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IFNγ-induced suppression of β-catenin signaling: evidence for roles of Akt and 14.3.3ζ.

Nava P, Kamekura R, Quirós M, Medina-Contreras O, Hamilton RW, Kolegraff KN, Koch S, Candelario A, Romo-Parra H, Laur O, Hilgarth RS, Denning TL, Parkos CA, Nusrat A - Mol. Biol. Cell (2014)

Bottom Line: Akt1 served as a bimodal switch that promotes or inhibits β-catenin transactivation in response to IFNγ stimulation.IFNγ initially promotes β-catenin transactivation through Akt-dependent C-terminal phosphorylation of β-catenin to promote its association with 14.3.3ζ.These results outline a dual function of Akt1 that suppresses IEC proliferation during intestinal inflammation.

View Article: PubMed Central - PubMed

Affiliation: Epithelial Pathobiology and Mucosal Inflammation Research Unit, Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, GA 30322 Departamento de Fisiología, Biofísica y Neurociencias, Centro de Investigación y de Estudios Avanzados, Instituto Politécnico Nacional, 07360 Mexico City, Mexico.

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Decreased IEC β-catenin transactivation in response to IFNγ is associated with phosphorylation of 14.3.3ζ at serine 58. (A) Regulation of β-catenin transactivation by 14.3.3ζ was analyzed in SW480 cells treated with IFNγ by TOPflash assay. Cells were transfected with 0.2 μg/ml vector expressing active β-catS33Y alone or in conjunction with 0.2 or 0.5 μg/ml 14.3.3ζ. IFNγ was added 12 h posttransfection and samples collected 24 h post cytokine treatment. Experiments were performed in triplicate in two different cell passages. Means ± SD of a representative experiment. (B) Phosphorylation status of 14.3.3ζ (Ser-58), β-catenin (Ser-552), Akt (Thr-308), and total protein levels of 14.3.3ζ was analyzed in SW480 cells exposed to IFNγ (12 h) by Western blot. Actin was used as a loading control. Densitometric analysis of p14.3.3ζ is shown in the graph (n = 3). (C) The expression of 14.3.3ζ and p14.3.3ζ in the intestinal colonic mucosa of mouse injected with IFNγ was analyzed by Western blot. Actin was used as a loading control. The distribution of 14.3.3ζ (D) and p14.3.3ζ (E) at the colonic crypts of C57BL/6N animals was analyzed by immunofluorescence. Bar, 10 μm. Nuclei are blue. Proliferating cells are marked with Ki67 (red). Crypt plane is marked by a discontinuous line. (F) PLA assays for 14.3.3ζ/β-catenin (green) and p14.3.3ζ/β-catenin (green) were performed in colonic mucosa of C57BL/6N animals. Scale bar, 5 μm. Nuclei are blue. (G) Immunofluorescence labeling for p14.3.3ζ (green) and β-catenin (red) and PLA assay for p14.3.3ζ/β-catenin (green) were performed in T84 cells exposed to IFNγ for 3 h. Scale bar, 10 μm. Nuclei are blue. (H) PLA assay for p14.3.3ζ/β-catenin (green) performed in T84 cells. High magnification of T84 cells exposed to IFNγ for 3 h. Scale bar, 2 μm. Nuclei are blue. (I) Overexpression of 14.3.3ζ mutants does not affect endogenous 14.3.3ζ protein levels. SW480 cells were transfected with 200 ng of plasmid expressing empty vector, 14.3.3ζ WT, 14.3.3ζ S58D, and 14.3.3ζ S58A overnight and 14.3.3ζ expression analyzed by Western blotting of whole-cell lysates. Black arrow marks the overexpressed proteins. (J) 14.3.3ζ S58A prevents inhibition of β-catenin transactivation in IECs exposed to IFNγ. The effect of 14.3.3ζ WT, 14.3.3ζ S58D, and 14.3.3ζ S58A on β-catenin transactivation mediated by IFNγ was evaluated by TOPflash luciferase assays in SKCO15 cells. IFNγ was added 12 h before cells were processed for the TOPflash luciferase assay. Values were normalized to empty vector. Transfections were performed in triplicate, and the means ± SD are shown (n = 3).
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Figure 3: Decreased IEC β-catenin transactivation in response to IFNγ is associated with phosphorylation of 14.3.3ζ at serine 58. (A) Regulation of β-catenin transactivation by 14.3.3ζ was analyzed in SW480 cells treated with IFNγ by TOPflash assay. Cells were transfected with 0.2 μg/ml vector expressing active β-catS33Y alone or in conjunction with 0.2 or 0.5 μg/ml 14.3.3ζ. IFNγ was added 12 h posttransfection and samples collected 24 h post cytokine treatment. Experiments were performed in triplicate in two different cell passages. Means ± SD of a representative experiment. (B) Phosphorylation status of 14.3.3ζ (Ser-58), β-catenin (Ser-552), Akt (Thr-308), and total protein levels of 14.3.3ζ was analyzed in SW480 cells exposed to IFNγ (12 h) by Western blot. Actin was used as a loading control. Densitometric analysis of p14.3.3ζ is shown in the graph (n = 3). (C) The expression of 14.3.3ζ and p14.3.3ζ in the intestinal colonic mucosa of mouse injected with IFNγ was analyzed by Western blot. Actin was used as a loading control. The distribution of 14.3.3ζ (D) and p14.3.3ζ (E) at the colonic crypts of C57BL/6N animals was analyzed by immunofluorescence. Bar, 10 μm. Nuclei are blue. Proliferating cells are marked with Ki67 (red). Crypt plane is marked by a discontinuous line. (F) PLA assays for 14.3.3ζ/β-catenin (green) and p14.3.3ζ/β-catenin (green) were performed in colonic mucosa of C57BL/6N animals. Scale bar, 5 μm. Nuclei are blue. (G) Immunofluorescence labeling for p14.3.3ζ (green) and β-catenin (red) and PLA assay for p14.3.3ζ/β-catenin (green) were performed in T84 cells exposed to IFNγ for 3 h. Scale bar, 10 μm. Nuclei are blue. (H) PLA assay for p14.3.3ζ/β-catenin (green) performed in T84 cells. High magnification of T84 cells exposed to IFNγ for 3 h. Scale bar, 2 μm. Nuclei are blue. (I) Overexpression of 14.3.3ζ mutants does not affect endogenous 14.3.3ζ protein levels. SW480 cells were transfected with 200 ng of plasmid expressing empty vector, 14.3.3ζ WT, 14.3.3ζ S58D, and 14.3.3ζ S58A overnight and 14.3.3ζ expression analyzed by Western blotting of whole-cell lysates. Black arrow marks the overexpressed proteins. (J) 14.3.3ζ S58A prevents inhibition of β-catenin transactivation in IECs exposed to IFNγ. The effect of 14.3.3ζ WT, 14.3.3ζ S58D, and 14.3.3ζ S58A on β-catenin transactivation mediated by IFNγ was evaluated by TOPflash luciferase assays in SKCO15 cells. IFNγ was added 12 h before cells were processed for the TOPflash luciferase assay. Values were normalized to empty vector. Transfections were performed in triplicate, and the means ± SD are shown (n = 3).

Mentions: The mechanism of inhibition of β-catenin signaling by 14.3.3ζ after IFNγ exposure was further investigated. TOPflash reporter assays demonstrated that expression of stabilized β-catenin (β-catS33Y) in SW480 cells increased TOPflash activity by ∼10-fold, and 14.3.3ζ overexpression alone induced an approximately twofold increase in β-catenin transactivation (Figure 3A). Coexpression of 14.3.3ζ and β-catS33Y did not have additive effects (unpublished data). However, when cells expressing β-catS33Y were treated with IFNγ for 24 h, β-catenin transactivation was reduced by ∼60%. Expression of increasing amounts of 14.3.3ζ had an additive effect on IFNγ-mediated suppression of β-catenin transactivation (Figure 3A). Unexpectedly, β-catenin transactivation was not affected in cells exposed to IFNγ for short periods of time (3–6 h), even in cells overexpressing 14.3.3ζ (unpublished data). These results suggested that inactivation of β-catenin transactivation by14.3.3ζ downstream of IFNγ requires additional posttranslational modifications.


IFNγ-induced suppression of β-catenin signaling: evidence for roles of Akt and 14.3.3ζ.

Nava P, Kamekura R, Quirós M, Medina-Contreras O, Hamilton RW, Kolegraff KN, Koch S, Candelario A, Romo-Parra H, Laur O, Hilgarth RS, Denning TL, Parkos CA, Nusrat A - Mol. Biol. Cell (2014)

Decreased IEC β-catenin transactivation in response to IFNγ is associated with phosphorylation of 14.3.3ζ at serine 58. (A) Regulation of β-catenin transactivation by 14.3.3ζ was analyzed in SW480 cells treated with IFNγ by TOPflash assay. Cells were transfected with 0.2 μg/ml vector expressing active β-catS33Y alone or in conjunction with 0.2 or 0.5 μg/ml 14.3.3ζ. IFNγ was added 12 h posttransfection and samples collected 24 h post cytokine treatment. Experiments were performed in triplicate in two different cell passages. Means ± SD of a representative experiment. (B) Phosphorylation status of 14.3.3ζ (Ser-58), β-catenin (Ser-552), Akt (Thr-308), and total protein levels of 14.3.3ζ was analyzed in SW480 cells exposed to IFNγ (12 h) by Western blot. Actin was used as a loading control. Densitometric analysis of p14.3.3ζ is shown in the graph (n = 3). (C) The expression of 14.3.3ζ and p14.3.3ζ in the intestinal colonic mucosa of mouse injected with IFNγ was analyzed by Western blot. Actin was used as a loading control. The distribution of 14.3.3ζ (D) and p14.3.3ζ (E) at the colonic crypts of C57BL/6N animals was analyzed by immunofluorescence. Bar, 10 μm. Nuclei are blue. Proliferating cells are marked with Ki67 (red). Crypt plane is marked by a discontinuous line. (F) PLA assays for 14.3.3ζ/β-catenin (green) and p14.3.3ζ/β-catenin (green) were performed in colonic mucosa of C57BL/6N animals. Scale bar, 5 μm. Nuclei are blue. (G) Immunofluorescence labeling for p14.3.3ζ (green) and β-catenin (red) and PLA assay for p14.3.3ζ/β-catenin (green) were performed in T84 cells exposed to IFNγ for 3 h. Scale bar, 10 μm. Nuclei are blue. (H) PLA assay for p14.3.3ζ/β-catenin (green) performed in T84 cells. High magnification of T84 cells exposed to IFNγ for 3 h. Scale bar, 2 μm. Nuclei are blue. (I) Overexpression of 14.3.3ζ mutants does not affect endogenous 14.3.3ζ protein levels. SW480 cells were transfected with 200 ng of plasmid expressing empty vector, 14.3.3ζ WT, 14.3.3ζ S58D, and 14.3.3ζ S58A overnight and 14.3.3ζ expression analyzed by Western blotting of whole-cell lysates. Black arrow marks the overexpressed proteins. (J) 14.3.3ζ S58A prevents inhibition of β-catenin transactivation in IECs exposed to IFNγ. The effect of 14.3.3ζ WT, 14.3.3ζ S58D, and 14.3.3ζ S58A on β-catenin transactivation mediated by IFNγ was evaluated by TOPflash luciferase assays in SKCO15 cells. IFNγ was added 12 h before cells were processed for the TOPflash luciferase assay. Values were normalized to empty vector. Transfections were performed in triplicate, and the means ± SD are shown (n = 3).
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Figure 3: Decreased IEC β-catenin transactivation in response to IFNγ is associated with phosphorylation of 14.3.3ζ at serine 58. (A) Regulation of β-catenin transactivation by 14.3.3ζ was analyzed in SW480 cells treated with IFNγ by TOPflash assay. Cells were transfected with 0.2 μg/ml vector expressing active β-catS33Y alone or in conjunction with 0.2 or 0.5 μg/ml 14.3.3ζ. IFNγ was added 12 h posttransfection and samples collected 24 h post cytokine treatment. Experiments were performed in triplicate in two different cell passages. Means ± SD of a representative experiment. (B) Phosphorylation status of 14.3.3ζ (Ser-58), β-catenin (Ser-552), Akt (Thr-308), and total protein levels of 14.3.3ζ was analyzed in SW480 cells exposed to IFNγ (12 h) by Western blot. Actin was used as a loading control. Densitometric analysis of p14.3.3ζ is shown in the graph (n = 3). (C) The expression of 14.3.3ζ and p14.3.3ζ in the intestinal colonic mucosa of mouse injected with IFNγ was analyzed by Western blot. Actin was used as a loading control. The distribution of 14.3.3ζ (D) and p14.3.3ζ (E) at the colonic crypts of C57BL/6N animals was analyzed by immunofluorescence. Bar, 10 μm. Nuclei are blue. Proliferating cells are marked with Ki67 (red). Crypt plane is marked by a discontinuous line. (F) PLA assays for 14.3.3ζ/β-catenin (green) and p14.3.3ζ/β-catenin (green) were performed in colonic mucosa of C57BL/6N animals. Scale bar, 5 μm. Nuclei are blue. (G) Immunofluorescence labeling for p14.3.3ζ (green) and β-catenin (red) and PLA assay for p14.3.3ζ/β-catenin (green) were performed in T84 cells exposed to IFNγ for 3 h. Scale bar, 10 μm. Nuclei are blue. (H) PLA assay for p14.3.3ζ/β-catenin (green) performed in T84 cells. High magnification of T84 cells exposed to IFNγ for 3 h. Scale bar, 2 μm. Nuclei are blue. (I) Overexpression of 14.3.3ζ mutants does not affect endogenous 14.3.3ζ protein levels. SW480 cells were transfected with 200 ng of plasmid expressing empty vector, 14.3.3ζ WT, 14.3.3ζ S58D, and 14.3.3ζ S58A overnight and 14.3.3ζ expression analyzed by Western blotting of whole-cell lysates. Black arrow marks the overexpressed proteins. (J) 14.3.3ζ S58A prevents inhibition of β-catenin transactivation in IECs exposed to IFNγ. The effect of 14.3.3ζ WT, 14.3.3ζ S58D, and 14.3.3ζ S58A on β-catenin transactivation mediated by IFNγ was evaluated by TOPflash luciferase assays in SKCO15 cells. IFNγ was added 12 h before cells were processed for the TOPflash luciferase assay. Values were normalized to empty vector. Transfections were performed in triplicate, and the means ± SD are shown (n = 3).
Mentions: The mechanism of inhibition of β-catenin signaling by 14.3.3ζ after IFNγ exposure was further investigated. TOPflash reporter assays demonstrated that expression of stabilized β-catenin (β-catS33Y) in SW480 cells increased TOPflash activity by ∼10-fold, and 14.3.3ζ overexpression alone induced an approximately twofold increase in β-catenin transactivation (Figure 3A). Coexpression of 14.3.3ζ and β-catS33Y did not have additive effects (unpublished data). However, when cells expressing β-catS33Y were treated with IFNγ for 24 h, β-catenin transactivation was reduced by ∼60%. Expression of increasing amounts of 14.3.3ζ had an additive effect on IFNγ-mediated suppression of β-catenin transactivation (Figure 3A). Unexpectedly, β-catenin transactivation was not affected in cells exposed to IFNγ for short periods of time (3–6 h), even in cells overexpressing 14.3.3ζ (unpublished data). These results suggested that inactivation of β-catenin transactivation by14.3.3ζ downstream of IFNγ requires additional posttranslational modifications.

Bottom Line: Akt1 served as a bimodal switch that promotes or inhibits β-catenin transactivation in response to IFNγ stimulation.IFNγ initially promotes β-catenin transactivation through Akt-dependent C-terminal phosphorylation of β-catenin to promote its association with 14.3.3ζ.These results outline a dual function of Akt1 that suppresses IEC proliferation during intestinal inflammation.

View Article: PubMed Central - PubMed

Affiliation: Epithelial Pathobiology and Mucosal Inflammation Research Unit, Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, GA 30322 Departamento de Fisiología, Biofísica y Neurociencias, Centro de Investigación y de Estudios Avanzados, Instituto Politécnico Nacional, 07360 Mexico City, Mexico.

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Related in: MedlinePlus