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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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Smad3 silencing has opposite effects on the expression of mesenchymal marker PAI-1 and on MF characteristics (SMA and cytoskeleton remodeling). (A) PAI-1 and SMA mRNA was measured by qPCR in control and Smad3-depleted cells (Fig. 5 C) and treated with vehicle or TGF-β for 3 or 6 h. Note that although Smad3 depletion strongly stimulates TGF-β–induced SMA mRNA expression, the effect is still much less (∼0.125) compared with LCM (Fig. 5 C), in accordance with the fact that TGF-β alone (as opposed to LCM) does not induce SMA expression even in Smad3-depleted cells. (B) Expression of PAI-1 and CTGF protein under two-hit condition in Smad3-containing and -depleted cells. Cells were transfected with NR or Smad3 siRNA for 24 h, treated for an additional 48 h as indicated, and processed for Western blotting. (C) Smad3 depletion induces reorganization of the cytoskeleton. Cells were transfected with NR (top) or Smad3 siRNA (bottom) for 48 h, and F-actin and focal adhesions were visualized by rhodamine phalloidin or staining for FAK, phosphorylated FAK (pFAK), paxillin, or actinin. Loss of Smad3 induces the formation of central stress fibers and thick, elongated focal adhesions in cells located at the edges of islands. (bottom) Higher magnification images are shown of boxed areas. Bars: (top) 30 µm; (bottom) 10 µm. Error bars indicate mean ± SEM.
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fig8: Smad3 silencing has opposite effects on the expression of mesenchymal marker PAI-1 and on MF characteristics (SMA and cytoskeleton remodeling). (A) PAI-1 and SMA mRNA was measured by qPCR in control and Smad3-depleted cells (Fig. 5 C) and treated with vehicle or TGF-β for 3 or 6 h. Note that although Smad3 depletion strongly stimulates TGF-β–induced SMA mRNA expression, the effect is still much less (∼0.125) compared with LCM (Fig. 5 C), in accordance with the fact that TGF-β alone (as opposed to LCM) does not induce SMA expression even in Smad3-depleted cells. (B) Expression of PAI-1 and CTGF protein under two-hit condition in Smad3-containing and -depleted cells. Cells were transfected with NR or Smad3 siRNA for 24 h, treated for an additional 48 h as indicated, and processed for Western blotting. (C) Smad3 depletion induces reorganization of the cytoskeleton. Cells were transfected with NR (top) or Smad3 siRNA (bottom) for 48 h, and F-actin and focal adhesions were visualized by rhodamine phalloidin or staining for FAK, phosphorylated FAK (pFAK), paxillin, or actinin. Loss of Smad3 induces the formation of central stress fibers and thick, elongated focal adhesions in cells located at the edges of islands. (bottom) Higher magnification images are shown of boxed areas. Bars: (top) 30 µm; (bottom) 10 µm. Error bars indicate mean ± SEM.

Mentions: Although our findings indicate a potent inhibitory role for Smad3 in the process of EMyT, Smad3 has been also implicated as a strong profibrotic transcription factor that contributes to EMT. To explain this apparent discrepancy, we considered that Smad3 might play distinct roles in the first (mesenchymal) and second (myogenic) phase of the process. To address this, we followed the impact of Smad3 knockdown on the transcription of PAI-1 (plasminogen activator inhibitor-1), a TGF-β–responsive, profibrogenic gene, and SMA, the hallmark of MFs. Smad3 silencing induced opposite responses to TGF-β in these genes (Fig. 8 A). Both the basal level of the PAI-1 mRNA and its TGF-β–induced rise were strongly suppressed. Accordingly, Smad3 siRNA reduced PAI-1 protein expression induced by TGF-β or the combined treatment (Fig. 8 B). Similarly, the absence of Smad3 prevented the LCM/TGF-β–induced up-regulation of connective tissue growth factor (CTGF), another mediator of EMT (Fig. 8 B). In contrast, Smad3 silencing resulted in a significant increase in SMA mRNA in nonstimulated cells, which was further augmented by TGF-β (Fig. 8 A). TGF-β failed to induce SMA mRNA in control cells, whereas it had a substantial effect in the absence of Smad3. These data indicate that Smad3 is essential for the expression of key proteins of mesenchymal transition, whereas it inhibits the myogenic reprogramming.


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)

Smad3 silencing has opposite effects on the expression of mesenchymal marker PAI-1 and on MF characteristics (SMA and cytoskeleton remodeling). (A) PAI-1 and SMA mRNA was measured by qPCR in control and Smad3-depleted cells (Fig. 5 C) and treated with vehicle or TGF-β for 3 or 6 h. Note that although Smad3 depletion strongly stimulates TGF-β–induced SMA mRNA expression, the effect is still much less (∼0.125) compared with LCM (Fig. 5 C), in accordance with the fact that TGF-β alone (as opposed to LCM) does not induce SMA expression even in Smad3-depleted cells. (B) Expression of PAI-1 and CTGF protein under two-hit condition in Smad3-containing and -depleted cells. Cells were transfected with NR or Smad3 siRNA for 24 h, treated for an additional 48 h as indicated, and processed for Western blotting. (C) Smad3 depletion induces reorganization of the cytoskeleton. Cells were transfected with NR (top) or Smad3 siRNA (bottom) for 48 h, and F-actin and focal adhesions were visualized by rhodamine phalloidin or staining for FAK, phosphorylated FAK (pFAK), paxillin, or actinin. Loss of Smad3 induces the formation of central stress fibers and thick, elongated focal adhesions in cells located at the edges of islands. (bottom) Higher magnification images are shown of boxed areas. Bars: (top) 30 µm; (bottom) 10 µm. Error bars indicate mean ± SEM.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2819691&req=5

fig8: Smad3 silencing has opposite effects on the expression of mesenchymal marker PAI-1 and on MF characteristics (SMA and cytoskeleton remodeling). (A) PAI-1 and SMA mRNA was measured by qPCR in control and Smad3-depleted cells (Fig. 5 C) and treated with vehicle or TGF-β for 3 or 6 h. Note that although Smad3 depletion strongly stimulates TGF-β–induced SMA mRNA expression, the effect is still much less (∼0.125) compared with LCM (Fig. 5 C), in accordance with the fact that TGF-β alone (as opposed to LCM) does not induce SMA expression even in Smad3-depleted cells. (B) Expression of PAI-1 and CTGF protein under two-hit condition in Smad3-containing and -depleted cells. Cells were transfected with NR or Smad3 siRNA for 24 h, treated for an additional 48 h as indicated, and processed for Western blotting. (C) Smad3 depletion induces reorganization of the cytoskeleton. Cells were transfected with NR (top) or Smad3 siRNA (bottom) for 48 h, and F-actin and focal adhesions were visualized by rhodamine phalloidin or staining for FAK, phosphorylated FAK (pFAK), paxillin, or actinin. Loss of Smad3 induces the formation of central stress fibers and thick, elongated focal adhesions in cells located at the edges of islands. (bottom) Higher magnification images are shown of boxed areas. Bars: (top) 30 µm; (bottom) 10 µm. Error bars indicate mean ± SEM.
Mentions: Although our findings indicate a potent inhibitory role for Smad3 in the process of EMyT, Smad3 has been also implicated as a strong profibrotic transcription factor that contributes to EMT. To explain this apparent discrepancy, we considered that Smad3 might play distinct roles in the first (mesenchymal) and second (myogenic) phase of the process. To address this, we followed the impact of Smad3 knockdown on the transcription of PAI-1 (plasminogen activator inhibitor-1), a TGF-β–responsive, profibrogenic gene, and SMA, the hallmark of MFs. Smad3 silencing induced opposite responses to TGF-β in these genes (Fig. 8 A). Both the basal level of the PAI-1 mRNA and its TGF-β–induced rise were strongly suppressed. Accordingly, Smad3 siRNA reduced PAI-1 protein expression induced by TGF-β or the combined treatment (Fig. 8 B). Similarly, the absence of Smad3 prevented the LCM/TGF-β–induced up-regulation of connective tissue growth factor (CTGF), another mediator of EMT (Fig. 8 B). In contrast, Smad3 silencing resulted in a significant increase in SMA mRNA in nonstimulated cells, which was further augmented by TGF-β (Fig. 8 A). TGF-β failed to induce SMA mRNA in control cells, whereas it had a substantial effect in the absence of Smad3. These data indicate that Smad3 is essential for the expression of key proteins of mesenchymal transition, whereas it inhibits the myogenic reprogramming.

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