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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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Effect of calcium on TGF-β1–mediated signaling and β-catenin–p-Smad2 complex formation. (A) Phase photographs of α3 wt cells cultured in either low (125 µM) or high (1.8 mM) calcium media showing cells cultured in high calcium media form tight cell–cell contact, whereas cells cultured in low calcium media have loose cell–cell contact. Bar, 50 µm. (B) Confocal microscopy of E-cadherin clusters in α3 wt cells cultured under either low (125 µM) or high (1.8 mM) calcium conditions. α3 wt cells cultured in high calcium media show that E-cadherin clusters occur mainly in cells at the periphery of the colony, whereas α3 wt cells cultured in low calcium media form E-cadherin clusters throughout the colony. The boxed areas are shown in higher magnifications in the bottom panels. Bar, 2 µm. (C) Serum-starved α3 wt and H245A mutant cells under high or low calcium conditions were stimulated with TGF-β1 for 48 h. Supernatants were removed from the plates and concentrated for zymography. α3 wt cells cultured in low calcium show higher MMP-9 levels than α3 wt cells cultured in high calcium after TGF-β1 stimulation. (D) Serum-starved α3 wt cells in high or low calcium media were stimulated with TGF-β1 for 48 h, and the lysates were blotted for α-SMA. α-SMA is only up-regulated in cells cultured in low calcium medium and not in high calcium condition. (E) Cells cultured under low calcium condition show increased β-catenin–p-Smad2 complex formation. α3 wt cells cultured in either high or low calcium medium were treated with TGF-β1, and the lysates were subjected to β-catenin IP followed by p-Smad2 immunoblotting. The β-catenin–p-Smad2 complex formation is only seen with cells cultured in low calcium condition and not in high calcium condition. All of the aforementioned experiments have been performed at least three times with similar results.
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fig6: Effect of calcium on TGF-β1–mediated signaling and β-catenin–p-Smad2 complex formation. (A) Phase photographs of α3 wt cells cultured in either low (125 µM) or high (1.8 mM) calcium media showing cells cultured in high calcium media form tight cell–cell contact, whereas cells cultured in low calcium media have loose cell–cell contact. Bar, 50 µm. (B) Confocal microscopy of E-cadherin clusters in α3 wt cells cultured under either low (125 µM) or high (1.8 mM) calcium conditions. α3 wt cells cultured in high calcium media show that E-cadherin clusters occur mainly in cells at the periphery of the colony, whereas α3 wt cells cultured in low calcium media form E-cadherin clusters throughout the colony. The boxed areas are shown in higher magnifications in the bottom panels. Bar, 2 µm. (C) Serum-starved α3 wt and H245A mutant cells under high or low calcium conditions were stimulated with TGF-β1 for 48 h. Supernatants were removed from the plates and concentrated for zymography. α3 wt cells cultured in low calcium show higher MMP-9 levels than α3 wt cells cultured in high calcium after TGF-β1 stimulation. (D) Serum-starved α3 wt cells in high or low calcium media were stimulated with TGF-β1 for 48 h, and the lysates were blotted for α-SMA. α-SMA is only up-regulated in cells cultured in low calcium medium and not in high calcium condition. (E) Cells cultured under low calcium condition show increased β-catenin–p-Smad2 complex formation. α3 wt cells cultured in either high or low calcium medium were treated with TGF-β1, and the lysates were subjected to β-catenin IP followed by p-Smad2 immunoblotting. The β-catenin–p-Smad2 complex formation is only seen with cells cultured in low calcium condition and not in high calcium condition. All of the aforementioned experiments have been performed at least three times with similar results.

Mentions: The finding of α3β1-dependent β-catenin–p-Smad2 complexes raised the intriguing possibility that formation of such complexes is regulated at the cell surface by determinants of an assembly of complexes containing α3β1, E-cadherin, and TGF-βR1. Therefore, we asked whether limiting the availability of either α3β1 or E-cadherin would influence the formation of β-catenin–p-Smad2 complexes and the subsequent transcriptional responses. To vary access of E-cadherin to TGF-β1 receptors, we used two approaches. First, we varied the culture medium calcium concentration. Under high calcium conditions (∼1.8 mM), α3 wt cells show a tight clustered phenotype (Fig. 6 A). Clustering of E-cadherin on live cells is mainly confined to the periphery of well-compacted epithelial cell islands (Fig. 6 B), and there are relatively little β-catenin–p-Smad2 complexes formed after TGF-β1 stimulation (Fig. 6 E). In contrast, under low calcium conditions (∼125 µM Ca2+), there is more extensive E-cadherin clustering (Fig. 6 B) and complex formation (Fig. 6 E). Again, there is a direct correlation between the degree of β-catenin–p-Smad complexes and the induction of MMP-9 (Fig. 6 C) and α-SMA (Fig. 6 D) after TGF-β1 stimulation. Note that the complexes are observed within 60 min of exposure of the cells to TGF-β1, whereas the protein responses are measured after 48 h. In a second approach, we varied the plating density of the epithelial cells 24 h before TGF-β1 stimulation. At a high density in which there is extensive E-cadherin–dependent cell–cell contact, little or no β-catenin–p-Smad complexes were observed (Fig. S5 B) and little α-SMA was induced (not depicted). At low plating density, i.e., when E-cadherin is unengaged, both transcriptional complexes and induction of α-SMA were robust, confirming the critical role of the extent of adherens junction formation as a regulator of cellular responses 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)

Effect of calcium on TGF-β1–mediated signaling and β-catenin–p-Smad2 complex formation. (A) Phase photographs of α3 wt cells cultured in either low (125 µM) or high (1.8 mM) calcium media showing cells cultured in high calcium media form tight cell–cell contact, whereas cells cultured in low calcium media have loose cell–cell contact. Bar, 50 µm. (B) Confocal microscopy of E-cadherin clusters in α3 wt cells cultured under either low (125 µM) or high (1.8 mM) calcium conditions. α3 wt cells cultured in high calcium media show that E-cadherin clusters occur mainly in cells at the periphery of the colony, whereas α3 wt cells cultured in low calcium media form E-cadherin clusters throughout the colony. The boxed areas are shown in higher magnifications in the bottom panels. Bar, 2 µm. (C) Serum-starved α3 wt and H245A mutant cells under high or low calcium conditions were stimulated with TGF-β1 for 48 h. Supernatants were removed from the plates and concentrated for zymography. α3 wt cells cultured in low calcium show higher MMP-9 levels than α3 wt cells cultured in high calcium after TGF-β1 stimulation. (D) Serum-starved α3 wt cells in high or low calcium media were stimulated with TGF-β1 for 48 h, and the lysates were blotted for α-SMA. α-SMA is only up-regulated in cells cultured in low calcium medium and not in high calcium condition. (E) Cells cultured under low calcium condition show increased β-catenin–p-Smad2 complex formation. α3 wt cells cultured in either high or low calcium medium were treated with TGF-β1, and the lysates were subjected to β-catenin IP followed by p-Smad2 immunoblotting. The β-catenin–p-Smad2 complex formation is only seen with cells cultured in low calcium condition and not in high calcium condition. All of the aforementioned experiments have been performed at least three times with similar results.
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Related In: Results  -  Collection

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fig6: Effect of calcium on TGF-β1–mediated signaling and β-catenin–p-Smad2 complex formation. (A) Phase photographs of α3 wt cells cultured in either low (125 µM) or high (1.8 mM) calcium media showing cells cultured in high calcium media form tight cell–cell contact, whereas cells cultured in low calcium media have loose cell–cell contact. Bar, 50 µm. (B) Confocal microscopy of E-cadherin clusters in α3 wt cells cultured under either low (125 µM) or high (1.8 mM) calcium conditions. α3 wt cells cultured in high calcium media show that E-cadherin clusters occur mainly in cells at the periphery of the colony, whereas α3 wt cells cultured in low calcium media form E-cadherin clusters throughout the colony. The boxed areas are shown in higher magnifications in the bottom panels. Bar, 2 µm. (C) Serum-starved α3 wt and H245A mutant cells under high or low calcium conditions were stimulated with TGF-β1 for 48 h. Supernatants were removed from the plates and concentrated for zymography. α3 wt cells cultured in low calcium show higher MMP-9 levels than α3 wt cells cultured in high calcium after TGF-β1 stimulation. (D) Serum-starved α3 wt cells in high or low calcium media were stimulated with TGF-β1 for 48 h, and the lysates were blotted for α-SMA. α-SMA is only up-regulated in cells cultured in low calcium medium and not in high calcium condition. (E) Cells cultured under low calcium condition show increased β-catenin–p-Smad2 complex formation. α3 wt cells cultured in either high or low calcium medium were treated with TGF-β1, and the lysates were subjected to β-catenin IP followed by p-Smad2 immunoblotting. The β-catenin–p-Smad2 complex formation is only seen with cells cultured in low calcium condition and not in high calcium condition. All of the aforementioned experiments have been performed at least three times with similar results.
Mentions: The finding of α3β1-dependent β-catenin–p-Smad2 complexes raised the intriguing possibility that formation of such complexes is regulated at the cell surface by determinants of an assembly of complexes containing α3β1, E-cadherin, and TGF-βR1. Therefore, we asked whether limiting the availability of either α3β1 or E-cadherin would influence the formation of β-catenin–p-Smad2 complexes and the subsequent transcriptional responses. To vary access of E-cadherin to TGF-β1 receptors, we used two approaches. First, we varied the culture medium calcium concentration. Under high calcium conditions (∼1.8 mM), α3 wt cells show a tight clustered phenotype (Fig. 6 A). Clustering of E-cadherin on live cells is mainly confined to the periphery of well-compacted epithelial cell islands (Fig. 6 B), and there are relatively little β-catenin–p-Smad2 complexes formed after TGF-β1 stimulation (Fig. 6 E). In contrast, under low calcium conditions (∼125 µM Ca2+), there is more extensive E-cadherin clustering (Fig. 6 B) and complex formation (Fig. 6 E). Again, there is a direct correlation between the degree of β-catenin–p-Smad complexes and the induction of MMP-9 (Fig. 6 C) and α-SMA (Fig. 6 D) after TGF-β1 stimulation. Note that the complexes are observed within 60 min of exposure of the cells to TGF-β1, whereas the protein responses are measured after 48 h. In a second approach, we varied the plating density of the epithelial cells 24 h before TGF-β1 stimulation. At a high density in which there is extensive E-cadherin–dependent cell–cell contact, little or no β-catenin–p-Smad complexes were observed (Fig. S5 B) and little α-SMA was induced (not depicted). At low plating density, i.e., when E-cadherin is unengaged, both transcriptional complexes and induction of α-SMA were robust, confirming the critical role of the extent of adherens junction formation as a regulator of cellular responses 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