisingly, expression of active TAZ induced a four- to fivefold activation of the SMA promoter and potentiated its activation to the same extent by the individual and combined effect of TGFβ and LCM, attaining an overall >40-fold stimulation under the two-hit conditions (Figure 5C). Moreover, overexpression of active TAZ inhibited the Smad3 promoter by 80% (Figure 5D). To test whether TAZ indeed regulates SMA and Smad3 mRNA expression in the context of the wound model, we transfected the cells with NR or TAZ-specific siRNA and measured the message for these proteins at both loci with or without TGFβ treatment. As expected, the specific siRNA efficiently suppressed the message for TAZ under all conditions (Figure 6A). Remarkably, down-regulation of TAZ completely eliminated the induction of SMA mRNA (Figure 6B) and prevented the loss of Smad3 mRNA (Figure 6C) in the TGFβ-exposed wound. To verify that these changes are reflected on the corresponding protein levels, we compared the expression of SMA and Smad3 protein in samples obtained from the intact untreated region with those collected from the TGFβ-treated wound area. Elimination of TAZ prevented both the expression of SMA and the down-regulation of Smad3 in the TGFβ-treated wound region (Figure 6D). Thus TAZ is an essential factor for the topical sensitivity of the contact-deprived epithelium in terms of both SMA expression and Smad3 suppression. However, it should be mentioned that TAZ alone is not sufficient to drive SMA expression, as indicated by the fact that neither wounding (which induces nuclear accumulation of TAZ) nor the expression of constitutively active nuclear TAZ induced detectable SMA protein expression. Nonetheless, consistent with the potentiation of the promoter (Figure 5C), expression of active TAZ rendered TGFβ capable of inducing SMA expression. Although TGFβ alone never provoked SMA expression in a confluent monolayer, it induced SMA expression in a significant fraction of active TAZ-transfected cells (Supplemental Figure S3, A and B).

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