IFNγ-induced suppression of β-catenin signaling: evidence for roles of Akt and 14.3.3ζ.
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.
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.Show MeSH
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Mentions: To understand the influence of sustained Akt/β-catenin activation on epithelial cell proliferation, we investigated the mechanism by which Akt controls β-catenin transactivation downstream of IFNγ. IECs express two isoforms of Akt, Akt1 and Akt2 (Brazil et al., 2004). Active Akt1 has been shown to directly phosphorylate β-catenin at serine 552 (pβ-cat552) to promote β-catenin association with an adaptor protein 14.3.3ζ, which, in turn, stabilizes and activates β-catenin (Tian et al., 2004). Because IFNγ influences IEC proliferation, we examined whether IFNγ exposure modulates association of β-catenin with 14.3.3ζ. Indeed, as shown in Figure 2A, injection of mice with IFNγ or addition of IFNγ to cultured IECs for 1 h resulted in an increase in coimmunoprecipitation of β-catenin with 14.3.3ζ in IECs. Furthermore, β-catenin that was complexed with 14.3.3ζ was phosphorylated at serine 552 (Figure 2A). In contrast, there was no effect on the association of both molecules after TNFα treatment in vivo (Figure 2A). Next we analyzed the effects of 14.3.3ζ on the stability of β-catenin, using an in vitro model. For these studies, we used CHO-K1 cells because they lack E-cadherin and express low levels of β-catenin due to its proteosomal degradation (Supplemental Figure S3). As shown in Figure 2B, expression of exogenous β-catenin in CHO-K1 cells along with increasing concentrations of 14.3.3ζ resulted in a corresponding increase in β-catenin and pβ-cat552. Under these conditions, we also observed a decrease in the phosphorylation β-catenin at serine 33 (pβ-cat33; Figure 2B), which has been shown to generate a recognition site for β-Trcp, resulting in the ubiquitination and degradation of soluble β-catenin (Liu et al., 1999). We then evaluated the role of Akt1 and 14.3.3ζ in regulating the stability of β-catenin. Consistent with the role of Akt1 and 14.3.3ζ in regulating β-catenin stability, small interfering RNA (siRNA)–mediated down-regulation of endogenous 14.3.3ζ or Akt1 resulted in reduced β-catenin protein levels (Supplemental Figure S4A). Furthermore, pharmacologic inhibition of Akt (Akt inhibitor VIII) also resulted in decreased β-catenin protein in control cells (Supplemental Figure S4B) and cells exposed to IFNγ for 12 h (unpublished data). Inhibition of Akt was verified by Western blot for pAkt308 (Supplemental Figure S4B). Moreover, as shown in Figure 2C, cytokine treatment or up-regulation of 14.3.3ζ resulted in increased endogenous β-catenin protein levels (3.6- and 3.4-fold increase, respectively). Consistent with previous reports (Tian et al., 2004), we observed that increased expression of 14.3.3ζ promoted β-catenin transactivation. Conversely, down-regulation of 14.3.3ζ inhibited β-catenin transactivation (Figure 2D). The influence of IFNγ treatment on localization of 14.3.3ζ and β-catenin was evaluated in IECs by immunofluorescence labeling. In control cells, immunofluorescence labeling demonstrated that β-catenin is evenly distributed in both nuclear and cytosolic compartments but is mainly present in the cytoplasm after treatment with IFNγ for 12 h (Figure 2E). Similarly, IFNγ treatment for 12 h induced accumulation of 14.3.3ζ in the cytoplasm (Figure 2F). Taken together, these findings suggested that 14.3.3ζ may play an important role in regulating the cellular distribution of β-catenin and its function after stimulation with IFNγ.
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.