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Inhibitors of endocytosis prevent Wnt/Wingless signalling by reducing the level of basal β-catenin/Armadillo.

Gagliardi M, Hernandez A, McGough IJ, Vincent JP - J. Cell. Sci. (2014)

Bottom Line: Here, we use specific inhibitors (Dynasore and Dyngo-4a) to confirm the essential role of endocytosis in Wnt/Wingless signalling in human and Drosophila cells.Moreover, we show that activation of signalling through chemical blockade of GSK3β is prevented by endocytosis inhibitors, suggesting that endocytosis impacts on Wnt/Wingless signalling downstream of the ligand-receptor complex.We propose that, through an unknown mechanism, endocytosis boosts the resting pool of β-catenin upon which GSK3β normally acts.

View Article: PubMed Central - PubMed

Affiliation: MRC's National Institute for Medical Research, The Ridgeway, Mill Hill, London NW71AA, UK.

Show MeSH
Endocytosis inhibitors cause a decrease in the level of Armadillo/β-catenin both in stimulated and unstimulated cells. (A) The level of Armadillo increased in S2R+ cells that had been treated with Wingless-conditioned medium (lane 2) or SB-216763 (lane 5). This was prevented by treatment with Dyngo-4a (lanes 3 and 6) or Dynasore (lanes 4 and 7). Lamin, Actin and Syntaxin levels were unaffected. (B) Dyngo-4a reversibly reduced the level of Armadillo in uninduced cells. (C) Dyngo-4a reduced signalling-induced accumulation of β-catenin in RKO cells. Cells were pre-incubated with SB-216763 to activate signalling and then exposed to a mixture of SB-216763 and Dyngo-4a. The total time of treatment with either drug is indicated. As in Drosophila cells, Dyngo-4a caused a decrease in β-catenin levels in unstimulated cells. A progressive decrease can be seen after 0.5, 1 and 2 hours of treatment with Dyngo-4a (no SB-216763) in lanes 9–11. (D) The effect of Dyngo-4a and SB-216763 on the level of various components of the Wnt pathway. LRP6, GSK3β and CK1α were largely unaffected, whereas the levels of APC and Axin1 dropped markedly 30–60 minutes after treatment with Dyngo-4a. SB-216763 caused an increase in the amount of β-catenin. This correlated with a decrease in phosphorylated β-catenin (pT42/S37/S33 β-catenin; lane 3), as expected because phosphorylated β-catenin reflects the activity of the degradation complex (Hernández et al., 2012). By contrast, the (mild) decrease in β-catenin caused by Dyngo-4a (lanes 7 and 8) is paralleled by a similar decrease in phosphorylated β-catenin, suggesting that Dyngo-4a impacts on the level of β-catenin through a mechanism that is independent of the destruction complex.
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f04: Endocytosis inhibitors cause a decrease in the level of Armadillo/β-catenin both in stimulated and unstimulated cells. (A) The level of Armadillo increased in S2R+ cells that had been treated with Wingless-conditioned medium (lane 2) or SB-216763 (lane 5). This was prevented by treatment with Dyngo-4a (lanes 3 and 6) or Dynasore (lanes 4 and 7). Lamin, Actin and Syntaxin levels were unaffected. (B) Dyngo-4a reversibly reduced the level of Armadillo in uninduced cells. (C) Dyngo-4a reduced signalling-induced accumulation of β-catenin in RKO cells. Cells were pre-incubated with SB-216763 to activate signalling and then exposed to a mixture of SB-216763 and Dyngo-4a. The total time of treatment with either drug is indicated. As in Drosophila cells, Dyngo-4a caused a decrease in β-catenin levels in unstimulated cells. A progressive decrease can be seen after 0.5, 1 and 2 hours of treatment with Dyngo-4a (no SB-216763) in lanes 9–11. (D) The effect of Dyngo-4a and SB-216763 on the level of various components of the Wnt pathway. LRP6, GSK3β and CK1α were largely unaffected, whereas the levels of APC and Axin1 dropped markedly 30–60 minutes after treatment with Dyngo-4a. SB-216763 caused an increase in the amount of β-catenin. This correlated with a decrease in phosphorylated β-catenin (pT42/S37/S33 β-catenin; lane 3), as expected because phosphorylated β-catenin reflects the activity of the degradation complex (Hernández et al., 2012). By contrast, the (mild) decrease in β-catenin caused by Dyngo-4a (lanes 7 and 8) is paralleled by a similar decrease in phosphorylated β-catenin, suggesting that Dyngo-4a impacts on the level of β-catenin through a mechanism that is independent of the destruction complex.

Mentions: Wingless signalling is activated by preventing GSK3β from phosphorylating β-catenin. Indeed, chemical inhibitors of GSK3β trigger signalling, even in the absence of formation of a Wnt–receptor complex. For example, LiCl, which has been known for a while to block GSK3β activity (Stambolic et al., 1996), causes marked accumulation of β-catenin in L cells, and this is suppressed by concomitant treatment with monodansylcadaverine, chlorpromazine or hypertonic sucrose (Blitzer and Nusse, 2006). This provided an early indication that endocytosis could modulate signalling at or below the level of GSK3β, i.e. downstream of the ligand–receptor complex. This finding is at odds with the sequestration hypothesis; therefore, we sought to confirm it with more specific inhibitors. As an inhibitor of GSK3β, we chose SB-216763, which activates TOPFlash in the absence of exogenous Wnt in human cells (Coghlan et al., 2000). We found that SB-216763 activates TOPFlash in Drosophila S2R+ cells in a similar manner and therefore used this compound for subsequent investigation [the effect of other potent GSK3 inhibitors, such as CHIR98014 (Naujok et al., 2014; Ring et al., 2003) was not investigated]. We found earlier that TOPFlash activation with SB-216763 was prevented by concomitant treatment with Dynasore or Dyngo-4a (Fig. 1A). We conclude that inhibitors of endocytosis are unlikely to block Wingless signalling through preventing internalisation of the ligand receptor complex. Instead, it appears that endocytosis impacts on a more downstream event. The most immediate downstream sign of Wnt/Wingless signalling is the accumulation of β-catenin/Armadillo. We therefore investigated the effects of Dynasore or Dyngo-4a on the level of Armadillo in S2R+ cells. As shown in Fig. 4A, a 30 minute pre-treatment with either drug prevented the levels of Armadillo from rising in response to Wingless-conditioned medium or to SB-216763. In fact, it appeared that both drugs caused the level of Armadillo to dip below that seen in otherwise unstimulated cells, whereas the amounts of Actin and Syntaxin remained unchanged. This suggests that endocytosis could be required to maintain a sufficient steady-state level of Armadillo in resting cells. Indeed, addition of Dyngo-4a to S2R+ cells without any treatment to stimulate signalling led to a marked decrease in Armadillo (Fig. 4B). Interestingly, this effect was reversible – in cells that had been treated for 2 hours with Dyngo-4a and then allowed to recover in normal culture medium, Armadillo levels rose back to normal (Fig. 4B), suggesting that the effect of Dyngo-4a is transient and that cell viability was not adversely affected.


Inhibitors of endocytosis prevent Wnt/Wingless signalling by reducing the level of basal β-catenin/Armadillo.

Gagliardi M, Hernandez A, McGough IJ, Vincent JP - J. Cell. Sci. (2014)

Endocytosis inhibitors cause a decrease in the level of Armadillo/β-catenin both in stimulated and unstimulated cells. (A) The level of Armadillo increased in S2R+ cells that had been treated with Wingless-conditioned medium (lane 2) or SB-216763 (lane 5). This was prevented by treatment with Dyngo-4a (lanes 3 and 6) or Dynasore (lanes 4 and 7). Lamin, Actin and Syntaxin levels were unaffected. (B) Dyngo-4a reversibly reduced the level of Armadillo in uninduced cells. (C) Dyngo-4a reduced signalling-induced accumulation of β-catenin in RKO cells. Cells were pre-incubated with SB-216763 to activate signalling and then exposed to a mixture of SB-216763 and Dyngo-4a. The total time of treatment with either drug is indicated. As in Drosophila cells, Dyngo-4a caused a decrease in β-catenin levels in unstimulated cells. A progressive decrease can be seen after 0.5, 1 and 2 hours of treatment with Dyngo-4a (no SB-216763) in lanes 9–11. (D) The effect of Dyngo-4a and SB-216763 on the level of various components of the Wnt pathway. LRP6, GSK3β and CK1α were largely unaffected, whereas the levels of APC and Axin1 dropped markedly 30–60 minutes after treatment with Dyngo-4a. SB-216763 caused an increase in the amount of β-catenin. This correlated with a decrease in phosphorylated β-catenin (pT42/S37/S33 β-catenin; lane 3), as expected because phosphorylated β-catenin reflects the activity of the degradation complex (Hernández et al., 2012). By contrast, the (mild) decrease in β-catenin caused by Dyngo-4a (lanes 7 and 8) is paralleled by a similar decrease in phosphorylated β-catenin, suggesting that Dyngo-4a impacts on the level of β-catenin through a mechanism that is independent of the destruction complex.
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Related In: Results  -  Collection

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Show All Figures
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f04: Endocytosis inhibitors cause a decrease in the level of Armadillo/β-catenin both in stimulated and unstimulated cells. (A) The level of Armadillo increased in S2R+ cells that had been treated with Wingless-conditioned medium (lane 2) or SB-216763 (lane 5). This was prevented by treatment with Dyngo-4a (lanes 3 and 6) or Dynasore (lanes 4 and 7). Lamin, Actin and Syntaxin levels were unaffected. (B) Dyngo-4a reversibly reduced the level of Armadillo in uninduced cells. (C) Dyngo-4a reduced signalling-induced accumulation of β-catenin in RKO cells. Cells were pre-incubated with SB-216763 to activate signalling and then exposed to a mixture of SB-216763 and Dyngo-4a. The total time of treatment with either drug is indicated. As in Drosophila cells, Dyngo-4a caused a decrease in β-catenin levels in unstimulated cells. A progressive decrease can be seen after 0.5, 1 and 2 hours of treatment with Dyngo-4a (no SB-216763) in lanes 9–11. (D) The effect of Dyngo-4a and SB-216763 on the level of various components of the Wnt pathway. LRP6, GSK3β and CK1α were largely unaffected, whereas the levels of APC and Axin1 dropped markedly 30–60 minutes after treatment with Dyngo-4a. SB-216763 caused an increase in the amount of β-catenin. This correlated with a decrease in phosphorylated β-catenin (pT42/S37/S33 β-catenin; lane 3), as expected because phosphorylated β-catenin reflects the activity of the degradation complex (Hernández et al., 2012). By contrast, the (mild) decrease in β-catenin caused by Dyngo-4a (lanes 7 and 8) is paralleled by a similar decrease in phosphorylated β-catenin, suggesting that Dyngo-4a impacts on the level of β-catenin through a mechanism that is independent of the destruction complex.
Mentions: Wingless signalling is activated by preventing GSK3β from phosphorylating β-catenin. Indeed, chemical inhibitors of GSK3β trigger signalling, even in the absence of formation of a Wnt–receptor complex. For example, LiCl, which has been known for a while to block GSK3β activity (Stambolic et al., 1996), causes marked accumulation of β-catenin in L cells, and this is suppressed by concomitant treatment with monodansylcadaverine, chlorpromazine or hypertonic sucrose (Blitzer and Nusse, 2006). This provided an early indication that endocytosis could modulate signalling at or below the level of GSK3β, i.e. downstream of the ligand–receptor complex. This finding is at odds with the sequestration hypothesis; therefore, we sought to confirm it with more specific inhibitors. As an inhibitor of GSK3β, we chose SB-216763, which activates TOPFlash in the absence of exogenous Wnt in human cells (Coghlan et al., 2000). We found that SB-216763 activates TOPFlash in Drosophila S2R+ cells in a similar manner and therefore used this compound for subsequent investigation [the effect of other potent GSK3 inhibitors, such as CHIR98014 (Naujok et al., 2014; Ring et al., 2003) was not investigated]. We found earlier that TOPFlash activation with SB-216763 was prevented by concomitant treatment with Dynasore or Dyngo-4a (Fig. 1A). We conclude that inhibitors of endocytosis are unlikely to block Wingless signalling through preventing internalisation of the ligand receptor complex. Instead, it appears that endocytosis impacts on a more downstream event. The most immediate downstream sign of Wnt/Wingless signalling is the accumulation of β-catenin/Armadillo. We therefore investigated the effects of Dynasore or Dyngo-4a on the level of Armadillo in S2R+ cells. As shown in Fig. 4A, a 30 minute pre-treatment with either drug prevented the levels of Armadillo from rising in response to Wingless-conditioned medium or to SB-216763. In fact, it appeared that both drugs caused the level of Armadillo to dip below that seen in otherwise unstimulated cells, whereas the amounts of Actin and Syntaxin remained unchanged. This suggests that endocytosis could be required to maintain a sufficient steady-state level of Armadillo in resting cells. Indeed, addition of Dyngo-4a to S2R+ cells without any treatment to stimulate signalling led to a marked decrease in Armadillo (Fig. 4B). Interestingly, this effect was reversible – in cells that had been treated for 2 hours with Dyngo-4a and then allowed to recover in normal culture medium, Armadillo levels rose back to normal (Fig. 4B), suggesting that the effect of Dyngo-4a is transient and that cell viability was not adversely affected.

Bottom Line: Here, we use specific inhibitors (Dynasore and Dyngo-4a) to confirm the essential role of endocytosis in Wnt/Wingless signalling in human and Drosophila cells.Moreover, we show that activation of signalling through chemical blockade of GSK3β is prevented by endocytosis inhibitors, suggesting that endocytosis impacts on Wnt/Wingless signalling downstream of the ligand-receptor complex.We propose that, through an unknown mechanism, endocytosis boosts the resting pool of β-catenin upon which GSK3β normally acts.

View Article: PubMed Central - PubMed

Affiliation: MRC's National Institute for Medical Research, The Ridgeway, Mill Hill, London NW71AA, UK.

Show MeSH