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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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The one-hit scenario. Smad3 silencing renders injury sufficient to induce MRTF-dependent SMA expression and potentiates the expression of other CArG-dependent genes. (A) Confluent monolayers were exposed to NR, Smad3, MRTF, or Smad3 + MRTF siRNAs for 24 h followed by 48 h treatment according to the two-hit scheme. Cell lysates were prepared and probed by Western blotting for the indicated proteins. (B) Human lung epithelial cells (BEAS-2B) and gingival primary fibroblasts (HGF) were transfected with NR or Smad3 siRNA and 24 h later treated with TGF-β for 48 h. Whole cell lysates were probed for the indicated proteins. (C) Smad2 silencing does not facilitate SMA expression. Cells were transfected with siRNA against Smad2 and treated as in A for 2 d. Whole cell lysates were probed. (D) Cells were transfected as indicated and left untreated or exposed to LCM and processed for Western blotting using antibodies against cofilin and SRF. (E) E-cadherin loss is less complete in Smad3 siRNA cells. E-cadherin blots were analyzed by densitometry and normalized to control values. Error bars indicate mean ± SEM.
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fig6: The one-hit scenario. Smad3 silencing renders injury sufficient to induce MRTF-dependent SMA expression and potentiates the expression of other CArG-dependent genes. (A) Confluent monolayers were exposed to NR, Smad3, MRTF, or Smad3 + MRTF siRNAs for 24 h followed by 48 h treatment according to the two-hit scheme. Cell lysates were prepared and probed by Western blotting for the indicated proteins. (B) Human lung epithelial cells (BEAS-2B) and gingival primary fibroblasts (HGF) were transfected with NR or Smad3 siRNA and 24 h later treated with TGF-β for 48 h. Whole cell lysates were probed for the indicated proteins. (C) Smad2 silencing does not facilitate SMA expression. Cells were transfected with siRNA against Smad2 and treated as in A for 2 d. Whole cell lysates were probed. (D) Cells were transfected as indicated and left untreated or exposed to LCM and processed for Western blotting using antibodies against cofilin and SRF. (E) E-cadherin loss is less complete in Smad3 siRNA cells. E-cadherin blots were analyzed by densitometry and normalized to control values. Error bars indicate mean ± SEM.

Mentions: To investigate whether a reduction in Smad3 indeed translates into elevated SMA protein levels, we compared the expression of SMA in the presence of control or Smad3 siRNA in cells treated according to the two-hit scheme (Fig. 6 A). Although in control cells SMA was just becoming detectable after a 48-h exposure to these stimuli (Masszi et al., 2004), in the Smad3 knockdown group, robust SMA expression occurred (Fig. 6 A). Moreover, in Smad3 down-regulated cells, LCM in itself was sufficient to provoke SMA protein expression. Because LCM alone never causes SMA expression in control cells, this striking observation implies that the absence of Smad3 makes TGF-β unnecessary for SMA expression and renders contact injury, as a single hit, sufficient for MF generation. Identical results were obtained when another Smad3-specific siRNA was used (unpublished data). To test whether SMA expression in Smad3-depleted cells still remained dependent on MRTF, cells were cotransfected with MRTF and Smad3 siRNAs. The absence of MRTF prevented SMA expression in the Smad3 knockdown cells as well, when LCM or LCM + TGF-β were used as stimuli (Fig. 6 A). This verifies that the absence of Smad3 did not divert the myogenic program to an alternate pathway; instead, it increased the efficiency of the MRTF-dependent mechanism. Importantly, the robust potentiation of SMA expression by the loss of Smad3 was also observed in BEAS-2B lung epithelial cells and human gingival fibroblasts (Fig. 6 B), implying that this is a general phenomenon. Smad2 silencing had no such effect (Fig. 6 C). The loss of Smad3 also facilitated the expression of cofilin and SRF, suggesting that Smad3 can also inhibit the expression of other CArGome proteins (Fig. 6 D). Finally, E-cadherin down-regulation was less robust in Smad3-depleted cells (Fig. 6, A and E), a finding consistent with (but less pronounced than) that reported by Morita et al. (2007a) in MDCK cells. Together, these results indicate that elimination of Smad3 strongly stimulates EMyT, or conversely, Smad3 acts as a break or delayer of MF generation.


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)

The one-hit scenario. Smad3 silencing renders injury sufficient to induce MRTF-dependent SMA expression and potentiates the expression of other CArG-dependent genes. (A) Confluent monolayers were exposed to NR, Smad3, MRTF, or Smad3 + MRTF siRNAs for 24 h followed by 48 h treatment according to the two-hit scheme. Cell lysates were prepared and probed by Western blotting for the indicated proteins. (B) Human lung epithelial cells (BEAS-2B) and gingival primary fibroblasts (HGF) were transfected with NR or Smad3 siRNA and 24 h later treated with TGF-β for 48 h. Whole cell lysates were probed for the indicated proteins. (C) Smad2 silencing does not facilitate SMA expression. Cells were transfected with siRNA against Smad2 and treated as in A for 2 d. Whole cell lysates were probed. (D) Cells were transfected as indicated and left untreated or exposed to LCM and processed for Western blotting using antibodies against cofilin and SRF. (E) E-cadherin loss is less complete in Smad3 siRNA cells. E-cadherin blots were analyzed by densitometry and normalized to control values. 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

fig6: The one-hit scenario. Smad3 silencing renders injury sufficient to induce MRTF-dependent SMA expression and potentiates the expression of other CArG-dependent genes. (A) Confluent monolayers were exposed to NR, Smad3, MRTF, or Smad3 + MRTF siRNAs for 24 h followed by 48 h treatment according to the two-hit scheme. Cell lysates were prepared and probed by Western blotting for the indicated proteins. (B) Human lung epithelial cells (BEAS-2B) and gingival primary fibroblasts (HGF) were transfected with NR or Smad3 siRNA and 24 h later treated with TGF-β for 48 h. Whole cell lysates were probed for the indicated proteins. (C) Smad2 silencing does not facilitate SMA expression. Cells were transfected with siRNA against Smad2 and treated as in A for 2 d. Whole cell lysates were probed. (D) Cells were transfected as indicated and left untreated or exposed to LCM and processed for Western blotting using antibodies against cofilin and SRF. (E) E-cadherin loss is less complete in Smad3 siRNA cells. E-cadherin blots were analyzed by densitometry and normalized to control values. Error bars indicate mean ± SEM.
Mentions: To investigate whether a reduction in Smad3 indeed translates into elevated SMA protein levels, we compared the expression of SMA in the presence of control or Smad3 siRNA in cells treated according to the two-hit scheme (Fig. 6 A). Although in control cells SMA was just becoming detectable after a 48-h exposure to these stimuli (Masszi et al., 2004), in the Smad3 knockdown group, robust SMA expression occurred (Fig. 6 A). Moreover, in Smad3 down-regulated cells, LCM in itself was sufficient to provoke SMA protein expression. Because LCM alone never causes SMA expression in control cells, this striking observation implies that the absence of Smad3 makes TGF-β unnecessary for SMA expression and renders contact injury, as a single hit, sufficient for MF generation. Identical results were obtained when another Smad3-specific siRNA was used (unpublished data). To test whether SMA expression in Smad3-depleted cells still remained dependent on MRTF, cells were cotransfected with MRTF and Smad3 siRNAs. The absence of MRTF prevented SMA expression in the Smad3 knockdown cells as well, when LCM or LCM + TGF-β were used as stimuli (Fig. 6 A). This verifies that the absence of Smad3 did not divert the myogenic program to an alternate pathway; instead, it increased the efficiency of the MRTF-dependent mechanism. Importantly, the robust potentiation of SMA expression by the loss of Smad3 was also observed in BEAS-2B lung epithelial cells and human gingival fibroblasts (Fig. 6 B), implying that this is a general phenomenon. Smad2 silencing had no such effect (Fig. 6 C). The loss of Smad3 also facilitated the expression of cofilin and SRF, suggesting that Smad3 can also inhibit the expression of other CArGome proteins (Fig. 6 D). Finally, E-cadherin down-regulation was less robust in Smad3-depleted cells (Fig. 6, A and E), a finding consistent with (but less pronounced than) that reported by Morita et al. (2007a) in MDCK cells. Together, these results indicate that elimination of Smad3 strongly stimulates EMyT, or conversely, Smad3 acts as a break or delayer of MF generation.

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