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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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Expression of Pak1-K299R down-regulates cadherin-6 and induces tubulogenesis. (A1–A3) In the presence of doxycycline (+dox), control cells form cysts with a normal spherical morphology, a central lumen, and cadherin-6 (A1, green in A3) and E-cadherin (A2, red in A3) at the basolateral membrane. (B1–B3) In the absence of doxycycline (−dox), Pak1-K299R is expressed and cysts form tubules and have lost expression of cadherin-6 (B1, green in B3) but not E-cadherin (B2, red in B3). Nuclei, stained with DAPI, are blue (A3, B3). Scale bar, 20 μm. (C) Western blot of lysates of control (+dox) and Pak1-K299R–expressing cells were stained for cadherin-6 and E-cadherin. GAPDH is a loading control.
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Figure 5: Expression of Pak1-K299R down-regulates cadherin-6 and induces tubulogenesis. (A1–A3) In the presence of doxycycline (+dox), control cells form cysts with a normal spherical morphology, a central lumen, and cadherin-6 (A1, green in A3) and E-cadherin (A2, red in A3) at the basolateral membrane. (B1–B3) In the absence of doxycycline (−dox), Pak1-K299R is expressed and cysts form tubules and have lost expression of cadherin-6 (B1, green in B3) but not E-cadherin (B2, red in B3). Nuclei, stained with DAPI, are blue (A3, B3). Scale bar, 20 μm. (C) Western blot of lysates of control (+dox) and Pak1-K299R–expressing cells were stained for cadherin-6 and E-cadherin. GAPDH is a loading control.

Mentions: When we compared the phenotypes of tubules induced by HGF and those in the cad6-KD cyst, we noticed morphological distinctions. In contrast to the HGF-induced tubules, the cad6-KD–induced tubules mainly exhibit either small extensions or large lumen-containing tubules but very few chains and cords (Figure 1C). We recently reported a remarkably similar phenotype in cells that inducibly express a kinase-dead, dominant-negative mutant of Pak1 (Pak1-K299R) (Hunter and Zegers, 2010). In this Tet-off system, Pak1-K299R is expressed under the control of the tetracycline-controlled transactivator. Expression of Pak1-K299R is suppressed by doxycycline and induced by removing doxycycline from the culture media. As we found previously, Pak1-K299R–expressing cysts (−dox) formed robust tubules, which maintained the lateral localization of E-cadherin (compare Figure 5, A2 to B2) (Hunter and Zegers, 2010). Cadherin-6, however, was completely lost from the membrane in all cells. This was likely due to down-regulation of cadherin-6 expression, as Western blot analysis showed a large decrease in the levels of cadherin-6 but not E-cadherin (Figure 5C). To analyze whether the loss of cadherin-6 was required for Pak1-K299R–induced tubules, we next restored cadherin-6 expression by stably expressing EGFP-tagged mouse cadherin-6 in these cells (Figure 6A). Examination of live cysts revealed that cadherin-6–EGFP expression did not alter cyst morphology in the absence of Pak1-K299R expression (+dox; compare Figure 6, B to D1) and that cadherin–EGFP exclusively localized to the lateral membranes (Figure 6D2). Cadherin-6–EGFP also localized laterally in Pak1-K299R–expressing cells (Figure 6C2) and strongly inhibited Pak1-K299R–induced tubules (−dox, compare Figure 6, C to E1). Quantification of the cyst phenotypes revealed that expression of cadherin-6–EGFP completely inhibited the increased tubulogenesis that was induced by Pak1-K299R expression (Figure 6F, compare +dox and −dox). Western blot analysis showed that at least one of the clones (K299R-cad6-EGFP#1) expressed equal levels of Pak1-K299R as compared with the parental cells, although expression levels of clone K299R-cad6-EGFP#2 were somewhat lower (Figure 6A). We previously found that Pak1-K299R clones with expression levels that were at least 50% lower than the clone shown in Figure 6, C and F, still induce robust tubules (M.Z., unpublished data), and even clones with Pak1-K299R expression that is below the detection limit in Western blots could induce tubules. The generally higher levels of tubulogenesis in the noninduced K299R-cad6-EGFP clones, although not statistically significant, may therefore be explained by “leakiness” of the Pak1-K299R expression in these clones. For the same reason, it is unlikely that the lower expression levels of Pak1-K299R in the K299R-cad6-EGFP#2 clone account for the inhibition of tubulogenesis. Rather, our data obtained with the two cadherin-6–overexpressing clones indicate that down-regulation of cadherin-6 is required for tubulogenesis in Pak1-K299R–expressing cysts.


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)

Expression of Pak1-K299R down-regulates cadherin-6 and induces tubulogenesis. (A1–A3) In the presence of doxycycline (+dox), control cells form cysts with a normal spherical morphology, a central lumen, and cadherin-6 (A1, green in A3) and E-cadherin (A2, red in A3) at the basolateral membrane. (B1–B3) In the absence of doxycycline (−dox), Pak1-K299R is expressed and cysts form tubules and have lost expression of cadherin-6 (B1, green in B3) but not E-cadherin (B2, red in B3). Nuclei, stained with DAPI, are blue (A3, B3). Scale bar, 20 μm. (C) Western blot of lysates of control (+dox) and Pak1-K299R–expressing cells were stained for cadherin-6 and E-cadherin. GAPDH is a loading control.
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Related In: Results  -  Collection

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Figure 5: Expression of Pak1-K299R down-regulates cadherin-6 and induces tubulogenesis. (A1–A3) In the presence of doxycycline (+dox), control cells form cysts with a normal spherical morphology, a central lumen, and cadherin-6 (A1, green in A3) and E-cadherin (A2, red in A3) at the basolateral membrane. (B1–B3) In the absence of doxycycline (−dox), Pak1-K299R is expressed and cysts form tubules and have lost expression of cadherin-6 (B1, green in B3) but not E-cadherin (B2, red in B3). Nuclei, stained with DAPI, are blue (A3, B3). Scale bar, 20 μm. (C) Western blot of lysates of control (+dox) and Pak1-K299R–expressing cells were stained for cadherin-6 and E-cadherin. GAPDH is a loading control.
Mentions: When we compared the phenotypes of tubules induced by HGF and those in the cad6-KD cyst, we noticed morphological distinctions. In contrast to the HGF-induced tubules, the cad6-KD–induced tubules mainly exhibit either small extensions or large lumen-containing tubules but very few chains and cords (Figure 1C). We recently reported a remarkably similar phenotype in cells that inducibly express a kinase-dead, dominant-negative mutant of Pak1 (Pak1-K299R) (Hunter and Zegers, 2010). In this Tet-off system, Pak1-K299R is expressed under the control of the tetracycline-controlled transactivator. Expression of Pak1-K299R is suppressed by doxycycline and induced by removing doxycycline from the culture media. As we found previously, Pak1-K299R–expressing cysts (−dox) formed robust tubules, which maintained the lateral localization of E-cadherin (compare Figure 5, A2 to B2) (Hunter and Zegers, 2010). Cadherin-6, however, was completely lost from the membrane in all cells. This was likely due to down-regulation of cadherin-6 expression, as Western blot analysis showed a large decrease in the levels of cadherin-6 but not E-cadherin (Figure 5C). To analyze whether the loss of cadherin-6 was required for Pak1-K299R–induced tubules, we next restored cadherin-6 expression by stably expressing EGFP-tagged mouse cadherin-6 in these cells (Figure 6A). Examination of live cysts revealed that cadherin-6–EGFP expression did not alter cyst morphology in the absence of Pak1-K299R expression (+dox; compare Figure 6, B to D1) and that cadherin–EGFP exclusively localized to the lateral membranes (Figure 6D2). Cadherin-6–EGFP also localized laterally in Pak1-K299R–expressing cells (Figure 6C2) and strongly inhibited Pak1-K299R–induced tubules (−dox, compare Figure 6, C to E1). Quantification of the cyst phenotypes revealed that expression of cadherin-6–EGFP completely inhibited the increased tubulogenesis that was induced by Pak1-K299R expression (Figure 6F, compare +dox and −dox). Western blot analysis showed that at least one of the clones (K299R-cad6-EGFP#1) expressed equal levels of Pak1-K299R as compared with the parental cells, although expression levels of clone K299R-cad6-EGFP#2 were somewhat lower (Figure 6A). We previously found that Pak1-K299R clones with expression levels that were at least 50% lower than the clone shown in Figure 6, C and F, still induce robust tubules (M.Z., unpublished data), and even clones with Pak1-K299R expression that is below the detection limit in Western blots could induce tubules. The generally higher levels of tubulogenesis in the noninduced K299R-cad6-EGFP clones, although not statistically significant, may therefore be explained by “leakiness” of the Pak1-K299R expression in these clones. For the same reason, it is unlikely that the lower expression levels of Pak1-K299R in the K299R-cad6-EGFP#2 clone account for the inhibition of tubulogenesis. Rather, our data obtained with the two cadherin-6–overexpressing clones indicate that down-regulation of cadherin-6 is required for tubulogenesis in Pak1-K299R–expressing cysts.

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