Tumor suppressor FOXO3 participates in the regulation of intestinal inflammation

Abstract

Inflammatory bowel disease (IBD), including Crohn's disease and ulcerative colitis, is characterized by chronic mucosal injury and the infiltration of inflammatory cells. Tumor suppressor FOXO3 regulates gene expression and its translocation to the cytosol leads to the abrogation of its transcriptional function. We have previously shown that bacterial infection regulates FOXO3 in intestinal epithelial cells and increases cytokine levels. As TNFalpha is a major contributor in intestinal inflammation, the aim of this study was to assess its effect on FOXO3 and FOXO3's contribution to intestinal inflammation in vitro and in vivo. TNFalpha induces the translocation of nuclear FOXO3 into the cytosol where it undergoes proteasomal degradation in human intestinal HT-29 cells. Proximally, the PI3K and IKK pathways mediate TNFalpha-induced FOXO3 phosphorylation. In FOXO3-silenced HT-29 cells, TNFalpha-induced IL-8 expression is increased approximately 83%. In vivo, Foxo3 is present in the nuclei and cytosol of colonic crypt epithelia. In DSS-induced colonic inflammation, Foxo3's nuclear localization is lost and it is only found in the cytosol. Consistent with a role for Foxo3 in colitis, Foxo3-deficient mice treated with DSS developed more severe colonic inflammation with an increased number of intraepithelial lymphocytes and PMNs infiltrated in the epithelia, than wild-type mice. In summary, TNFalpha inactivates FOXO3 in intestinal epithelia through the PI3K and IKK pathways and FOXO3 inactivation leads to the upregulation of IL-8 in vitro; in vivo Foxo3 is in the cytosol of inflamed colonic epithelia and Foxo3 deficiency leads to severe intestinal inflammation.

Figure 1

TNFα induces FOXO3 translocation in HT-29 cells. (a) HT-29 cells, control and treated with TNFα were fixed, immunofluorescent stained for FOXO3 and images were taken with matched exposures. In control cells, FOXO3 is localized in the nucleus. During TNFα treatment, nuclear FOXO3 translocates to the cytosol, suggesting its inactivation (× 60 magnification). This experiment was repeated three independent times. (b) Nuclear (8 μg) and cytosolic (40 μg) proteins from HT-29 cells, control and TNFα treated for 3 h, were separated on SDS-PAGE and immunoblotted for FOXO3. Immunoblots reveal decreased nuclear and increased cytosolic amounts of FOXO3 after TNFα treatment. Immunoblots were reprobed with antibodies against Oct-1 for nuclear extracts and actin for cytosolic extracts as a control. Densitometric analysis shows a significant differences (*) between groups (P<0.05).

Figure 2

TNFα treatment of HT-29 cells induces degradation of FOXO3 by proteasome. (a) The total proteins from HT-29 cells, control and treated with TNFα for various time points, were separated on SDS-PAGE and immunoblotted for FOXO3 and actin. (b) The HT-29 monolayers were pre-incubated with proteasome inhibitor MG132 for 1 h and then treated with TNFα for various time points. Protein was separated on SDS-PAGE and immunoblotted for FOXO3 and actin. Each experiment was performed in triplicate and the densitometric analysis shows significant (*) degradation of FOXO3 during the course of TNFα treatment compared with untreated cells and protection with MG132 (P<0.05).

Figure 3

TNFα-induced FOXO3 phosphorylation is PI3K dependent. The HT-29 cells; with or without pretreatment with PI3K inhibitor (LY294002) were incubated with TNFα. Protein was separated on SDS-PAGE and immunoprobed with an antibody against phosphorylated FOXO3 at the Thr32 PI3K-dependent site and actin. The immunoblot is representative of three independent experiments (three samples were used per experimental group). Densitometric analysis shows a significant increase (*) in phosphorylated FOXO3 after TNFα treatment, which is attenuated in the presence of LY294002 (**) (P<0.05).

Figure 4

TNFα-induced inactivation of FOXO3 is controlled by IKK. (a) Total proteins from untreated and TNFα-treated cells were separated on SDS-PAGE and immunoprobed with an antibody against phosphorylated FOXO3 at the Ser644 IKK-dependent position. Immunoblots were also re-probed with an antibody against actin. (b) The HT-29 monolayers were pre-treated with the IKK inhibitor, PS1145, and induced with TNFα for various time points. Protein was separated on SDS-PAGE and immunoprobed with an antibody against total FOXO3 and actin. The graphs represent the densitometric analysis showing a significant decrease of FOXO3 (*) after TNFα treatment (n = 3, P<0.05) and protection of degradation with the IKK inhibitor. (c) Protein from the monolayers pretreated with PS1145 and TNFα was separated and immunoprobed against phosphorylated FOXO3 at Thr32 PI3K-dependent site and actin. The densitometric analysis shows a significant increase (*) in phosphorylated FOXO3 after TNFα treatment, which was not attenuated in the presence of PS1145.

Figure 5

FOXO3 is involved in the regulation of TNFα-induced IL-8 expression. (a). The monolayers were treated with TNFα for a period of 6 h and media was collected for IL-8 quantification. (b) Representative immunoblot of three independent experiments showing efficiency of FOXO3 knock down. The densitometric analysis shows significant (*) knock down of FOXO3 after 48 h, n = 3, P<0.05. (c) The monolayers with silent FOXO3 were treated with TNFα for 4 h and media was collected for IL-8 quantification. The graph represents the average IL-8 ratio of three independent experiments and the asterisk represents a significant difference (n = 4, P<0.05).

Figure 6

Foxo3 status in colonic epithelia of mice with DSS-induced inflammation. (a) Colonic tissue from C57BL/J6 mice, control and treated with DSS were immunohistostained for Foxo3. Immunohistostaining revealed cytoplasmic Foxo3 localization in inflamed colonic epithelia. (b) Colonic tissue from Foxo3-deficient mice is immunohistostained for Foxo3 as a control (× 20 magnification: bar 100 μm; inset × 63 magnification: bar 40 μm).

Figure 7

Foxo3 deficiency leads to severe intestinal inflammation. Foxo3 knockout (KO) and wild-type (WT) mice (n= 7 from each group) were treated with 2.5% DSS for 5 days and left 2 days to recover. (a) Measuring blood in stool revealed that KO mice have more bleeding than WT mice (P<0.05). (b) Graph represents inflammation scoring index between KO and WT mice (P<0.05). (c) H&E staining revealed mild inflammation in WT and severe inflammation in KO colon (× 20 magnification: bar 100 μm). (d and e) Graphs represent the number of lymphocytes and PMNs in colonic epithelia enumerated in five different fields. Asterisk represents significant differences between WT and KO mice (P<0.05).