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Fate-determining mechanisms in epithelial-myofibroblast transition: major inhibitory role for Smad3.

Masszi A, Speight P, Charbonney E, Lodyga M, Nakano H, Szászi K, Kapus A - J. Cell Biol. (2010)

Bottom Line: Because the SMA promoter harbors both MRTF-responsive CC(A/T)-rich GG element (CArG) boxes and TGF-beta-responsive Smad-binding elements, we hypothesized that the myogenic program is mobilized by a synergy between MRTF and Smad3.Furthermore, Smad3 is degraded under two-hit conditions, thereby liberating the myogenic program.Thus, Smad3 is a critical timer/delayer of MF commitment in the epithelium, and EMyT can be dissected into Smad3-promoted (mesenchymal) and Smad3-inhibited (myogenic) phases.

View Article: PubMed Central - HTML - PubMed

Affiliation: Keenan Research Centre, Li Ka Shing Knowledge Institute, University of Toronto, Toronto, Ontario M5B 1W8, Canada.

ABSTRACT
Epithelial-myofibroblast (MF) transition (EMyT) is a critical process in organ fibrosis, leading to alpha-smooth muscle actin (SMA) expression in the epithelium. The mechanism underlying the activation of this myogenic program is unknown. We have shown previously that both injury to intercellular contacts and transforming growth factor beta (TGF-beta) are indispensable for SMA expression (two-hit model) and that contact disruption induces nuclear translocation of myocardin-related transcription factor (MRTF). Because the SMA promoter harbors both MRTF-responsive CC(A/T)-rich GG element (CArG) boxes and TGF-beta-responsive Smad-binding elements, we hypothesized that the myogenic program is mobilized by a synergy between MRTF and Smad3. In this study, we show that the synergy between injury and TGF-beta exclusively requires CArG elements. Surprisingly, Smad3 inhibits MRTF-driven activation of the SMA promoter, and Smad3 silencing renders injury sufficient to induce SMA expression. Furthermore, Smad3 is degraded under two-hit conditions, thereby liberating the myogenic program. Thus, Smad3 is a critical timer/delayer of MF commitment in the epithelium, and EMyT can be dissected into Smad3-promoted (mesenchymal) and Smad3-inhibited (myogenic) phases.

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Reduced Smad3 expression robustly facilitates SMA promoter activity, SMA mRNA expression, and the interaction between MRTF and the endogenous SMA promoter. (A) Relative Smad3 levels were determined by Western blotting after LCM or combined treatment at various times (0–48 h) and plotted against the corresponding SMA promoter activity measured in parallel after the same treatments. Each point represents the means of three determinations. Power function fit resulted in a hyperbola. (inset) Linearization of the relationship using the reciprocal of the promoter activity. (B) Efficiency of Smad3 silencing. (C) Smad3 silencing strongly potentiates LCM-induced mRNA expression. Cells were treated with NR or Smad3 siRNA and left untreated or exposed to LCM for 3 or 6 h and processed for RNA extraction. SMA mRNA was determined by qPCR and normalized to GAPDH. Data are expressed as fold change (logarithmic scale) compared with control. (D) Smad3 silencing enhances MRTF binding to the SMA promoter. Cells were transfected as in B, exposed to normal medium or LCM for 1 h, and processed for ChIP assays using anti-MRTF antibody. (top) Antibody-associated SMA promoter signals were quantified by qPCR (shown as fold cycle threshold change). (middle) Amplicons shown on agarose gels after 30 PCR cycles from the input and the MRTF antibody precipitates. (bottom) Western blots verifying Smad3 down-regulation. Error bars indicate mean ± SEM.
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fig5: Reduced Smad3 expression robustly facilitates SMA promoter activity, SMA mRNA expression, and the interaction between MRTF and the endogenous SMA promoter. (A) Relative Smad3 levels were determined by Western blotting after LCM or combined treatment at various times (0–48 h) and plotted against the corresponding SMA promoter activity measured in parallel after the same treatments. Each point represents the means of three determinations. Power function fit resulted in a hyperbola. (inset) Linearization of the relationship using the reciprocal of the promoter activity. (B) Efficiency of Smad3 silencing. (C) Smad3 silencing strongly potentiates LCM-induced mRNA expression. Cells were treated with NR or Smad3 siRNA and left untreated or exposed to LCM for 3 or 6 h and processed for RNA extraction. SMA mRNA was determined by qPCR and normalized to GAPDH. Data are expressed as fold change (logarithmic scale) compared with control. (D) Smad3 silencing enhances MRTF binding to the SMA promoter. Cells were transfected as in B, exposed to normal medium or LCM for 1 h, and processed for ChIP assays using anti-MRTF antibody. (top) Antibody-associated SMA promoter signals were quantified by qPCR (shown as fold cycle threshold change). (middle) Amplicons shown on agarose gels after 30 PCR cycles from the input and the MRTF antibody precipitates. (bottom) Western blots verifying Smad3 down-regulation. Error bars indicate mean ± SEM.

Mentions: So far, we showed that Smad3 overexpression inhibits the effect of MRTF and that Smad3 degrades in the two-hit model. In the following experiments, we sought to examine whether decreasing Smad3 levels indeed play a role in the genetic reprogramming during EMyT. We first determined whether the level of Smad3 degradation, as observed in the two-hit model, correlates with the ensuing SMA promoter activation. To this end, we treated the cells according to the two-hit scheme (LCM, TGF-β, or both) and prepared lysates at various times (2, 6, 12, 24, and 48 h) after stimulation. Smad3 expression was determined in each sample by Western blotting as in Fig. 4 A. In parallel experiments, cells had been transfected with the 765-bp SMA-Luc reporter and treated as for the Western blots, after which the activity of the SMA promoter was measured. Having obtained these two datasets, we plotted the activation of the SMA promoter against the level of the corresponding Smad3 expression (Fig. 5 A). The resulting function was best fitted with a hyperbola (see also the linearized form; r2 = 0.93), signifying a reciprocal relationship between the level of Smad3 and the corresponding promoter response.


Fate-determining mechanisms in epithelial-myofibroblast transition: major inhibitory role for Smad3.

Masszi A, Speight P, Charbonney E, Lodyga M, Nakano H, Szászi K, Kapus A - J. Cell Biol. (2010)

Reduced Smad3 expression robustly facilitates SMA promoter activity, SMA mRNA expression, and the interaction between MRTF and the endogenous SMA promoter. (A) Relative Smad3 levels were determined by Western blotting after LCM or combined treatment at various times (0–48 h) and plotted against the corresponding SMA promoter activity measured in parallel after the same treatments. Each point represents the means of three determinations. Power function fit resulted in a hyperbola. (inset) Linearization of the relationship using the reciprocal of the promoter activity. (B) Efficiency of Smad3 silencing. (C) Smad3 silencing strongly potentiates LCM-induced mRNA expression. Cells were treated with NR or Smad3 siRNA and left untreated or exposed to LCM for 3 or 6 h and processed for RNA extraction. SMA mRNA was determined by qPCR and normalized to GAPDH. Data are expressed as fold change (logarithmic scale) compared with control. (D) Smad3 silencing enhances MRTF binding to the SMA promoter. Cells were transfected as in B, exposed to normal medium or LCM for 1 h, and processed for ChIP assays using anti-MRTF antibody. (top) Antibody-associated SMA promoter signals were quantified by qPCR (shown as fold cycle threshold change). (middle) Amplicons shown on agarose gels after 30 PCR cycles from the input and the MRTF antibody precipitates. (bottom) Western blots verifying Smad3 down-regulation. Error bars indicate mean ± SEM.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2819691&req=5

fig5: Reduced Smad3 expression robustly facilitates SMA promoter activity, SMA mRNA expression, and the interaction between MRTF and the endogenous SMA promoter. (A) Relative Smad3 levels were determined by Western blotting after LCM or combined treatment at various times (0–48 h) and plotted against the corresponding SMA promoter activity measured in parallel after the same treatments. Each point represents the means of three determinations. Power function fit resulted in a hyperbola. (inset) Linearization of the relationship using the reciprocal of the promoter activity. (B) Efficiency of Smad3 silencing. (C) Smad3 silencing strongly potentiates LCM-induced mRNA expression. Cells were treated with NR or Smad3 siRNA and left untreated or exposed to LCM for 3 or 6 h and processed for RNA extraction. SMA mRNA was determined by qPCR and normalized to GAPDH. Data are expressed as fold change (logarithmic scale) compared with control. (D) Smad3 silencing enhances MRTF binding to the SMA promoter. Cells were transfected as in B, exposed to normal medium or LCM for 1 h, and processed for ChIP assays using anti-MRTF antibody. (top) Antibody-associated SMA promoter signals were quantified by qPCR (shown as fold cycle threshold change). (middle) Amplicons shown on agarose gels after 30 PCR cycles from the input and the MRTF antibody precipitates. (bottom) Western blots verifying Smad3 down-regulation. Error bars indicate mean ± SEM.
Mentions: So far, we showed that Smad3 overexpression inhibits the effect of MRTF and that Smad3 degrades in the two-hit model. In the following experiments, we sought to examine whether decreasing Smad3 levels indeed play a role in the genetic reprogramming during EMyT. We first determined whether the level of Smad3 degradation, as observed in the two-hit model, correlates with the ensuing SMA promoter activation. To this end, we treated the cells according to the two-hit scheme (LCM, TGF-β, or both) and prepared lysates at various times (2, 6, 12, 24, and 48 h) after stimulation. Smad3 expression was determined in each sample by Western blotting as in Fig. 4 A. In parallel experiments, cells had been transfected with the 765-bp SMA-Luc reporter and treated as for the Western blots, after which the activity of the SMA promoter was measured. Having obtained these two datasets, we plotted the activation of the SMA promoter against the level of the corresponding Smad3 expression (Fig. 5 A). The resulting function was best fitted with a hyperbola (see also the linearized form; r2 = 0.93), signifying a reciprocal relationship between the level of Smad3 and the corresponding promoter response.

Bottom Line: Because the SMA promoter harbors both MRTF-responsive CC(A/T)-rich GG element (CArG) boxes and TGF-beta-responsive Smad-binding elements, we hypothesized that the myogenic program is mobilized by a synergy between MRTF and Smad3.Furthermore, Smad3 is degraded under two-hit conditions, thereby liberating the myogenic program.Thus, Smad3 is a critical timer/delayer of MF commitment in the epithelium, and EMyT can be dissected into Smad3-promoted (mesenchymal) and Smad3-inhibited (myogenic) phases.

View Article: PubMed Central - HTML - PubMed

Affiliation: Keenan Research Centre, Li Ka Shing Knowledge Institute, University of Toronto, Toronto, Ontario M5B 1W8, Canada.

ABSTRACT
Epithelial-myofibroblast (MF) transition (EMyT) is a critical process in organ fibrosis, leading to alpha-smooth muscle actin (SMA) expression in the epithelium. The mechanism underlying the activation of this myogenic program is unknown. We have shown previously that both injury to intercellular contacts and transforming growth factor beta (TGF-beta) are indispensable for SMA expression (two-hit model) and that contact disruption induces nuclear translocation of myocardin-related transcription factor (MRTF). Because the SMA promoter harbors both MRTF-responsive CC(A/T)-rich GG element (CArG) boxes and TGF-beta-responsive Smad-binding elements, we hypothesized that the myogenic program is mobilized by a synergy between MRTF and Smad3. In this study, we show that the synergy between injury and TGF-beta exclusively requires CArG elements. Surprisingly, Smad3 inhibits MRTF-driven activation of the SMA promoter, and Smad3 silencing renders injury sufficient to induce SMA expression. Furthermore, Smad3 is degraded under two-hit conditions, thereby liberating the myogenic program. Thus, Smad3 is a critical timer/delayer of MF commitment in the epithelium, and EMyT can be dissected into Smad3-promoted (mesenchymal) and Smad3-inhibited (myogenic) phases.

Show MeSH
Related in: MedlinePlus