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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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IFNγ induces transient transactivation of β-catenin in IECs. Effects of IFNγ in β-catenin transactivation (A) and Akt/β-catenin signaling pathway in vitro (B) and in vivo (C) were evaluated by TOPflash assays and Western blot analysis in SW480 cells and colonic mucosa of C57BL/6J mice, respectively. IFNγ was added 3–24 h before SW480 cells were processed. C57BL/6J mice were injected intraperitoneally with IFNγ for 2 h or vehicle alone (mouse serum albumin [MSA]). Transfections were performed in triplicate, and the means ± SD are shown (n = 3). Specific antibodies against pAkt308 and pβ-cat552 for Akt signaling pathway activation were used. Pan-Akt antibody was used to detect Akt total levels. Actin was used as a loading control. (D) The effect of IFNγ in IEC proliferation was evaluated by analyzing pHist3 expression in the mucosa of mice exposed to the cytokine for 2 and 96 h. Bar graph obtained of the densitometric analysis. pHist3 levels were normalized to actin.
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Figure 1: IFNγ induces transient transactivation of β-catenin in IECs. Effects of IFNγ in β-catenin transactivation (A) and Akt/β-catenin signaling pathway in vitro (B) and in vivo (C) were evaluated by TOPflash assays and Western blot analysis in SW480 cells and colonic mucosa of C57BL/6J mice, respectively. IFNγ was added 3–24 h before SW480 cells were processed. C57BL/6J mice were injected intraperitoneally with IFNγ for 2 h or vehicle alone (mouse serum albumin [MSA]). Transfections were performed in triplicate, and the means ± SD are shown (n = 3). Specific antibodies against pAkt308 and pβ-cat552 for Akt signaling pathway activation were used. Pan-Akt antibody was used to detect Akt total levels. Actin was used as a loading control. (D) The effect of IFNγ in IEC proliferation was evaluated by analyzing pHist3 expression in the mucosa of mice exposed to the cytokine for 2 and 96 h. Bar graph obtained of the densitometric analysis. pHist3 levels were normalized to actin.

Mentions: To understand the mechanisms by which of β-catenin activity and epithelial proliferation are influenced by IFNγ, we used complementary in vitro and in vivo approaches. Using a cultured intestinal epithelial cell line SW480, we evaluated the influence of IFNγ treatment on β-catenin transactivation by measuring TOPflash reporter activity (Korinek et al., 1997). IFNγ exposure resulted in an initial increase in β-catenin transactivation (3 h), followed by a steady decline over the next 24 h of cytokine treatment, suggesting transient β-catenin transactivation by the cytokine (Figure 1A). In agreement with our previous results using a model intestinal epithelial cell line, T84 (Nava et al., 2010), exposure of epithelial cells to IFNγ resulted in a biphasic proliferative response, with an initial increase in 5-ethynyl-2′-deoxyuridine incorporation (6–12 h), followed by reduced proliferation at 24 h (Supplemental Figure S1A). Although decreased activation of classical Wnt/β-catenin signaling was observed after IFNγ treatment (as we previously reported; Nava et al., 2010), increased activation of the Akt/β-catenin signaling was observed, as shown by the presence of high levels of pAkt308 and its downstream target protein, pβ-cat552 (Figure 1B and Supplemental Figure S1B). Moreover, Western blot analysis of colonic mucosal lysates of C57BL/6 mice that had received intraperitoneal injections of IFNγ also revealed increased activation of Akt/β-catenin signaling within 2 h, an effect that was also observed when mice were continuously exposed to IFNγ for 96 h (Figure 1C and Supplemental Figure S2). However, as shown in Figure 1D, increased IEC cell proliferation was only observed 2 h after IFNγ treatment. In fact, continuous exposure of IECs to IFNγ for 96 h resulted in a clear reduction in cell proliferation (Figure 1D). These findings suggested that sustained activation of Akt/β-catenin downstream of IFNγ signaling exerts biological effects that extend beyond increasing epithelial cell proliferation during inflammation.


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)

IFNγ induces transient transactivation of β-catenin in IECs. Effects of IFNγ in β-catenin transactivation (A) and Akt/β-catenin signaling pathway in vitro (B) and in vivo (C) were evaluated by TOPflash assays and Western blot analysis in SW480 cells and colonic mucosa of C57BL/6J mice, respectively. IFNγ was added 3–24 h before SW480 cells were processed. C57BL/6J mice were injected intraperitoneally with IFNγ for 2 h or vehicle alone (mouse serum albumin [MSA]). Transfections were performed in triplicate, and the means ± SD are shown (n = 3). Specific antibodies against pAkt308 and pβ-cat552 for Akt signaling pathway activation were used. Pan-Akt antibody was used to detect Akt total levels. Actin was used as a loading control. (D) The effect of IFNγ in IEC proliferation was evaluated by analyzing pHist3 expression in the mucosa of mice exposed to the cytokine for 2 and 96 h. Bar graph obtained of the densitometric analysis. pHist3 levels were normalized to actin.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Figure 1: IFNγ induces transient transactivation of β-catenin in IECs. Effects of IFNγ in β-catenin transactivation (A) and Akt/β-catenin signaling pathway in vitro (B) and in vivo (C) were evaluated by TOPflash assays and Western blot analysis in SW480 cells and colonic mucosa of C57BL/6J mice, respectively. IFNγ was added 3–24 h before SW480 cells were processed. C57BL/6J mice were injected intraperitoneally with IFNγ for 2 h or vehicle alone (mouse serum albumin [MSA]). Transfections were performed in triplicate, and the means ± SD are shown (n = 3). Specific antibodies against pAkt308 and pβ-cat552 for Akt signaling pathway activation were used. Pan-Akt antibody was used to detect Akt total levels. Actin was used as a loading control. (D) The effect of IFNγ in IEC proliferation was evaluated by analyzing pHist3 expression in the mucosa of mice exposed to the cytokine for 2 and 96 h. Bar graph obtained of the densitometric analysis. pHist3 levels were normalized to actin.
Mentions: To understand the mechanisms by which of β-catenin activity and epithelial proliferation are influenced by IFNγ, we used complementary in vitro and in vivo approaches. Using a cultured intestinal epithelial cell line SW480, we evaluated the influence of IFNγ treatment on β-catenin transactivation by measuring TOPflash reporter activity (Korinek et al., 1997). IFNγ exposure resulted in an initial increase in β-catenin transactivation (3 h), followed by a steady decline over the next 24 h of cytokine treatment, suggesting transient β-catenin transactivation by the cytokine (Figure 1A). In agreement with our previous results using a model intestinal epithelial cell line, T84 (Nava et al., 2010), exposure of epithelial cells to IFNγ resulted in a biphasic proliferative response, with an initial increase in 5-ethynyl-2′-deoxyuridine incorporation (6–12 h), followed by reduced proliferation at 24 h (Supplemental Figure S1A). Although decreased activation of classical Wnt/β-catenin signaling was observed after IFNγ treatment (as we previously reported; Nava et al., 2010), increased activation of the Akt/β-catenin signaling was observed, as shown by the presence of high levels of pAkt308 and its downstream target protein, pβ-cat552 (Figure 1B and Supplemental Figure S1B). Moreover, Western blot analysis of colonic mucosal lysates of C57BL/6 mice that had received intraperitoneal injections of IFNγ also revealed increased activation of Akt/β-catenin signaling within 2 h, an effect that was also observed when mice were continuously exposed to IFNγ for 96 h (Figure 1C and Supplemental Figure S2). However, as shown in Figure 1D, increased IEC cell proliferation was only observed 2 h after IFNγ treatment. In fact, continuous exposure of IECs to IFNγ for 96 h resulted in a clear reduction in cell proliferation (Figure 1D). These findings suggested that sustained activation of Akt/β-catenin downstream of IFNγ signaling exerts biological effects that extend beyond increasing epithelial cell proliferation during inflammation.

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