Differential topical susceptibility to TGFβ in intact and injured regions of the epithelium: key role in myofibroblast transition

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.

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.

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).

FIGURE 3:

β-Catenin is essential for maintaining MRTF stability and mRNA expression in the wound. (A) Cells were transfected with NR or β-catenin–specific siRNA and treated with or without TGFβ for 72 h. Samples collected from the wound edge or the intact area were probed for the indicated proteins. (B) SMA mRNA analysis performed in samples prepared as in A. (C) To assess the time dependence of MRTF down-regulation in β-catenin–depleted cells, cultures were transfected as in A and treated with TGFβ for 24 or 72 h. MRTF expression, normalized to GAPDH, was quantified by densitometry. (D) After transfection with NR or β-catenin siRNA as in A, cultures were exposed to TGFβ for 24 h. Cells collected from indicated areas were subjected to immunoprecipitation using an anti-MRTF antibody. The precipitates were probed for MRTF and coprecipitating GSK-3β and Smad3. The graph shows densitometric quantification of the cosedimented GSK-3β normalized to the precipitated MRTF (mean ± SEM; n = 3). (E) MRTF mRNA analysis performed in samples prepared as in A.

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.

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.

FIGURE 6:

TAZ plays an important role in wound-restricted SMA expression and Smad3 down-regulation. Cells cultured in the context of the wound model were transfected with NR or TAZ-specific siRNA. After 24 h they were either left untreated or exposed to TGFβ for 72 h, and samples corresponding to indicated conditions and regions were analyzed by quantitative PCR for TAZ (A), SMA (B), and Smad3 (C) mRNA. (D) The effect of TAZ silencing on SMA and Smad3 protein expression in the wound model. Monolayers were treated as described, and samples corresponding to I and W+T conditions were subjected to Western blotting for the indicated proteins. The graph shown below represents the densitometric analysis of the GAPDH-normalized Smad3 levels under the indicated conditions (mean ± SEM; n = 4).

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.