M1840 Activity of Foxo Family Transcriptional Factors is Diminished in Inflamed Intestinal Epithelia

Abstract

FOXO1, FOXO3, FOXO4, and FOXO6 members belong to the FOXO family of Forkhead transcriptional factors. Active FOXO proteins are localized in the nucleus and are responsible for regulating the expression of specific target genes that modulate the metabolic state, cell cycle progression, the mitotic program or cellular apoptosis. We have been the first to demonstarate the significant role of FOXO3 in intestinal inflammation; showing: (i) proinflammatory stimuli inhibit FOXO3 activity; (ii) attenuated FOXO3 activity leads to increased expression of cytokines in intestinal cells by down regulating NF-κB inhibitor IκBα; (iii) Foxo3 activity is impaerd in mouse inflamed colon; and (iv) Foxo3 deficnecy increases intestinal susceptiblity to induced inflammation. Hence, we hypothesize that other FOXO family members may regulate intestinal inflammation and serve as a pharmacological target for treatment of intestinal inflammation. The Aim of this study was to assess the activity of FOXO family members in inflamed intestinal epithelia. Methods: For In Vitro studies, intestinal epithelial HT29 cells were employed. The effect of TNFα (10 ng/ml) and LPS (E. coli LPS serotype 0111:B4) (100 μg/ml) on FOXO localization was determined by immunofluorescent staining. For In Vivo study, C57BL/6J mice were treated with 2.5% DSS for 5 days and left for 2 days to recover. Inflammation was assessed by H&E staining and FOXO localization was assessed by immunohistostaining. Results: For In Vitro study, HT29 cells, with or without LPS and TNFα treatment, were examined for localization of FOXO family members. In control (untreated) HT29 cells, FOXO proteins are localized in the nucleus, while LPS and TNFα treatment induced retention of FOXO proteins in the cytosol. This suggests that the FOXO transcriptional activity occurred in the nucleus and the cytosolic retention in the presence of proinflammtory stimuli abrogates the activity. Next, we examined localization of Foxo proteins in control and inflamed mouse colonic epithelia. In control colonic epithelia, there was prominent nuclear localization of Foxo proteins along the crypt, with cytosolic and nuclear staining in proliferating cells in the base of the crypt. In inflamed colonic epithelia Foxo proteins were localized exclusively in the cytosol. This confirmed In Vitro study where in inflamed intestinal epithelia the transcriptional activity of Foxo proteins was impaired. Conclusion: Inflamed intestinal environment abrogates transcriptional activity of all FOXO members and provides the foundation necessary to consider the FOXO proteins as a pharmacological target for the treatment of intestinal inflammation.