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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

Locus-specific differences in mRNA expression for smooth muscle actin and its key regulators in the intact and wound-adjacent regions of the epithelium. (A) Schematic representation of the wound model used to obtain cell populations from the intact (I) or cell contact-deprived, that is, “wound” (W) area of a monolayer. See Materials and Methods for details. (B) Cells were grown to confluence on the entire tissue culture dish except in a defined region, where growth was blocked by a surgical tape (“wound”) as shown in A. Cultures were then left untreated or exposed to TGFβ for 72 h, and then narrow (2 mm) strips were isolated from the intact and wound edge regions of control (I and W) or TGFβ-treated (I+T and W+T) monolayers. Samples were processed for RNA extractions and quantitative PCR analysis for the indicated genes. Values were normalized to the housekeeping gene GAPDH. Data are expressed as fold change compared with the normalized mRNA levels obtained in the intact region of the control (I) (mean ± SEM from at least four independent experiments). (C) The Smad3/β-catenin mRNA ratio plotted against the corresponding SMA mRNA level. Note the reciprocal relationship between these parameters.
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Figure 1: Locus-specific differences in mRNA expression for smooth muscle actin and its key regulators in the intact and wound-adjacent regions of the epithelium. (A) Schematic representation of the wound model used to obtain cell populations from the intact (I) or cell contact-deprived, that is, “wound” (W) area of a monolayer. See Materials and Methods for details. (B) Cells were grown to confluence on the entire tissue culture dish except in a defined region, where growth was blocked by a surgical tape (“wound”) as shown in A. Cultures were then left untreated or exposed to TGFβ for 72 h, and then narrow (2 mm) strips were isolated from the intact and wound edge regions of control (I and W) or TGFβ-treated (I+T and W+T) monolayers. Samples were processed for RNA extractions and quantitative PCR analysis for the indicated genes. Values were normalized to the housekeeping gene GAPDH. Data are expressed as fold change compared with the normalized mRNA levels obtained in the intact region of the control (I) (mean ± SEM from at least four independent experiments). (C) The Smad3/β-catenin mRNA ratio plotted against the corresponding SMA mRNA level. Note the reciprocal relationship between these parameters.

Mentions: To assess the topical susceptibility of the intact and “injured” parts of the epithelium to TGFβ and study the role of cell contacts in this phenomenon, we generated a model system in which changes in gene and protein expression can be studied simultaneously at these two loci. We grew cells to confluence in a tissue culture dish, at the middle of which the formation of intercellular contacts was prevented by surgical tape (Figure 1A). This method allowed us to establish a well-defined “wound” model (or gap model), characterized by the absence of intercellular contacts without the additional effects of cellular breakage and the efflux of intracellular constituents. Once confluent, the monolayer was either left untreated or exposed to TGFβ, and at given times 2-mm-wide cellular strips were collected from regions either directly adjacent to the wound (referred to as wound cells [W]) or from the intact area (I cells, >10 mm away from the wound). In this way, four different sample types were obtained, designated as I (intact), W (wound), I+T (intact area exposed to TGFβ), and W+T (wound exposed to TGFβ).


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)

Locus-specific differences in mRNA expression for smooth muscle actin and its key regulators in the intact and wound-adjacent regions of the epithelium. (A) Schematic representation of the wound model used to obtain cell populations from the intact (I) or cell contact-deprived, that is, “wound” (W) area of a monolayer. See Materials and Methods for details. (B) Cells were grown to confluence on the entire tissue culture dish except in a defined region, where growth was blocked by a surgical tape (“wound”) as shown in A. Cultures were then left untreated or exposed to TGFβ for 72 h, and then narrow (2 mm) strips were isolated from the intact and wound edge regions of control (I and W) or TGFβ-treated (I+T and W+T) monolayers. Samples were processed for RNA extractions and quantitative PCR analysis for the indicated genes. Values were normalized to the housekeeping gene GAPDH. Data are expressed as fold change compared with the normalized mRNA levels obtained in the intact region of the control (I) (mean ± SEM from at least four independent experiments). (C) The Smad3/β-catenin mRNA ratio plotted against the corresponding SMA mRNA level. Note the reciprocal relationship between these parameters.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 1: Locus-specific differences in mRNA expression for smooth muscle actin and its key regulators in the intact and wound-adjacent regions of the epithelium. (A) Schematic representation of the wound model used to obtain cell populations from the intact (I) or cell contact-deprived, that is, “wound” (W) area of a monolayer. See Materials and Methods for details. (B) Cells were grown to confluence on the entire tissue culture dish except in a defined region, where growth was blocked by a surgical tape (“wound”) as shown in A. Cultures were then left untreated or exposed to TGFβ for 72 h, and then narrow (2 mm) strips were isolated from the intact and wound edge regions of control (I and W) or TGFβ-treated (I+T and W+T) monolayers. Samples were processed for RNA extractions and quantitative PCR analysis for the indicated genes. Values were normalized to the housekeeping gene GAPDH. Data are expressed as fold change compared with the normalized mRNA levels obtained in the intact region of the control (I) (mean ± SEM from at least four independent experiments). (C) The Smad3/β-catenin mRNA ratio plotted against the corresponding SMA mRNA level. Note the reciprocal relationship between these parameters.
Mentions: To assess the topical susceptibility of the intact and “injured” parts of the epithelium to TGFβ and study the role of cell contacts in this phenomenon, we generated a model system in which changes in gene and protein expression can be studied simultaneously at these two loci. We grew cells to confluence in a tissue culture dish, at the middle of which the formation of intercellular contacts was prevented by surgical tape (Figure 1A). This method allowed us to establish a well-defined “wound” model (or gap model), characterized by the absence of intercellular contacts without the additional effects of cellular breakage and the efflux of intracellular constituents. Once confluent, the monolayer was either left untreated or exposed to TGFβ, and at given times 2-mm-wide cellular strips were collected from regions either directly adjacent to the wound (referred to as wound cells [W]) or from the intact area (I cells, >10 mm away from the wound). In this way, four different sample types were obtained, designated as I (intact), W (wound), I+T (intact area exposed to TGFβ), and W+T (wound exposed to TGFβ).

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