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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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Knockdown of E-cadherin or cadherin-6 induces distinct phenotypes. MDCK cells expressing a nontargeting control vector or inducible shRNA against E-cadherin, cadherin-6, or both were grown in the presence of doxycycline to induce shRNA expression. (A) Western blots from lysates of confluent cells that were induced for 2 d to express a nontargeting shRNA (control) or different shRNAs against cadherin-6 (cad6-KD1, cad6-KD2), E-cadherin (Ecad-KD1, Ecad-KD2), or both (6/E-KD1, 6/E-KD2). (B–E) Cell–cell adhesion as analyzed by an aggregation assay in control (B), cad6-KD1 (C), Ecad-KD1 (D), or 6/E-KD1 (E) cells. (F–I) Control cells or cadherin knockdown cells were grown as cysts in collagen gels. Phase contrast images were taken 13 d after plating. Control cells (F) form cysts with a spherical phenotype and a hollow lumen. Cad6-KD cysts (G) develop large tubules (arrows) and some narrow extensions (arrowhead), whereas Ecad-KD cysts (H) form spherical cysts with filled lumens. 6/E-KD cysts (I) have both tubules and filled lumens (arrow). Scale bar, 50 μm. (J) Percentage of cysts with tubules and filled lumens in cad6-KD1, cad6-KD2 (left), Ecad-KD1, Ecad-KD2 (middle), and 6/E-KD1, 6/E-KD2 cysts (right). Data represent mean ± SD. N = 3.
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Figure 1: Knockdown of E-cadherin or cadherin-6 induces distinct phenotypes. MDCK cells expressing a nontargeting control vector or inducible shRNA against E-cadherin, cadherin-6, or both were grown in the presence of doxycycline to induce shRNA expression. (A) Western blots from lysates of confluent cells that were induced for 2 d to express a nontargeting shRNA (control) or different shRNAs against cadherin-6 (cad6-KD1, cad6-KD2), E-cadherin (Ecad-KD1, Ecad-KD2), or both (6/E-KD1, 6/E-KD2). (B–E) Cell–cell adhesion as analyzed by an aggregation assay in control (B), cad6-KD1 (C), Ecad-KD1 (D), or 6/E-KD1 (E) cells. (F–I) Control cells or cadherin knockdown cells were grown as cysts in collagen gels. Phase contrast images were taken 13 d after plating. Control cells (F) form cysts with a spherical phenotype and a hollow lumen. Cad6-KD cysts (G) develop large tubules (arrows) and some narrow extensions (arrowhead), whereas Ecad-KD cysts (H) form spherical cysts with filled lumens. 6/E-KD cysts (I) have both tubules and filled lumens (arrow). Scale bar, 50 μm. (J) Percentage of cysts with tubules and filled lumens in cad6-KD1, cad6-KD2 (left), Ecad-KD1, Ecad-KD2 (middle), and 6/E-KD1, 6/E-KD2 cysts (right). Data represent mean ± SD. N = 3.

Mentions: To determine the roles of cadherin-6 and E-cadherin in epithelial morphogenesis in 3D cell culture, we created cell lines that conditionally express shRNA specific to cadherin-6 (cad6-KD), E-cadherin (Ecad-KD), or both (6/E-KD). We used two different targeting sequences specific for either cadherin and selected clones in which doxycycline-induced expression of the shRNAs effectively and specifically down-regulated cadherin-6 or E-cadherin protein expression by at least 95% (Figure 1A). We chose one clone for each hairpin to cadherin-6 (cad6-KD1, cad6-KD2) and E-cadherin (Ecad-KD1, Ecad-KD2) for all subsequent experiments. As conditional shRNA expression was not completely tight in all clones, we used cells that conditionally express a nontargeting shRNA as control. Western blot analysis showed that the loss of cadherin-6 was not compensated by increased protein levels of E-cadherin in any of cad6-KD clones we established. Some of the Ecad-KD clones we generated had slightly elevated (20–40%) cadherin-6 levels (unpublished data), but this was not the case for the clones we selected for our experiments (Ecad-KD1 and Ecad-KD2; Figure 1A). Loss of either cadherin isoform did not result in a loss of cell–cell adhesion as judged by an aggregation assay (compare control in Figure 1, B to C and D). In contrast, knockdown of both cadherins severely disrupted cell–cell adhesion in this assay (Figure 1E).


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)

Knockdown of E-cadherin or cadherin-6 induces distinct phenotypes. MDCK cells expressing a nontargeting control vector or inducible shRNA against E-cadherin, cadherin-6, or both were grown in the presence of doxycycline to induce shRNA expression. (A) Western blots from lysates of confluent cells that were induced for 2 d to express a nontargeting shRNA (control) or different shRNAs against cadherin-6 (cad6-KD1, cad6-KD2), E-cadherin (Ecad-KD1, Ecad-KD2), or both (6/E-KD1, 6/E-KD2). (B–E) Cell–cell adhesion as analyzed by an aggregation assay in control (B), cad6-KD1 (C), Ecad-KD1 (D), or 6/E-KD1 (E) cells. (F–I) Control cells or cadherin knockdown cells were grown as cysts in collagen gels. Phase contrast images were taken 13 d after plating. Control cells (F) form cysts with a spherical phenotype and a hollow lumen. Cad6-KD cysts (G) develop large tubules (arrows) and some narrow extensions (arrowhead), whereas Ecad-KD cysts (H) form spherical cysts with filled lumens. 6/E-KD cysts (I) have both tubules and filled lumens (arrow). Scale bar, 50 μm. (J) Percentage of cysts with tubules and filled lumens in cad6-KD1, cad6-KD2 (left), Ecad-KD1, Ecad-KD2 (middle), and 6/E-KD1, 6/E-KD2 cysts (right). Data represent mean ± SD. N = 3.
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Related In: Results  -  Collection

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Figure 1: Knockdown of E-cadherin or cadherin-6 induces distinct phenotypes. MDCK cells expressing a nontargeting control vector or inducible shRNA against E-cadherin, cadherin-6, or both were grown in the presence of doxycycline to induce shRNA expression. (A) Western blots from lysates of confluent cells that were induced for 2 d to express a nontargeting shRNA (control) or different shRNAs against cadherin-6 (cad6-KD1, cad6-KD2), E-cadherin (Ecad-KD1, Ecad-KD2), or both (6/E-KD1, 6/E-KD2). (B–E) Cell–cell adhesion as analyzed by an aggregation assay in control (B), cad6-KD1 (C), Ecad-KD1 (D), or 6/E-KD1 (E) cells. (F–I) Control cells or cadherin knockdown cells were grown as cysts in collagen gels. Phase contrast images were taken 13 d after plating. Control cells (F) form cysts with a spherical phenotype and a hollow lumen. Cad6-KD cysts (G) develop large tubules (arrows) and some narrow extensions (arrowhead), whereas Ecad-KD cysts (H) form spherical cysts with filled lumens. 6/E-KD cysts (I) have both tubules and filled lumens (arrow). Scale bar, 50 μm. (J) Percentage of cysts with tubules and filled lumens in cad6-KD1, cad6-KD2 (left), Ecad-KD1, Ecad-KD2 (middle), and 6/E-KD1, 6/E-KD2 cysts (right). Data represent mean ± SD. N = 3.
Mentions: To determine the roles of cadherin-6 and E-cadherin in epithelial morphogenesis in 3D cell culture, we created cell lines that conditionally express shRNA specific to cadherin-6 (cad6-KD), E-cadherin (Ecad-KD), or both (6/E-KD). We used two different targeting sequences specific for either cadherin and selected clones in which doxycycline-induced expression of the shRNAs effectively and specifically down-regulated cadherin-6 or E-cadherin protein expression by at least 95% (Figure 1A). We chose one clone for each hairpin to cadherin-6 (cad6-KD1, cad6-KD2) and E-cadherin (Ecad-KD1, Ecad-KD2) for all subsequent experiments. As conditional shRNA expression was not completely tight in all clones, we used cells that conditionally express a nontargeting shRNA as control. Western blot analysis showed that the loss of cadherin-6 was not compensated by increased protein levels of E-cadherin in any of cad6-KD clones we established. Some of the Ecad-KD clones we generated had slightly elevated (20–40%) cadherin-6 levels (unpublished data), but this was not the case for the clones we selected for our experiments (Ecad-KD1 and Ecad-KD2; Figure 1A). Loss of either cadherin isoform did not result in a loss of cell–cell adhesion as judged by an aggregation assay (compare control in Figure 1, B to C and D). In contrast, knockdown of both cadherins severely disrupted cell–cell adhesion in this assay (Figure 1E).

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