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EPB41L5 functions to post-transcriptionally regulate cadherin and integrin during epithelial-mesenchymal transition.

Hirano M, Hashimoto S, Yonemura S, Sabe H, Aizawa S - J. Cell Biol. (2008)

Bottom Line: Moreover, cell attachment, spreading, and mobility are greatly reduced by EPB41L5 deficiency.Gene transcription regulation during EMT occurs normally at the mRNA level; EPB41L5 siRNA does not affect either the decrease in E-cadherin or the increase in integrin expression.However, at the protein level, the decrease in E-cadherin and increase in integrin are inhibited in both EPB41L5 siRNA-treated NMuMG cells and mutant mesoderm.

View Article: PubMed Central - PubMed

Affiliation: Laboratory for Vertebrate Body Plan, Center for Developmental Biology, RIKEN Kobe, Chuo-ku, Kobe 650-0047, Japan.

ABSTRACT
EPB41L5 belongs to the band 4.1 superfamily. We investigate here the involvement of EPB41L5 in epithelial-mesenchymal transition (EMT) during mouse gastrulation. EPB41L5 expression is induced during TGFbeta-stimulated EMT, whereas silencing of EPB41L5 by siRNA inhibits this transition. In EPB41L5 mutants, cell-cell adhesion is enhanced, and EMT is greatly impaired during gastrulation. Moreover, cell attachment, spreading, and mobility are greatly reduced by EPB41L5 deficiency. Gene transcription regulation during EMT occurs normally at the mRNA level; EPB41L5 siRNA does not affect either the decrease in E-cadherin or the increase in integrin expression. However, at the protein level, the decrease in E-cadherin and increase in integrin are inhibited in both EPB41L5 siRNA-treated NMuMG cells and mutant mesoderm. We find that EPB41L5 binds p120ctn through its N-terminal FERM domain, inhibiting p120ctn-E-cadherin binding. EPB41L5 overexpression causes E-cadherin relocalization into Rab5-positive vesicles in epithelial cells. At the same time, EPB41L5 binds to paxillin through its C terminus, enhancing integrin/paxillin association, thereby stimulating focal adhesion formation.

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EPB41L5 interaction with E-cadherin–associated adhesion components. (a) EPB41L5 binding to Arf1, Arf6, Hakai, and p120 catenin. Each expression vector harboring Arf1, Arf6, Hakai, or p120 catenin (p120ctn) linked with flag was transfected into Cos7 cells 12 h after plating, and the cells were harvested 24 h later. The bottom panel gives the Western blotting for the expression of each transgene in total cell lysates of respective transfectants. In the top panel the lysates were immunoprecipitated with the anti-EPB41L5 antibody, and the presence or absence of each molecule in the immunoprecipitates was blotted with anti-flag antibody. (b) EPB41L5 binding to p120ctn, α-catenin (αctn) and β-catenin (βctn). The left panel gives the expression level of each catenin in TGFβ-treated NMuMG cells. In the other two lanes the cell lysates were immunoprecipitated with rabbit IgG (middle) or rabbit anti-EPB41L5 antibody (right), and the presence of each catenin in the precipitates was blotted with the antibody against each catenin. (c) EPB41L5 binding to p120 catenin. Myc, myc-linked full-length p120ctn, myc-linked p120ctn that lacks N-terminal (ΔN), or myc-linked p120ctn lacking C-terminal armadillo repeats (ΔC) was transfected into Cos7 cells as described in panel a, cell lysates were immunoprecipitated with anti-myc antibody, and the presence or absence of EPB41L5 in the precipitates was blotted with anti-EPB41L5 antibody. (d) p120 catenin binding to EPB41L5. HA, HA-linked full-length EPB41L5, HA-linked EPB41L5 that lacks N-terminal FERM domain (ΔN), or HA-linked EPB41L5 lacking C-terminal (ΔC) was transfected into Cos7 cells, cell lysates were immunoprecipitated with anti-HA antibody, and the presence or absence of p120ctn in the precipitates was blotted with anti-p120ctn antibody. See Fig. 1 A for the site of the EPB41L5 truncation. (e) Effects of EPB41L5 on the binding between E-cadherin and p120ctn. 1 μg HA-linked E-cadherin and myc-linked p120ctn were cotransfected with 0, 1, 2.5, or 5 μg GFP-linked full-length EPB41L5 or with 5 μg GFP-linked EPB41L5 that lacks the N-terminal (ΔN). The bottom panel gives the amount of E-cadherin in total cell lysates. The lysates were immunoprecipitated with anti-myc antibody for p120ctn and the amount of E-cadherin in the precipitates was blotted with anti-HA antibody. (f) Effects of EPB41L5 that lacks C-terminal but retains N-terminal FERM domain on the binding between E-cadherin and p120ctn. (g) Effects of EPB41L5 on the binding between N-cadherin and p120ctn. The binding assay in f and g was conducted as described in panel e.
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fig6: EPB41L5 interaction with E-cadherin–associated adhesion components. (a) EPB41L5 binding to Arf1, Arf6, Hakai, and p120 catenin. Each expression vector harboring Arf1, Arf6, Hakai, or p120 catenin (p120ctn) linked with flag was transfected into Cos7 cells 12 h after plating, and the cells were harvested 24 h later. The bottom panel gives the Western blotting for the expression of each transgene in total cell lysates of respective transfectants. In the top panel the lysates were immunoprecipitated with the anti-EPB41L5 antibody, and the presence or absence of each molecule in the immunoprecipitates was blotted with anti-flag antibody. (b) EPB41L5 binding to p120ctn, α-catenin (αctn) and β-catenin (βctn). The left panel gives the expression level of each catenin in TGFβ-treated NMuMG cells. In the other two lanes the cell lysates were immunoprecipitated with rabbit IgG (middle) or rabbit anti-EPB41L5 antibody (right), and the presence of each catenin in the precipitates was blotted with the antibody against each catenin. (c) EPB41L5 binding to p120 catenin. Myc, myc-linked full-length p120ctn, myc-linked p120ctn that lacks N-terminal (ΔN), or myc-linked p120ctn lacking C-terminal armadillo repeats (ΔC) was transfected into Cos7 cells as described in panel a, cell lysates were immunoprecipitated with anti-myc antibody, and the presence or absence of EPB41L5 in the precipitates was blotted with anti-EPB41L5 antibody. (d) p120 catenin binding to EPB41L5. HA, HA-linked full-length EPB41L5, HA-linked EPB41L5 that lacks N-terminal FERM domain (ΔN), or HA-linked EPB41L5 lacking C-terminal (ΔC) was transfected into Cos7 cells, cell lysates were immunoprecipitated with anti-HA antibody, and the presence or absence of p120ctn in the precipitates was blotted with anti-p120ctn antibody. See Fig. 1 A for the site of the EPB41L5 truncation. (e) Effects of EPB41L5 on the binding between E-cadherin and p120ctn. 1 μg HA-linked E-cadherin and myc-linked p120ctn were cotransfected with 0, 1, 2.5, or 5 μg GFP-linked full-length EPB41L5 or with 5 μg GFP-linked EPB41L5 that lacks the N-terminal (ΔN). The bottom panel gives the amount of E-cadherin in total cell lysates. The lysates were immunoprecipitated with anti-myc antibody for p120ctn and the amount of E-cadherin in the precipitates was blotted with anti-HA antibody. (f) Effects of EPB41L5 that lacks C-terminal but retains N-terminal FERM domain on the binding between E-cadherin and p120ctn. (g) Effects of EPB41L5 on the binding between N-cadherin and p120ctn. The binding assay in f and g was conducted as described in panel e.

Mentions: EPB41L5 functions in EMT of NMuMG cells by TGFβ. (A) EPB41L5 expression in NMuMG cells. (a) Schematic representation of EPB41L5. Red represents FERM domain. An arrow indicates the point of truncation in the analyses of Fig. 6 (d–f), Fig. 7 (e–l), Fig. 8 (d), and Fig. 9 (Bc and d). The underline indicates the location of the sequences used as an antigen to raise an antibody. Arrowheads (F, R) indicate the locations of primers for RT-PCR analyses in b. (b and c) The increase in EPB41L5 expression with the TGFβ treatment assayed by RT-PCR (b) at 48 h and Western blotting (c) at the indicated time. (d) Effect of EPB41L5 siRNA treatment on the EPB41L5 protein induction by TGFβ. The cells were transfected with control or EPB41L5 siRNA and cultured in the absence or presence of TGFβ for 48 h. (B) Inhibition of TGFβ-induced EMT of NMuMG cells by EPB41L5 siRNA. EPB41L5 (red) is scarce in the confluent epithelial NMuMG cells in the absence of TGFβ; E-cadherin (green) is abundant at cell–cell contact sites (a and b). In “mesenchymal” cells induced by TGFβ, EPB41L5 is abundantly present with no E-cadherin expression (c and d). EPB41L5 siRNA inhibits the transformation into “mesenchyme” by TGFβ (e), retaining E-cadherin at cell–cell contact sites (f). Bars, 20 μm.


EPB41L5 functions to post-transcriptionally regulate cadherin and integrin during epithelial-mesenchymal transition.

Hirano M, Hashimoto S, Yonemura S, Sabe H, Aizawa S - J. Cell Biol. (2008)

EPB41L5 interaction with E-cadherin–associated adhesion components. (a) EPB41L5 binding to Arf1, Arf6, Hakai, and p120 catenin. Each expression vector harboring Arf1, Arf6, Hakai, or p120 catenin (p120ctn) linked with flag was transfected into Cos7 cells 12 h after plating, and the cells were harvested 24 h later. The bottom panel gives the Western blotting for the expression of each transgene in total cell lysates of respective transfectants. In the top panel the lysates were immunoprecipitated with the anti-EPB41L5 antibody, and the presence or absence of each molecule in the immunoprecipitates was blotted with anti-flag antibody. (b) EPB41L5 binding to p120ctn, α-catenin (αctn) and β-catenin (βctn). The left panel gives the expression level of each catenin in TGFβ-treated NMuMG cells. In the other two lanes the cell lysates were immunoprecipitated with rabbit IgG (middle) or rabbit anti-EPB41L5 antibody (right), and the presence of each catenin in the precipitates was blotted with the antibody against each catenin. (c) EPB41L5 binding to p120 catenin. Myc, myc-linked full-length p120ctn, myc-linked p120ctn that lacks N-terminal (ΔN), or myc-linked p120ctn lacking C-terminal armadillo repeats (ΔC) was transfected into Cos7 cells as described in panel a, cell lysates were immunoprecipitated with anti-myc antibody, and the presence or absence of EPB41L5 in the precipitates was blotted with anti-EPB41L5 antibody. (d) p120 catenin binding to EPB41L5. HA, HA-linked full-length EPB41L5, HA-linked EPB41L5 that lacks N-terminal FERM domain (ΔN), or HA-linked EPB41L5 lacking C-terminal (ΔC) was transfected into Cos7 cells, cell lysates were immunoprecipitated with anti-HA antibody, and the presence or absence of p120ctn in the precipitates was blotted with anti-p120ctn antibody. See Fig. 1 A for the site of the EPB41L5 truncation. (e) Effects of EPB41L5 on the binding between E-cadherin and p120ctn. 1 μg HA-linked E-cadherin and myc-linked p120ctn were cotransfected with 0, 1, 2.5, or 5 μg GFP-linked full-length EPB41L5 or with 5 μg GFP-linked EPB41L5 that lacks the N-terminal (ΔN). The bottom panel gives the amount of E-cadherin in total cell lysates. The lysates were immunoprecipitated with anti-myc antibody for p120ctn and the amount of E-cadherin in the precipitates was blotted with anti-HA antibody. (f) Effects of EPB41L5 that lacks C-terminal but retains N-terminal FERM domain on the binding between E-cadherin and p120ctn. (g) Effects of EPB41L5 on the binding between N-cadherin and p120ctn. The binding assay in f and g was conducted as described in panel e.
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fig6: EPB41L5 interaction with E-cadherin–associated adhesion components. (a) EPB41L5 binding to Arf1, Arf6, Hakai, and p120 catenin. Each expression vector harboring Arf1, Arf6, Hakai, or p120 catenin (p120ctn) linked with flag was transfected into Cos7 cells 12 h after plating, and the cells were harvested 24 h later. The bottom panel gives the Western blotting for the expression of each transgene in total cell lysates of respective transfectants. In the top panel the lysates were immunoprecipitated with the anti-EPB41L5 antibody, and the presence or absence of each molecule in the immunoprecipitates was blotted with anti-flag antibody. (b) EPB41L5 binding to p120ctn, α-catenin (αctn) and β-catenin (βctn). The left panel gives the expression level of each catenin in TGFβ-treated NMuMG cells. In the other two lanes the cell lysates were immunoprecipitated with rabbit IgG (middle) or rabbit anti-EPB41L5 antibody (right), and the presence of each catenin in the precipitates was blotted with the antibody against each catenin. (c) EPB41L5 binding to p120 catenin. Myc, myc-linked full-length p120ctn, myc-linked p120ctn that lacks N-terminal (ΔN), or myc-linked p120ctn lacking C-terminal armadillo repeats (ΔC) was transfected into Cos7 cells as described in panel a, cell lysates were immunoprecipitated with anti-myc antibody, and the presence or absence of EPB41L5 in the precipitates was blotted with anti-EPB41L5 antibody. (d) p120 catenin binding to EPB41L5. HA, HA-linked full-length EPB41L5, HA-linked EPB41L5 that lacks N-terminal FERM domain (ΔN), or HA-linked EPB41L5 lacking C-terminal (ΔC) was transfected into Cos7 cells, cell lysates were immunoprecipitated with anti-HA antibody, and the presence or absence of p120ctn in the precipitates was blotted with anti-p120ctn antibody. See Fig. 1 A for the site of the EPB41L5 truncation. (e) Effects of EPB41L5 on the binding between E-cadherin and p120ctn. 1 μg HA-linked E-cadherin and myc-linked p120ctn were cotransfected with 0, 1, 2.5, or 5 μg GFP-linked full-length EPB41L5 or with 5 μg GFP-linked EPB41L5 that lacks the N-terminal (ΔN). The bottom panel gives the amount of E-cadherin in total cell lysates. The lysates were immunoprecipitated with anti-myc antibody for p120ctn and the amount of E-cadherin in the precipitates was blotted with anti-HA antibody. (f) Effects of EPB41L5 that lacks C-terminal but retains N-terminal FERM domain on the binding between E-cadherin and p120ctn. (g) Effects of EPB41L5 on the binding between N-cadherin and p120ctn. The binding assay in f and g was conducted as described in panel e.
Mentions: EPB41L5 functions in EMT of NMuMG cells by TGFβ. (A) EPB41L5 expression in NMuMG cells. (a) Schematic representation of EPB41L5. Red represents FERM domain. An arrow indicates the point of truncation in the analyses of Fig. 6 (d–f), Fig. 7 (e–l), Fig. 8 (d), and Fig. 9 (Bc and d). The underline indicates the location of the sequences used as an antigen to raise an antibody. Arrowheads (F, R) indicate the locations of primers for RT-PCR analyses in b. (b and c) The increase in EPB41L5 expression with the TGFβ treatment assayed by RT-PCR (b) at 48 h and Western blotting (c) at the indicated time. (d) Effect of EPB41L5 siRNA treatment on the EPB41L5 protein induction by TGFβ. The cells were transfected with control or EPB41L5 siRNA and cultured in the absence or presence of TGFβ for 48 h. (B) Inhibition of TGFβ-induced EMT of NMuMG cells by EPB41L5 siRNA. EPB41L5 (red) is scarce in the confluent epithelial NMuMG cells in the absence of TGFβ; E-cadherin (green) is abundant at cell–cell contact sites (a and b). In “mesenchymal” cells induced by TGFβ, EPB41L5 is abundantly present with no E-cadherin expression (c and d). EPB41L5 siRNA inhibits the transformation into “mesenchyme” by TGFβ (e), retaining E-cadherin at cell–cell contact sites (f). Bars, 20 μm.

Bottom Line: Moreover, cell attachment, spreading, and mobility are greatly reduced by EPB41L5 deficiency.Gene transcription regulation during EMT occurs normally at the mRNA level; EPB41L5 siRNA does not affect either the decrease in E-cadherin or the increase in integrin expression.However, at the protein level, the decrease in E-cadherin and increase in integrin are inhibited in both EPB41L5 siRNA-treated NMuMG cells and mutant mesoderm.

View Article: PubMed Central - PubMed

Affiliation: Laboratory for Vertebrate Body Plan, Center for Developmental Biology, RIKEN Kobe, Chuo-ku, Kobe 650-0047, Japan.

ABSTRACT
EPB41L5 belongs to the band 4.1 superfamily. We investigate here the involvement of EPB41L5 in epithelial-mesenchymal transition (EMT) during mouse gastrulation. EPB41L5 expression is induced during TGFbeta-stimulated EMT, whereas silencing of EPB41L5 by siRNA inhibits this transition. In EPB41L5 mutants, cell-cell adhesion is enhanced, and EMT is greatly impaired during gastrulation. Moreover, cell attachment, spreading, and mobility are greatly reduced by EPB41L5 deficiency. Gene transcription regulation during EMT occurs normally at the mRNA level; EPB41L5 siRNA does not affect either the decrease in E-cadherin or the increase in integrin expression. However, at the protein level, the decrease in E-cadherin and increase in integrin are inhibited in both EPB41L5 siRNA-treated NMuMG cells and mutant mesoderm. We find that EPB41L5 binds p120ctn through its N-terminal FERM domain, inhibiting p120ctn-E-cadherin binding. EPB41L5 overexpression causes E-cadherin relocalization into Rab5-positive vesicles in epithelial cells. At the same time, EPB41L5 binds to paxillin through its C terminus, enhancing integrin/paxillin association, thereby stimulating focal adhesion formation.

Show MeSH
Related in: MedlinePlus