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Integrin alpha3beta1-dependent beta-catenin phosphorylation links epithelial Smad signaling to cell contacts.

Kim Y, Kugler MC, Wei Y, Kim KK, Li X, Brumwell AN, Chapman HA - J. Cell Biol. (2009)

Bottom Line: Injury-initiated epithelial to mesenchymal transition (EMT) depends on contextual signals from the extracellular matrix, suggesting a role for integrin signaling.A mechanism for this defect was explored in alpha3- cells reconstituted with wild-type (wt) alpha3 or point mutants unable to engage laminin 5 (G163A) or epithelial cadherin (E-cadherin; H245A).These findings demonstrate that alpha3beta1 coordinates cross talk between beta-catenin and Smad signaling pathways as a function of extracellular contact cues and thereby regulates responses to TGF-beta1 activation.

View Article: PubMed Central - PubMed

Affiliation: Pulmonary and Critical Care Division, Department of Medicine, and Cardiovascular Research Institute, University of California, San Francisco, San Francisco, CA 94143, USA.

ABSTRACT
Injury-initiated epithelial to mesenchymal transition (EMT) depends on contextual signals from the extracellular matrix, suggesting a role for integrin signaling. Primary epithelial cells deficient in their prominent laminin receptor, alpha3beta1, were found to have a markedly blunted EMT response to TGF-beta1. A mechanism for this defect was explored in alpha3- cells reconstituted with wild-type (wt) alpha3 or point mutants unable to engage laminin 5 (G163A) or epithelial cadherin (E-cadherin; H245A). After TGF-beta1 stimulation, wt epithelial cells but not cells expressing the H245A mutant internalize complexes of E-cadherin and TGF-beta1 receptors, generate phospho-Smad2 (p-Smad2)-pY654-beta-catenin complexes, and up-regulate mesenchymal target genes. Although Smad2 phosphorylation is normal, p-Smad2-pY654-beta-catenin complexes do not form in the absence of alpha3 or when alpha3beta1 is mainly engaged on laminin 5 or E-cadherin in adherens junctions, leading to attenuated EMT. These findings demonstrate that alpha3beta1 coordinates cross talk between beta-catenin and Smad signaling pathways as a function of extracellular contact cues and thereby regulates responses to TGF-beta1 activation.

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β-Catenin–p-Smad2 complex formation requires wt α3 and is essential for TGF-β1–mediated up-regulation of mesenchymal genes. (A) β-Catenin effectively immunoprecipitates p-Smad2 in α3 wt cells but not in α3−/− or H245A mutant cells after TGF-β1 stimulation. (B) Inhibition of β-catenin by expression of a dominant-negative β-catenin (dnβ-cat; left) or an shRNA against β-catenin (shβ-cat; right) in α3 wt cells inhibits TGF-β1–induced α-SMA but not Smad2 phosphorylation compared with wt α3 cells transfected with vector alone, nonsilencing control shRNA (shctl), or left nontransfected. (C) Tyrosine phosphorylation of β-catenin and formation of the pY654–β-catenin–p-Smad2 complex is only detected in α3 wt cells treated with TGF-β1 but not in α3−/− or H245A mutant cells. (D) Tyrosine phosphorylation of β-catenin (β-cat) and formation of the pY654–β-catenin–p-Smad2 complex in α3 wt cells requires endocytosis. α3 wt cells untreated or exposed to 300 µM of the clathrin inhibitor MDC were stimulated with TGF-β1 and analyzed for pY654–β-catenin–p-Smad2 complexes by co-IP. The supernatant from pY654–β-catenin IP was subsequently immunoprecipitated with total β-catenin antibody. Lane 3 shows p-Smad associated with pY654–β-catenin, and lane 5 shows the remainder of p-Smad2 on β-catenin after pY654–β-catenin depletion. GAPDH, glyceraldehyde 3-phosphate dehydrogenase. All of the aforementioned experiments have been performed at least three times with similar results.
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fig5: β-Catenin–p-Smad2 complex formation requires wt α3 and is essential for TGF-β1–mediated up-regulation of mesenchymal genes. (A) β-Catenin effectively immunoprecipitates p-Smad2 in α3 wt cells but not in α3−/− or H245A mutant cells after TGF-β1 stimulation. (B) Inhibition of β-catenin by expression of a dominant-negative β-catenin (dnβ-cat; left) or an shRNA against β-catenin (shβ-cat; right) in α3 wt cells inhibits TGF-β1–induced α-SMA but not Smad2 phosphorylation compared with wt α3 cells transfected with vector alone, nonsilencing control shRNA (shctl), or left nontransfected. (C) Tyrosine phosphorylation of β-catenin and formation of the pY654–β-catenin–p-Smad2 complex is only detected in α3 wt cells treated with TGF-β1 but not in α3−/− or H245A mutant cells. (D) Tyrosine phosphorylation of β-catenin (β-cat) and formation of the pY654–β-catenin–p-Smad2 complex in α3 wt cells requires endocytosis. α3 wt cells untreated or exposed to 300 µM of the clathrin inhibitor MDC were stimulated with TGF-β1 and analyzed for pY654–β-catenin–p-Smad2 complexes by co-IP. The supernatant from pY654–β-catenin IP was subsequently immunoprecipitated with total β-catenin antibody. Lane 3 shows p-Smad associated with pY654–β-catenin, and lane 5 shows the remainder of p-Smad2 on β-catenin after pY654–β-catenin depletion. GAPDH, glyceraldehyde 3-phosphate dehydrogenase. All of the aforementioned experiments have been performed at least three times with similar results.

Mentions: As shown in Fig. 5 A, epithelial cells expressing wt α3 formed clear β-catenin–p-Smad2 complexes 1 h after TGF-β1 stimulation, whereas α3- or H245A mutant cells failed to support formation of this transcriptional complex. The appearance of β-catenin–p-Smad2 complexes did not result in canonical β-catenin signaling as judged by the lack of response of the TOPFlash T cell factor/catenin reporter to TGF-β1 in transiently transfected α3 wt cells (unpublished data), which is consistent with prior evidence that TOPFlash is not activated directly by TGF-β1 stimulation in other cell types (Labbe et al., 2000). Also, we could not detect β-catenin in the nucleus by direct immunostaining at any time point after TGF-β1 stimulation. However, the catenin is functionally involved in TGF-β1 signaling, which is indicated by near-complete suppression of induction of α-SMA and PAI-1 protein in α3 wt cells stably transfected with a dominant-negative version of β-catenin (Fig. 5 B, left). To further test this point, α3-expressing cells were stably transfected with short hairpin RNA (shRNA) blocking β-catenin expression or a nonblocking shRNA, and the response to TGF-β1 was again assessed. Compared with control, knockdown of β-catenin expression by >50% markedly attenuated the α-SMA response to TGF-β1 without decreasing the p-Smad2 response (Fig. 5 B, right), confirming the importance of β-catenin in the cellular response to TGF-β1 signaling.


Integrin alpha3beta1-dependent beta-catenin phosphorylation links epithelial Smad signaling to cell contacts.

Kim Y, Kugler MC, Wei Y, Kim KK, Li X, Brumwell AN, Chapman HA - J. Cell Biol. (2009)

β-Catenin–p-Smad2 complex formation requires wt α3 and is essential for TGF-β1–mediated up-regulation of mesenchymal genes. (A) β-Catenin effectively immunoprecipitates p-Smad2 in α3 wt cells but not in α3−/− or H245A mutant cells after TGF-β1 stimulation. (B) Inhibition of β-catenin by expression of a dominant-negative β-catenin (dnβ-cat; left) or an shRNA against β-catenin (shβ-cat; right) in α3 wt cells inhibits TGF-β1–induced α-SMA but not Smad2 phosphorylation compared with wt α3 cells transfected with vector alone, nonsilencing control shRNA (shctl), or left nontransfected. (C) Tyrosine phosphorylation of β-catenin and formation of the pY654–β-catenin–p-Smad2 complex is only detected in α3 wt cells treated with TGF-β1 but not in α3−/− or H245A mutant cells. (D) Tyrosine phosphorylation of β-catenin (β-cat) and formation of the pY654–β-catenin–p-Smad2 complex in α3 wt cells requires endocytosis. α3 wt cells untreated or exposed to 300 µM of the clathrin inhibitor MDC were stimulated with TGF-β1 and analyzed for pY654–β-catenin–p-Smad2 complexes by co-IP. The supernatant from pY654–β-catenin IP was subsequently immunoprecipitated with total β-catenin antibody. Lane 3 shows p-Smad associated with pY654–β-catenin, and lane 5 shows the remainder of p-Smad2 on β-catenin after pY654–β-catenin depletion. GAPDH, glyceraldehyde 3-phosphate dehydrogenase. All of the aforementioned experiments have been performed at least three times with similar results.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2654298&req=5

fig5: β-Catenin–p-Smad2 complex formation requires wt α3 and is essential for TGF-β1–mediated up-regulation of mesenchymal genes. (A) β-Catenin effectively immunoprecipitates p-Smad2 in α3 wt cells but not in α3−/− or H245A mutant cells after TGF-β1 stimulation. (B) Inhibition of β-catenin by expression of a dominant-negative β-catenin (dnβ-cat; left) or an shRNA against β-catenin (shβ-cat; right) in α3 wt cells inhibits TGF-β1–induced α-SMA but not Smad2 phosphorylation compared with wt α3 cells transfected with vector alone, nonsilencing control shRNA (shctl), or left nontransfected. (C) Tyrosine phosphorylation of β-catenin and formation of the pY654–β-catenin–p-Smad2 complex is only detected in α3 wt cells treated with TGF-β1 but not in α3−/− or H245A mutant cells. (D) Tyrosine phosphorylation of β-catenin (β-cat) and formation of the pY654–β-catenin–p-Smad2 complex in α3 wt cells requires endocytosis. α3 wt cells untreated or exposed to 300 µM of the clathrin inhibitor MDC were stimulated with TGF-β1 and analyzed for pY654–β-catenin–p-Smad2 complexes by co-IP. The supernatant from pY654–β-catenin IP was subsequently immunoprecipitated with total β-catenin antibody. Lane 3 shows p-Smad associated with pY654–β-catenin, and lane 5 shows the remainder of p-Smad2 on β-catenin after pY654–β-catenin depletion. GAPDH, glyceraldehyde 3-phosphate dehydrogenase. All of the aforementioned experiments have been performed at least three times with similar results.
Mentions: As shown in Fig. 5 A, epithelial cells expressing wt α3 formed clear β-catenin–p-Smad2 complexes 1 h after TGF-β1 stimulation, whereas α3- or H245A mutant cells failed to support formation of this transcriptional complex. The appearance of β-catenin–p-Smad2 complexes did not result in canonical β-catenin signaling as judged by the lack of response of the TOPFlash T cell factor/catenin reporter to TGF-β1 in transiently transfected α3 wt cells (unpublished data), which is consistent with prior evidence that TOPFlash is not activated directly by TGF-β1 stimulation in other cell types (Labbe et al., 2000). Also, we could not detect β-catenin in the nucleus by direct immunostaining at any time point after TGF-β1 stimulation. However, the catenin is functionally involved in TGF-β1 signaling, which is indicated by near-complete suppression of induction of α-SMA and PAI-1 protein in α3 wt cells stably transfected with a dominant-negative version of β-catenin (Fig. 5 B, left). To further test this point, α3-expressing cells were stably transfected with short hairpin RNA (shRNA) blocking β-catenin expression or a nonblocking shRNA, and the response to TGF-β1 was again assessed. Compared with control, knockdown of β-catenin expression by >50% markedly attenuated the α-SMA response to TGF-β1 without decreasing the p-Smad2 response (Fig. 5 B, right), confirming the importance of β-catenin in the cellular response to TGF-β1 signaling.

Bottom Line: Injury-initiated epithelial to mesenchymal transition (EMT) depends on contextual signals from the extracellular matrix, suggesting a role for integrin signaling.A mechanism for this defect was explored in alpha3- cells reconstituted with wild-type (wt) alpha3 or point mutants unable to engage laminin 5 (G163A) or epithelial cadherin (E-cadherin; H245A).These findings demonstrate that alpha3beta1 coordinates cross talk between beta-catenin and Smad signaling pathways as a function of extracellular contact cues and thereby regulates responses to TGF-beta1 activation.

View Article: PubMed Central - PubMed

Affiliation: Pulmonary and Critical Care Division, Department of Medicine, and Cardiovascular Research Institute, University of California, San Francisco, San Francisco, CA 94143, USA.

ABSTRACT
Injury-initiated epithelial to mesenchymal transition (EMT) depends on contextual signals from the extracellular matrix, suggesting a role for integrin signaling. Primary epithelial cells deficient in their prominent laminin receptor, alpha3beta1, were found to have a markedly blunted EMT response to TGF-beta1. A mechanism for this defect was explored in alpha3- cells reconstituted with wild-type (wt) alpha3 or point mutants unable to engage laminin 5 (G163A) or epithelial cadherin (E-cadherin; H245A). After TGF-beta1 stimulation, wt epithelial cells but not cells expressing the H245A mutant internalize complexes of E-cadherin and TGF-beta1 receptors, generate phospho-Smad2 (p-Smad2)-pY654-beta-catenin complexes, and up-regulate mesenchymal target genes. Although Smad2 phosphorylation is normal, p-Smad2-pY654-beta-catenin complexes do not form in the absence of alpha3 or when alpha3beta1 is mainly engaged on laminin 5 or E-cadherin in adherens junctions, leading to attenuated EMT. These findings demonstrate that alpha3beta1 coordinates cross talk between beta-catenin and Smad signaling pathways as a function of extracellular contact cues and thereby regulates responses to TGF-beta1 activation.

Show MeSH
Related in: MedlinePlus