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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 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.
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fig1: 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.

Mentions: Erythrocyte band 4.1 superfamily proteins have been suggested to play roles in epithelial integrity, and we had chosen epithelial NMuMG cells to examine EPB41L5 functions. However, EPB41L5 was barely detectable in confluent epithelial NMuMG cells (Fig. 1, Ab, c, and Bb). Epithelial NMuMG cells are transformed into mesenchyme-like cells (“mesenchyme”) by treatment with TGFβ (Miettinen et al., 1994). With this transition, the EPB41L5 expression was markedly elevated (Fig. 1, Ab, c, and Bd). In addition, EPB41L5 siRNA inhibited the EMT of NMuMG cells by TGFβ. At the mRNA level, even when the EPB41L5 protein expression was inhibited by the siRNA (Fig. 1 Ad), E-cadherin expression was reduced and β1-integrin, Snail, and Twist expression were all enhanced normally by TGFβ (Fig. 2, Aa and Ba). At the protein level, however, E-cadherin expression was retained and β1-integrin expression was not elevated significantly (Fig. 2, Ab and Bb). EPB41L5 siRNA did not affect the increase in N-cadherin expression by TGFβ; nor did it affect α-catenin, β-catenin, p120ctn, or paxillin expression (Fig. 2 A). Concomitantly, EPB41L5 siRNA-treated NMuMG cells did not undergo morphological transformation into “mesenchyme” by TGFβ, retaining E-cadherin at the sites of cell–cell contact (Fig. 1, Be and f).


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 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.
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fig1: 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.
Mentions: Erythrocyte band 4.1 superfamily proteins have been suggested to play roles in epithelial integrity, and we had chosen epithelial NMuMG cells to examine EPB41L5 functions. However, EPB41L5 was barely detectable in confluent epithelial NMuMG cells (Fig. 1, Ab, c, and Bb). Epithelial NMuMG cells are transformed into mesenchyme-like cells (“mesenchyme”) by treatment with TGFβ (Miettinen et al., 1994). With this transition, the EPB41L5 expression was markedly elevated (Fig. 1, Ab, c, and Bd). In addition, EPB41L5 siRNA inhibited the EMT of NMuMG cells by TGFβ. At the mRNA level, even when the EPB41L5 protein expression was inhibited by the siRNA (Fig. 1 Ad), E-cadherin expression was reduced and β1-integrin, Snail, and Twist expression were all enhanced normally by TGFβ (Fig. 2, Aa and Ba). At the protein level, however, E-cadherin expression was retained and β1-integrin expression was not elevated significantly (Fig. 2, Ab and Bb). EPB41L5 siRNA did not affect the increase in N-cadherin expression by TGFβ; nor did it affect α-catenin, β-catenin, p120ctn, or paxillin expression (Fig. 2 A). Concomitantly, EPB41L5 siRNA-treated NMuMG cells did not undergo morphological transformation into “mesenchyme” by TGFβ, retaining E-cadherin at the sites of cell–cell contact (Fig. 1, Be and f).

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