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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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Regulation of β-catenin–p-Smad2 complex formation by extracellular matrices. (A) α3β1 engagement on Ln5 limits the formation of β-catenin–p-Smad2 complex formation. Lysates of TGF-β1–stimulated α3 wt cells from either Fn- or Ln5-coated plates were subject to β-catenin IP followed by p-Smad2 immunoblotting. The β-catenin–p-Smad2 complex formation is only seen with cells plated on Fn and not with Ln5. (B) Ln5 does not limit formation of β-catenin–p-Smad2 complexes in cells unable to engage Ln5 through α3β1 (G163A mutant cells). Lysates of TGF-β1–stimulated G163A mutant cells from either Fn- or Ln5-coated plates were subjected to β-catenin IP followed by p-Smad2 immunoblotting. The β-catenin–p-Smad2 complex formation is seen with cells plated on both Fn and Ln5. (C) α3β1 engagement on Ln5 suppresses TGF-β1–induced α-SMA up-regulation. Up-regulation of α-SMA is suppressed in α3 wt cells plated on Ln5 but not in G163A mutant cells on Ln5. The aforementioned experiments have been performed three times with similar results.
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fig7: Regulation of β-catenin–p-Smad2 complex formation by extracellular matrices. (A) α3β1 engagement on Ln5 limits the formation of β-catenin–p-Smad2 complex formation. Lysates of TGF-β1–stimulated α3 wt cells from either Fn- or Ln5-coated plates were subject to β-catenin IP followed by p-Smad2 immunoblotting. The β-catenin–p-Smad2 complex formation is only seen with cells plated on Fn and not with Ln5. (B) Ln5 does not limit formation of β-catenin–p-Smad2 complexes in cells unable to engage Ln5 through α3β1 (G163A mutant cells). Lysates of TGF-β1–stimulated G163A mutant cells from either Fn- or Ln5-coated plates were subjected to β-catenin IP followed by p-Smad2 immunoblotting. The β-catenin–p-Smad2 complex formation is seen with cells plated on both Fn and Ln5. (C) α3β1 engagement on Ln5 suppresses TGF-β1–induced α-SMA up-regulation. Up-regulation of α-SMA is suppressed in α3 wt cells plated on Ln5 but not in G163A mutant cells on Ln5. The aforementioned experiments have been performed three times with similar results.

Mentions: When α3 wt cells were cultured on Ln5, sequestering α3β1 to the basal surface, β-catenin–p-Smad2 complexes failed to form (Fig. 7 A), and the α-SMA transcriptional response to TGF-β1 was again attenuated (Fig. 7 C, left). In contrast, on purified Fn, complex formation and α-SMA induction is robust. To further test the role of Ln5 engagement in regulating TGF-β1 responses, we repeated the experiments using G163A mutant α3-expressing cells that are unable to engage Ln5, and thus, Ln5 would be predicted to have less influence on the TGF-β1 responses of these cells. Indeed, β-catenin–p-Smad2 complexes were readily observed when G163A cells were plated onto either Fn or Ln5 (Fig. 7 B), and the α-SMA induction was equivalent on either matrix (Fig. 7 C, right). Collectively, these data indicate that the α3β1–E-cadherin–TGF-βR1 membrane complexes can coordinate cues from cell–matrix and cell–cell interactions to determine the cellular response to TGF-β1.


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)

Regulation of β-catenin–p-Smad2 complex formation by extracellular matrices. (A) α3β1 engagement on Ln5 limits the formation of β-catenin–p-Smad2 complex formation. Lysates of TGF-β1–stimulated α3 wt cells from either Fn- or Ln5-coated plates were subject to β-catenin IP followed by p-Smad2 immunoblotting. The β-catenin–p-Smad2 complex formation is only seen with cells plated on Fn and not with Ln5. (B) Ln5 does not limit formation of β-catenin–p-Smad2 complexes in cells unable to engage Ln5 through α3β1 (G163A mutant cells). Lysates of TGF-β1–stimulated G163A mutant cells from either Fn- or Ln5-coated plates were subjected to β-catenin IP followed by p-Smad2 immunoblotting. The β-catenin–p-Smad2 complex formation is seen with cells plated on both Fn and Ln5. (C) α3β1 engagement on Ln5 suppresses TGF-β1–induced α-SMA up-regulation. Up-regulation of α-SMA is suppressed in α3 wt cells plated on Ln5 but not in G163A mutant cells on Ln5. The aforementioned experiments have been performed three times with similar results.
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Related In: Results  -  Collection

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fig7: Regulation of β-catenin–p-Smad2 complex formation by extracellular matrices. (A) α3β1 engagement on Ln5 limits the formation of β-catenin–p-Smad2 complex formation. Lysates of TGF-β1–stimulated α3 wt cells from either Fn- or Ln5-coated plates were subject to β-catenin IP followed by p-Smad2 immunoblotting. The β-catenin–p-Smad2 complex formation is only seen with cells plated on Fn and not with Ln5. (B) Ln5 does not limit formation of β-catenin–p-Smad2 complexes in cells unable to engage Ln5 through α3β1 (G163A mutant cells). Lysates of TGF-β1–stimulated G163A mutant cells from either Fn- or Ln5-coated plates were subjected to β-catenin IP followed by p-Smad2 immunoblotting. The β-catenin–p-Smad2 complex formation is seen with cells plated on both Fn and Ln5. (C) α3β1 engagement on Ln5 suppresses TGF-β1–induced α-SMA up-regulation. Up-regulation of α-SMA is suppressed in α3 wt cells plated on Ln5 but not in G163A mutant cells on Ln5. The aforementioned experiments have been performed three times with similar results.
Mentions: When α3 wt cells were cultured on Ln5, sequestering α3β1 to the basal surface, β-catenin–p-Smad2 complexes failed to form (Fig. 7 A), and the α-SMA transcriptional response to TGF-β1 was again attenuated (Fig. 7 C, left). In contrast, on purified Fn, complex formation and α-SMA induction is robust. To further test the role of Ln5 engagement in regulating TGF-β1 responses, we repeated the experiments using G163A mutant α3-expressing cells that are unable to engage Ln5, and thus, Ln5 would be predicted to have less influence on the TGF-β1 responses of these cells. Indeed, β-catenin–p-Smad2 complexes were readily observed when G163A cells were plated onto either Fn or Ln5 (Fig. 7 B), and the α-SMA induction was equivalent on either matrix (Fig. 7 C, right). Collectively, these data indicate that the α3β1–E-cadherin–TGF-βR1 membrane complexes can coordinate cues from cell–matrix and cell–cell interactions to determine the cellular response to TGF-β1.

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