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Tumor suppressor FOXO3 participates in the regulation of
intestinal inflammation
Lobke Snoeks1, Christopher R Weber2, Kaarin Wasland1, Jerrold R Turner2, Charles
Vainder1, Wentao Qi1, and Suzana D Savkovic1
1Division of Gastroenterology, Department of Medicine, North Shore University Research
Institute, Evanston, IL, USA
2Department of Pathology, University of Chicago, Chicago, IL, USA
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 TNFα 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. TNFα 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 TNFα-induced
FOXO3 phosphorylation. In FOXO3-silenced HT-29 cells, TNFα-induced IL-8 expression is
increased ∼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, TNFα
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.
Keywords
FOXO3; inflammation; intestinal epithelia; signaling; TNFα
In the intestinal tissue of inflammatory bowel disease (IBD) patients with the active disease,
many proinflammatory cytokines are increased, which are imperative in maintaining
inflammatory responses.1 One of the cytokines that drives intestinal inflammation is tumor
necrosis factor-α (TNFα).2–4 TNFα-blocking agents significantly reduce intestinal
inflammation in the majority of patients with active IBD.5
TNFα binds to cell membrane TNFα receptors (TNFR1 and TNFR2),6–8 stimulating
signaling cascades that lead to the activation of nuclear factor kappaB (NF-κB).9 From
Correspondence: Dr SD Savkovic PhD, Department of Medicine, Division of Gastroenterology, NorthShore University Research
Institute, 1001 University Place; Room 314, Evanston, IL 60201, USA. SSavkovic@northshore.org.
Disclosure/Conflict of Interest: The authors declare no conflict of interest.
NIH Public Access
Author Manuscript
Lab Invest. Author manuscript; available in PMC 2011 March 4.
Published in final edited form as:
Lab Invest
. 2009 September ; 89(9): 1053–1062. doi:10.1038/labinvest.2009.66.
NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript
TNFRs, the intracellular signaling pathways, that include several receptor adapters, activate
the inhibitory kappaB (IκB) kinase (IKK) complex. Activated IKK phosphorylates IκB
proteins causing IκB degradation and the release of NF-κB to freely translocate to the
nucleus.10 Nuclear NF-κB regulates genes that are involved in inflammation, cell survival,
and proliferation.11,12
The tumor suppressor FOXO3 belongs to the family of Forkhead transcriptional factors.13
FOXO3 is located in the nucleus regulating the expression of specific target genes involved
in the regulation of cell cycle progression, metabolic state, and cellular apoptosis.13,14
Phosphorylation of FOXO3 can occur by upstream pathways that include PI3K and/or IKK;
14,15 phosphorylated FOXO3 translocates to the cytosol and becomes inactive.16 Cytosolic
FOXO3 might attach to 14–3–3 proteins or be degraded in the proteasome.15–19
Foxo3-deficient mice develop spontaneous, multisystemic inflammatory syndrome,
accompanied by an increased cytokine production, an increased NF-κB activation, and
hyperactivation of T cells.20 In T cells, the overexpressed Foxo3 inhibits NF-κB and
cytokine expression.20 We have demonstrated that bacterial infection inactivates FOXO3 in
the intestinal epithelia and that this contributes to an increased cytokine expression;21 hence,
we hypothesize that FOXO3 is involved in the regulation of intestinal inflammation. We
have shown here that TNFα inactivates FOXO3 in HT-29 cells through the PI3K and IKK
pathways, which additionally increase IL-8. In mice, where colonic inflammation was
induced with dextran sulfate sodium (DSS), Foxo3 is also inactive. Foxo3-deficient mice, in
response to DSS, developed more severe intestinal inflammation compared with the wild-
type mice. Altogether, our results suggest that tumor suppressor FOXO3 has an important
role in the regulation of intestinal inflammation in vitro and in vivo.
Materials and Methods
Tissue Culture
Human intestinal epithelial HT-29 cells (ATCC, Manassas, VA, USA; passages 10–20)
were cultured in complete McCoy's 5A media (Sigma-Aldrich, St Louis, MO, USA)
containing 10% fetal bovine serum (Gibco, Carlsbad, CA, USA) and Penicillin–
Streptomycin (Gibco). The cells were kept in an incubator at 37°C, with 5% CO2 and then
they were plated in 6- or 12-well plates. When the monolayers reached confluency of ∼70%,
cells were serum-starved overnight before the experimental procedures were performed.
Semi-confluent HT-29 monolayers were stimulated with 10 ng/ml TNFα (R&D,
Minneapolis, MN, USA) for different lengths of time to study the effect on FOXO3 and
IL-8.
Immunofluorescent Staining
For immunofluorescent staining, HT-29 cells were grown on coverslips. After treatment, the
cells were washed with PBS and fixed with 3.7% paraformaldehyde. The fixed cells were
washed with PBS and permeabilized with 0.2% Triton-X in PBS followed by blocking in
2.5% bovine serum albumin. The cells were incubated with a primary FOXO3 antibody
(Upstate, Billerica, MA, USA) for 1 h, followed by washing and incubation with a
secondary antibody for 1 h (Alexa Fluor 488 Molecular Probes, Eugene, OR, USA). The
coverslips with cells were mounted on microscope slides (Mounting Solution antifade
reagent, Molecular Probes). The results were assessed using a Nikon Opti-Photo
microscope. The images were captured using a Spot RT-slider camera (Diagnostic
Instruments, Sterling Heights, MI, USA) and managed by Image Pro software (Media
Cybernetics, San Diego, CA, USA).
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Inhibitor Studies
To study different pathways in FOXO3 regulation, several pharmacological inhibitors were
applied. To inhibit the IKK pathway, we utilized its specific inhibitor PS1145 (30 μM)
(Sigma). To study the role of the PI3K pathway inhibitors, wortmannin (200 nM)
(Calbiochem, Gibbstown, NJ, USA) and LY294002, (30 μM) (Calbiochem) were used. The
proteasome inhibitor MG132 (20 μM) (Calbiochem) was applied to study FOXO3
proteasome-dependent degradation. The serum-starved HT-29 monolayers were pre-
incubated with an inhibitor for 1 h after which the monolayers were treated with TNFα for
different lengths of time.
Protein Extraction
After treatment, the HT-29 monolayers were washed with PBS (Gibco) and protein was
extracted by applying cell lysis buffer (Cell Signaling, Danvers, MA, USA), containing a
protease inhibitor cocktail (Sigma). Protein concentrations were determined using the Quick
Start Bradford Protein Assay kit (Biorad, Hercules, CA, USA) as described by the
manufacturer. The protein extracts were stored at −20°C until further processing. The
nuclear proteins were extracted using the NE-PER Pierce extraction kit (Pierce, Rockford,
IL, USA) according to the manufacturer's protocol and stored at −80°C.
Immunoblots
Equal amounts of protein (40 μg) per sample were separated on 10% Sodium Dodecyl
Sulfate polyacrylamide (SDS-PAGE) gels. Protein was transferred to nitrocellulose
membranes (Biorad). The immunoblots were probed with appropriate antibodies against
FOXO3 (Upstate), anti-phospho-Thr32-FOXO3 (Upstate), anti-phospho-Ser644-FOXO3
(generous gift of Dr Hu), and actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA) all
according to the manufacturers' protocols. The secondary antibodies were horseradish
peroxidase-conjugated (Chemicon, Temecula, CA, USA). The protein bands were detected
by using the ECL Plus western blotting detection system (GE Healthcare, Amersham, UK).
SiRNA Experiments
To study the effects of FOXO3 on TNFα-induced IL-8 in intestinal epithelial cells, we
transiently knocked-out FOXO3 by applying siRNA. The HT-29 cells were transfected with
two different oligos, designed to silence FOXO3 (30 nM) or a negative control oligo
(Invitrogen, Carlsbad, CA, USA). Oligos were introduced into the cells using Lipofectamine
RNAiMax (Invitrogen) following the manufacturers protocol. Initially, 48 h post-
transfection showed optimal knock down of FOXO3. Therefore, 36 h after transfection with
siRNA, the monolayers of HT-29 cells were serum deprived overnight and sequentially
treated with TNFα for 4 h. The media was collected and stored at −20°C until it was used
for IL-8 quantification. Whole cell lysates were used as a control for knock down of FOXO3
with western blotting.
IL-8 Quantification
To quantify IL-8, the media collected from monolayers was used for IL-8 ELISA (R&D)
according to the manufacturer's protocol.
Statistical Analysis
These data were represented as the mean±s.d. The student's t-test was used to compare these
data and the differences were considered significant when the P-values were ≤0.05.
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Animal Studies
For an in vivo study, colonic inflammation was induced by introducing 2.5% DSS (MP
Biomedicals, LLS, Solon, OH, USA) into the drinking water of C57BL/6J mice (6–8 weeks
old) for 5 days, followed by two recovery days. Body weight was monitored during the
course of the treatment. Gastrointestinal bleeding was evaluated using Coulter Hemoccult
(Fisher, Pittsburgh, PA, USA). After 7 days, the animals were euthanized and the colonic
tissue was prepared for histopathological studies.
Furthermore, to study the in vivo role of FOXO3 in colonic inflammation, we utilized 4 to 6-
week-old Foxo3-deficient mice kindly provided by Dr Stanford Peng (Roche Palo Alto
LLC, Palo Alto, CA, USA). The genotypes of the breeds were determined by PCR on tail
DNA using primers according to Lin et al.20
All animals were kept in the Biological Resources Laboratory at the University of Illinois at
a Chicago facility and all experimental procedures were performed in compliance with the
local protocols and guidelines approved by the local ethical committee.
Histological Analysis
Colonic tissue was formalin-fixed and paraffin-embedded. The tissue sections (5-μm thick)
were stained with routine hematoxylin and eosin (H&E) staining. The distribution of Foxo3
in colonic tissue was assessed as we described before.21 The degree of inflammation was
evaluated according to the following criteria: (0) completely uninvolved, no architectural
distortion or infiltrates; (1) architectural distortion, increased lamina propria lymphs, no
activity; (2) increased lamina propria granulocytes without definite intraepithelial
granulocytes (ie, no activity); (3) intraepithelial granulocytes (ie, activity) without crypt
abscesses; (4) crypt abscesses in less than 50% of crypts; (5) crypt abscesses in greater than
50% of crypts or erosion/ulceration.
Results
TNFα Regulates FOXO3 in HT-29 Cells
Nuclear FOXO3 is involved in the regulation of transcription; signaling by growth factors
and other stimuli results in FOXO3 translocation from the nucleus to the cytosol and
terminates FOXO3 gene regulation.16–18 This process can be modeled in human colonic
epithelial HT-29 cells, where FOXO3 is localized in the nucleus but translocated to the
cytosol after bacterial infection.21 To address the effect of cytokines on this process,
FOXO3 localization was examined in HT-29 cells treated with TNFα. FOXO3 translocated
from the nucleus to the cytosol during the first 30 min of TNFα treatment (Figure 1a). An
immunoblot analysis of subcellular fractions confirmed that the amount of nuclear FOXO3
decreases, whereas the amount of cytosolic FOXO3 increases after TNFα stimulation
(Figure 1b). This data suggests that FOXO3 becomes inactive after TNFα treatment of the
HT-29 cells.
It was shown that cytosolic FOXO3 might degrade by proteasome.15 Therefore, we
examined the fate of cytosolic FOXO3 in HT-29 cells treated with TNFα. Figure 2a shows
that TNFα induces FOXO3 degradation in colonic HT-29 cells. To further define the
mechanism of TNFα-induced FOXO3 degradation, we used the proteasome inhibitor
MG132. The pretreatment of HT-29 cells with MG132 caused a small, statistically
insignificant, increase in total FOXO3 and treatment with TNFα of MG132-pretreated
HT-29 cells, blocked FOXO3 degradation (Figure 2b). In summary, TNFα induced the
degradation of FOXO3 and that the degradation is proteasome mediated in HT-29 cells.
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TNFα Regulates FOXO3 in HT-29 Cells via PI3K and IKK
The translocation of FOXO3 from the nucleus to the cytosol occurs after its
phosphorylation.16 The PI3K pathway mediates the phosphorylation of FOXO3 in many cell
types.16 We have previously reported that LPS-induced FOXO3 phosphorylation in
intestinal epithelial cells is through the PI3K pathway.21 Therefore, we assessed the role of
PI3K in the phosphorylation of FOXO3 in intestinal epithelial cells treated with TNFα. We
used an antibody that recognizes FOXO3 phosphorylation at Thr32, which is known to be
phosphorylated by the PI3K pathway.16 TNFα increased FOXO3 phosphorylation at Thr32
by 3.1±0.8-fold in the first 30 min of treatment (Figure 3). The pharmacological PI3K
inhibitors, wortmannin or LY294002, were used to assess the contribution of the PI3K
pathway in TNFα-induced FOXO3 phosphorylation. The inhibition of the PI3K pathway
with these inhibitors blocked the phosphorylation of Akt, which is responsible for the
phosphorylation of FOXO313 (data not shown). Both inhibitors blocked TNFα-induced
FOXO3 phosphorylation in HT-29 cells (Figure 3 shows inhibition with LY294002). Also,
the inhibition of PI3K-blocked TNFα-induced degradation of Foxo3 (data not shown). This
data shows that TNFα-induced FOXO3 phosphorylation and degradation in HT-29 cells is
controlled proximally by the PI3K pathway.
Hu reported that FOXO3 might be phosphorylated by IKK in breast cancer cells.15 To
examine the contribution of IKK in TNFα-dependent FOXO3 inactivation we employed an
antibody that recognizes FOXO3 phosphorylation at Ser644, which is only phosphorylated
by IKK.15 The TNFα treatment of HT-29 cells increased FOXO3 Ser644 phosphorylation
2.7±0.2-fold within 30 min (Figure 4a). To further examine the role of the IKK pathway in
TNFα-induced FOXO3 regulation in HT-29 cells, we used a specific IKK-inhibitor,
PS1145.22,23 We confirmed the specificity of IKK inhibition by PS1145 by showing the
attenuation of p65 phosphorylation24 (data not shown). Pretreatment of HT-29 cells with
PS1145 almost completely blocked TNFα-induced FOXO3 degradation (Figure 4b). These
data suggest that IKK participates in the regulation of FOXO3 in HT-29 cells treated with
TNFα. The inhibition of IKK did not change PI3K-dependent phosphorylation of FOXO3
(Figure 4c), suggesting that FOXO3 regulation by PI3K is independent of IKK in HT-29
cells treated with TNFα.
FOXO3 is Involved in the Regulation of TNFα-Induced IL-8
IL-8 is a proinflammatory chemokine that is a strong chemo-attractant for neutrophils and
lymphocytes.25–27 In the tissue of IBD patients, IL-8 is significantly upregulated 27–30 and
the level of IL-8 is in direct proportion to the degree of inflammation.30 Cultured intestinal
epithelial cells secrete IL-8 following TNFα treatment.31,32 Under the conditions used in
this study, HT-29 cells increased IL-8 secretion 20-fold within 6 h of TNFα treatment
(control: 23±2 pg/ml; TNFα: 456±13 pg/ml) (Figure 5a). To assess the contribution of
FOXO3 to TNFα-induced IL-8 expression in intestinal HT-29 cells, we performed siRNA
experiments. The efficiency of FOXO3 silencing by siRNA in HT-29 cells was ∼90%
(Figure 5b). The silencing of FOXO3 increased the basal level of IL-8 in HT-29 monolayers
insignificantly. IL-8 was increased on average 83% in monolayers with silenced FOXO3
compared with IL-8 in monolayers with negative siRNA control treated with TNFα for 4 h
(TNFα (−) control: 304±18 pg/ml; TNFα siRNA: 544±110 pg/ml) (Figure 5c). This data is
consistent with our previously published data where an inactive FOXO3 contributes to the
upregulation of IL-8 during bacterial infection.21
Cytosolic FOXO3 is Associated with Colonic Inflammation
To further examine the role of FOXO3 in in vivo intestinal inflammation, we used a mouse
model. Mouse colonic inflammation was stimulated by introducing 2.5% DSS in drinking
water. DSS treatment elevates cytokines and triggers the infiltration of inflammatory cells in
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the colon.33,34 Strong nuclear staining of Foxo3 was detected in mouse colonic epithelial
cells, and slight cytosolic staining was found in crypt cells.21 In DSS-treated mice, Foxo3
was detected primarily in the cytosol of the colonic epithelia with no nuclear staining
(Figure 6a and b), suggesting that Foxo3 is not active in the inflamed colonic epithelia.
These findings correlate with in vitro data where Foxo3 translocation into the cytosol was
detected. The degradation of Foxo3 in vivo was not apparent; we hypothesize that in
intestinal tissue Foxo3 may also degrade but at this particular time point of DSS treatment
degradation was not noticeable.
Foxo3 Deficiency Leads to Increased Inflammation in the DDS Model
To further assess the role of Foxo3 in intestinal inflammation in vivo, the Foxo3-deficient
mice were employed. Although Lin reported that these animals displayed spontaneous
inflammation in several organs,20 the intestinal tissue did not show signs of inflammation
over a period of 2 months (Figure 7c). Wild-type and Foxo3-deficient mice from the same
colony, treated with DSS, did not show significant differences in weight loss (WT: 21±4 g,
KO: 21±2 g); however, there was increased blood in the stool in Foxo3-deficient mice
(Figure 7a) suggesting that the inflammatory process might be more active. A histological
examination showed that the injury was significantly less severe and the recovery was
enhanced in the wild-type mice relative to Foxo3-deficient mice. Although mild active
inflammation was present in the wild-type mice, severe inflammation was present in the
Foxo3-deficient mice (Figure 7b). The ulceration was minimal in wild-type mice, involved
small areas, and was accompanied by evidence of mucosal healing (Figure 7c). In contrast,
there were broad areas of mucosal ulceration with only limited healing present in Foxo3-
deficient mice. In Foxo3-deficient mice the lamina propria was expanded with a mixed
population of lymphocytes and polymorphonuclear neutrophils (PMNs), especially in areas
close to ulcerations. In the intestinal epithelia, the number of lymphocytes and PMNs was
increased 2- and 5-fold, respectively (lymphocytes: WT 4.1±1.4; KO 7.6±3.2, PMN: WT
0.9±1.1; KO 6.4 ±2.7) (Figure 7d and e). These data show that Foxo3 deficiency results in
severe disease in response to DSS.
Discussion
In the pathogenesis of inflammatory diseases, cytokines have an important role in
maintaining the inflammatory response. The regulation of cytokines is better understood
today; however, further elucidation of the mechanisms of cytokine regulation is needed to
develop more specific and effective therapies. Here, we have shown that TNFα induces
FOXO3 phosphorylation, translocation, and degradation in HT-29 cells. TNFα-mediated
FOXO3 degradation is proteasome dependent. Proximally, TNFα used the PI3K and IKK
pathways to regulate FOXO3 in HT-29 cells. In addition, our in vitro data revealed that
silenced FOXO3 leads to dramatically increased TNFα-induced IL-8 expression in HT-29
cells, which is in agreement with our previously published data.21 In vivo, in the mouse DSS
colitis model, Foxo3 is localized primarily in the cytosol of the intestinal epithelia,
suggesting that Foxo3 is inactive. Also, Foxo3-deficient mice developed a more severe
intestinal inflammation compared with wild-type mice in response to the DSS treatment.
Altogether, our results suggest that tumor suppressor FOXO3 regulates intestinal
inflammation in vitro and in vivo.
FOXO3 is localized in the nucleus-controlling gene expression both directly and indirectly.
13 In certain tumor cells, the inactivated cytosolic FOXO3 contributes to the pathogenesis
and development of tumors by modulating proliferation and apoptosis.35–37 Our in vivo data
revealed that Foxo3 is localized in the cytosol in the inflamed colon and that Foxo3
deficiency additionally promotes the inflammation. We previously demonstrated that
FOXO3 knock down leads to the reduced inhibitory IκBα in intestinal epithelial cells.21
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Therefore, we concluded that the inactive cytosolic FOXO3 further promotes intestinal
inflammation. Kristof et al38 have shown that LPS controls FOXO3 in human lung epithelial
cells, thus regulating iNOS and contributing to inflammation. In macrophages, HIV
infection keeps FOXO3 in the nucleus, which further contributes to the apoptosis of infected
cells.39 Thus FOXO3 has very different, but important, roles in the regulation of cellular
events depending on the cell type and pathological conditions.
FOXO3 is controlled proximally by the PI3K and IKK pathways.13,15 Our data show that
TNFα regulates FOXO3 by both PI3K and IKK pathways in human intestinal HT-29 cells.
Previously, we have shown that bacterial infection regulates FOXO3 via PI3K, whereas the
role of IKK was insignificant.21 Thus it appears that the proximal pathways differ depending
on the stimulus. At this moment, the exact relationship between the PI3K and IKK
pathways, with regard to TNFα-dependent FOXO3 regulation in intestinal epithelial cells, is
unclear. The inhibition of IKK did not affect PI3K-dependent FOXO3 phosphorylation,
whereas we were unable to clarify how inhibition of PI3K affects IKK-dependent FOXO3
phosphorylation. We speculate that the PI3K and IKK pathways regulate FOXO3
independently. Sudheerkumar et al40 have demonstrated that TNFα can independently
activate IKK and PI3K pathways in human glioma cells. Agarwal et al41 showed that
simultaneous activation of the IKK and PI3K pathways regulate both NF-κB and β-catenin
to facilitate tumor progression. We hypothesize that TNFα facilitates the inflammation of
the intestinal epithelia by activating the PI3K and IKK pathways that regulate both NF-κB
and FOXO3. We speculate that TNFα activates two separate but interrelated signaling
pathways and that both are necessary for the full inactivation of FOXO3, which further
promotes inflammation. One set of signals activates the IKK complex to phosphorylate
FOXO3 and to initially liberate NF-κB and drive the immediate cytokine expression. The
second set of signals uses the PI3K mechanism to additionally phosphorylate FOXO3 and
keep it out of the nucleus so that the de novo IκBα synthesis is repressed and the
transcription of pro-inflammatory genes is uninterrupted.
Foxo3 deficiency in mice leads to spontaneous T-cell activation, cytokine production and a
mild lymphoproliferation. In Foxo3-deficient mice, this leads to an autoimmune syndrome
with spontaneous inflammation in multiple organs, while having no reported intestinal
changes.20 The intestinal epithelia have an important role in separating the unsterile from the
sterile environment and thus, the infiltration of immune cells is tightly controlled. Intestinal
inflammation might occur after the intestinal epithelia ‘sense’ the environmental changes
and send signals to summon immune cells to infiltrate the tissue. Therefore, we speculate
that the role of Foxo3 in the intestinal tissue is specific. In an inflamed colon, Foxo3 is
distributed in the cytosol of the intestinal epithelia, suggesting that Foxo3 is inactive. We
hypothesize that the localization of FOXO3 will correlate with the severity of inflammation
in the DSS model and that with mild inflammation, Foxo3 will be distributed between the
cytosol and the nucleus. In cultured intestinal cells TNFα induces cytosolic FOXO3
degradation, which was not apparent in the intestinal epithelia of the colon. The intestinal
epithelial cells in tissue permanently proliferate and differentiate, which is critical for
normal growth, development, and disease prevention. FOXO3 also regulates the
proliferation of these cells, thus we hypothesize that FOXO3 remains in the cytosol without
significant degradation and is ready to go back to the nucleus to regulate other functions.
Foxo3-deficient mice develop more severe inflammatory responses to DSS compared with
wild-type mice. We show in vitro that FOXO3 deficiency leads to the attenuation of
inhibitory IκBα,21 directly linking FOXO3 with inflammation. A similar role of Foxo3 was
observed in T cells. In mouse T cells deficient in Foxo3, NF-κB activation is unrestrained
and there are diminished levels of IκBs.20 It is possible that for colonic inflammation in the
DSS model, lymphocytes deficient in Foxo3 are in part responsible. However, the fact that
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Foxo3-deficient lymphocytes alone do not spontaneously infiltrate colonic tissue suggest
that Foxo3 has the primary role in intestinal epithelia. In B cells and PMNs, Foxo3 has less
of an effect on the NF-κB pathway, but it does regulate cell survival and proliferation.42,43
Foxo3-deficient mice were resistant to induced arthritis due to increased apoptosis of the
Foxo3-deficient PMNs.44 On the contrary, our data showed that in colonic epithelia PMN
accumulation and crypt abscesses are increased in Foxo3-deficient mice. We proposed two
possible scenarios: (a) infiltrated Foxo3-deficient PMNs in the intestinal tissue are resistant
to apoptosis; and (b) due to the large number of PMNs infiltrated in the colon the apoptotic
nature on Foxo3-deficient PMN cells is not enough to eliminate them. Yet the role of Foxo3
in infiltrated inflammatory cells in the colon is still unclear as well as FOXO3's role in the
healing of inflamed intestinal tissue. We need to further address these questions.
In summary, these data indicate that FOXO3 has an important role in controlling and
facilitating intestinal inflammation. Furthermore, FOXO3 should be considered as a
potential therapeutic target to treat IBD.
Acknowledgments
We thank Dr Hemant Roy, Dr Michael Goldberg, and Dr Ramesh Wali for their helpful assistance in preparation of
the article. We also thank Dr Stanford Peng (Roche Palo Alto LLC, Palo Alto, CA, USA) for providing the Foxo3-
deficient mice to establish our breeding colony. This work was in part supported by a Senior Investigator Award
from the Crohn's and Colitis Foundation of America (CCFA no. 1953) and North Shore University Health System
Pilot Study grant. Lobke Snoeks received a Fulbright scholarship for her visiting fellowship and Christopher Weber
received an NIH F32DK082134 fellowship award.
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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).
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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).
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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).
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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.
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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).
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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).
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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).
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