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Distinct roles of cadherin-6 and E-cadherin in tubulogenesis and lumen formation.

Jia L, Liu F, Hansen SH, Ter Beest MB, Zegers MM - Mol. Biol. Cell (2011)

Bottom Line: Specifically, we find that cadherin-6 functions as an inhibitor of tubulogenesis, whereas E-cadherin is required for lumen formation.Ablation of cadherin-6 leads to the spontaneous formation of tubules, which depends on increased phosphoinositide 3-kinase (PI3K) activity.In contrast, loss of E-cadherin inhibits lumen formation by a mechanism independent of PI3K.

View Article: PubMed Central - PubMed

Affiliation: Department of Surgery, Committee on Cancer Biology, University of Chicago, IL 60637, USA.

ABSTRACT
Classic cadherins are important regulators of tissue morphogenesis. The predominant cadherin in epithelial cells, E-cadherin, has been extensively studied because of its critical role in normal epithelial development and carcinogenesis. Epithelial cells may also coexpress other cadherins, but their roles are less clear. The Madin Darby canine kidney (MDCK) cell line has been a popular mammalian model to investigate the role of E-cadherin in epithelial polarization and tubulogenesis. However, MDCK cells also express relatively high levels of cadherin-6, and it is unclear whether the functions of this cadherin are redundant to those of E-cadherin. We investigate the specific roles of both cadherins using a knockdown approach. Although we find that both cadherins are able to form adherens junctions at the basolateral surface, we show that they have specific and mutually exclusive roles in epithelial morphogenesis. Specifically, we find that cadherin-6 functions as an inhibitor of tubulogenesis, whereas E-cadherin is required for lumen formation. Ablation of cadherin-6 leads to the spontaneous formation of tubules, which depends on increased phosphoinositide 3-kinase (PI3K) activity. In contrast, loss of E-cadherin inhibits lumen formation by a mechanism independent of PI3K.

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Specific knockdown of cadherin-6 or E-cadherin in 3D culture. (A1–D3) Control cysts (A1–A3) or cysts of MDCK cells expressing shRNA against cadherin-6 (B1–B3), E-cadherin (C1–C3), or both (D1–D3) were grown in the presence of doxycycline in 3D. Cysts were fixed and stained for E-cadherin and cadherin-6 at 13 d after plating. In control cysts, both cadherin-6 (A1) and E-cadherin (A2) localize at the lateral membrane. In cad6-KD cysts, cadherin-6 is lost from the lateral membrane (B1) without affecting expression or localization of E-cadherin (B2). Ecad-KD cysts have largely reduced E-cadherin (C2) at the basolateral surface, but expression or localization of cadherin-6 is not affected (C1). 6/E-KD cysts have lost expression of both cadherin-6 (D1) and E-cadherin (D2). Merged images (A3, B3, C3, D3) show cadherin-6 in green and E-cadherin in red. Nuclei, stained with 4’,6-diamidino-2-phenylindole (DAPI), are blue. Scale bar, 20 μm.
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Figure 2: Specific knockdown of cadherin-6 or E-cadherin in 3D culture. (A1–D3) Control cysts (A1–A3) or cysts of MDCK cells expressing shRNA against cadherin-6 (B1–B3), E-cadherin (C1–C3), or both (D1–D3) were grown in the presence of doxycycline in 3D. Cysts were fixed and stained for E-cadherin and cadherin-6 at 13 d after plating. In control cysts, both cadherin-6 (A1) and E-cadherin (A2) localize at the lateral membrane. In cad6-KD cysts, cadherin-6 is lost from the lateral membrane (B1) without affecting expression or localization of E-cadherin (B2). Ecad-KD cysts have largely reduced E-cadherin (C2) at the basolateral surface, but expression or localization of cadherin-6 is not affected (C1). 6/E-KD cysts have lost expression of both cadherin-6 (D1) and E-cadherin (D2). Merged images (A3, B3, C3, D3) show cadherin-6 in green and E-cadherin in red. Nuclei, stained with 4’,6-diamidino-2-phenylindole (DAPI), are blue. Scale bar, 20 μm.

Mentions: Confocal immunofluorescence microscopy confirmed that cadherin-6 and/or E-cadherin were effectively down-regulated in the cad6-KD, Ecad-KD, and 6/E-KD cysts (Figure 2, A–D). We next analyzed how cadherin knockdown affected cell polarization. Knockdown of cadherin-6 did not alter the basolateral localization of E-cadherin (compare Figure 2, A2 to B2) and vice versa (compare Figure 2, A1 to C1), and in both cases, β-catenin was still localized to the basolateral membrane (Figure 3, B1, C1). This indicates that cadherin-6 and E-cadherin play redundant roles in recruiting β-catenin to the basolateral membrane. The absence of basolateral β-catenin in 6/E-KD cysts (Figure 3D1) suggests that knockdown of both E-cadherin and cadherin-6 is sufficient to disrupt adherens junctions and that this defect is not compensated for by other cadherins. Cells in MDCK cysts orient their apical surface toward the central lumen. Apical polarization was not altered in cad6-KD cells, as judged by staining for the apical marker protein gp135/podocalyxin and the tight junction marker ZO-1 (compare Figure 3, A3, A4 to B3, B4). Ecad-KD cysts had multiple small lumens (Figure 3C3), which were not discernible by phase-contrast imaging (Figure 1D). Only cells facing these lumens exhibited apical-basolateral polarization of β-catenin, ZO-1, and gp135 (Figure 3, C1–C4). Apical-basolateral polarization was completely disrupted in 6/E-KD cysts, as judged by disorganized or loss of staining of β-catenin, ZO-1, and gp135 (Figure 3, D1–D4).


Distinct roles of cadherin-6 and E-cadherin in tubulogenesis and lumen formation.

Jia L, Liu F, Hansen SH, Ter Beest MB, Zegers MM - Mol. Biol. Cell (2011)

Specific knockdown of cadherin-6 or E-cadherin in 3D culture. (A1–D3) Control cysts (A1–A3) or cysts of MDCK cells expressing shRNA against cadherin-6 (B1–B3), E-cadherin (C1–C3), or both (D1–D3) were grown in the presence of doxycycline in 3D. Cysts were fixed and stained for E-cadherin and cadherin-6 at 13 d after plating. In control cysts, both cadherin-6 (A1) and E-cadherin (A2) localize at the lateral membrane. In cad6-KD cysts, cadherin-6 is lost from the lateral membrane (B1) without affecting expression or localization of E-cadherin (B2). Ecad-KD cysts have largely reduced E-cadherin (C2) at the basolateral surface, but expression or localization of cadherin-6 is not affected (C1). 6/E-KD cysts have lost expression of both cadherin-6 (D1) and E-cadherin (D2). Merged images (A3, B3, C3, D3) show cadherin-6 in green and E-cadherin in red. Nuclei, stained with 4’,6-diamidino-2-phenylindole (DAPI), are blue. Scale bar, 20 μm.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Figure 2: Specific knockdown of cadherin-6 or E-cadherin in 3D culture. (A1–D3) Control cysts (A1–A3) or cysts of MDCK cells expressing shRNA against cadherin-6 (B1–B3), E-cadherin (C1–C3), or both (D1–D3) were grown in the presence of doxycycline in 3D. Cysts were fixed and stained for E-cadherin and cadherin-6 at 13 d after plating. In control cysts, both cadherin-6 (A1) and E-cadherin (A2) localize at the lateral membrane. In cad6-KD cysts, cadherin-6 is lost from the lateral membrane (B1) without affecting expression or localization of E-cadherin (B2). Ecad-KD cysts have largely reduced E-cadherin (C2) at the basolateral surface, but expression or localization of cadherin-6 is not affected (C1). 6/E-KD cysts have lost expression of both cadherin-6 (D1) and E-cadherin (D2). Merged images (A3, B3, C3, D3) show cadherin-6 in green and E-cadherin in red. Nuclei, stained with 4’,6-diamidino-2-phenylindole (DAPI), are blue. Scale bar, 20 μm.
Mentions: Confocal immunofluorescence microscopy confirmed that cadherin-6 and/or E-cadherin were effectively down-regulated in the cad6-KD, Ecad-KD, and 6/E-KD cysts (Figure 2, A–D). We next analyzed how cadherin knockdown affected cell polarization. Knockdown of cadherin-6 did not alter the basolateral localization of E-cadherin (compare Figure 2, A2 to B2) and vice versa (compare Figure 2, A1 to C1), and in both cases, β-catenin was still localized to the basolateral membrane (Figure 3, B1, C1). This indicates that cadherin-6 and E-cadherin play redundant roles in recruiting β-catenin to the basolateral membrane. The absence of basolateral β-catenin in 6/E-KD cysts (Figure 3D1) suggests that knockdown of both E-cadherin and cadherin-6 is sufficient to disrupt adherens junctions and that this defect is not compensated for by other cadherins. Cells in MDCK cysts orient their apical surface toward the central lumen. Apical polarization was not altered in cad6-KD cells, as judged by staining for the apical marker protein gp135/podocalyxin and the tight junction marker ZO-1 (compare Figure 3, A3, A4 to B3, B4). Ecad-KD cysts had multiple small lumens (Figure 3C3), which were not discernible by phase-contrast imaging (Figure 1D). Only cells facing these lumens exhibited apical-basolateral polarization of β-catenin, ZO-1, and gp135 (Figure 3, C1–C4). Apical-basolateral polarization was completely disrupted in 6/E-KD cysts, as judged by disorganized or loss of staining of β-catenin, ZO-1, and gp135 (Figure 3, D1–D4).

Bottom Line: Specifically, we find that cadherin-6 functions as an inhibitor of tubulogenesis, whereas E-cadherin is required for lumen formation.Ablation of cadherin-6 leads to the spontaneous formation of tubules, which depends on increased phosphoinositide 3-kinase (PI3K) activity.In contrast, loss of E-cadherin inhibits lumen formation by a mechanism independent of PI3K.

View Article: PubMed Central - PubMed

Affiliation: Department of Surgery, Committee on Cancer Biology, University of Chicago, IL 60637, USA.

ABSTRACT
Classic cadherins are important regulators of tissue morphogenesis. The predominant cadherin in epithelial cells, E-cadherin, has been extensively studied because of its critical role in normal epithelial development and carcinogenesis. Epithelial cells may also coexpress other cadherins, but their roles are less clear. The Madin Darby canine kidney (MDCK) cell line has been a popular mammalian model to investigate the role of E-cadherin in epithelial polarization and tubulogenesis. However, MDCK cells also express relatively high levels of cadherin-6, and it is unclear whether the functions of this cadherin are redundant to those of E-cadherin. We investigate the specific roles of both cadherins using a knockdown approach. Although we find that both cadherins are able to form adherens junctions at the basolateral surface, we show that they have specific and mutually exclusive roles in epithelial morphogenesis. Specifically, we find that cadherin-6 functions as an inhibitor of tubulogenesis, whereas E-cadherin is required for lumen formation. Ablation of cadherin-6 leads to the spontaneous formation of tubules, which depends on increased phosphoinositide 3-kinase (PI3K) activity. In contrast, loss of E-cadherin inhibits lumen formation by a mechanism independent of PI3K.

Show MeSH
Related in: MedlinePlus