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Nutritional Immunology

Functional Diversity of Flavonoids in the Inhibition of the Proinflammatory NF-B, IRF, and Akt Signaling Pathways in Murine Intestinal Epithelial Cells

Else Kroener-Fresenius-Centre for Experimental Nutritional Medicine, Technical University of Munich, Am Forum 5, 85350 Freising-Weihenstephan, Germany

² To whom correspondence should be addressed. E-mail: haller{at}wzw.tum.de.

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
The molecular understanding of nutritional factors in the process of host factor–mediated activation of the intestinal epithelium may play an important role in the assessment of adjunct nutritional therapy for chronic intestinal inflammation. We characterized the molecular mechanisms of flavonoids including apigenin, luteolin, genistein, 3'-hydroxy-flavone, and flavone in inhibiting tumor necrosis factor- (TNF)-induced interferon-induced protein (IP)-10 gene expression in the murine intestinal epithelial cell (IEC) line Mode-K. We demonstrated that 3'-hydroxy-flavone but not the chemical core structure flavone blocked TNF-–induced nuclear factor (NF)-B transcriptional activity and IP-10 expression at the level of NF-B/IB phosphorylation/degradation by inhibiting IB kinase activity. Although 3'-hydroxy-flavone effectively triggered p38 mitogen-activated protein kinase signaling and late caspase-3 cleavage, the induction of apoptotic cell death in TNF-activated IEC was not the primary mechanism inhibiting NF-B transcriptional activity and IP-10 expression. In addition to the compound-specific inhibition of TNF-induced NF-B DNA binding and NF-B transcriptional activity, apigenin and luteolin selectively blocked Akt phosphorylation/activity. The ability of these polyphenolic compounds to target various signal transduction pathways was further supported by the observation that luteolin and 3'-hydroxy-flavone selectively induced interferon regulatory factor (IRF)-1 degradation. Finally, we showed that genistein blocked IP-10 but not IL-6 expression through NF-B, IRF, and Akt independent mechanisms, demonstrating the functional diversity of flavonoids in inhibiting proinflammatory processes in IEC. In conclusion, we provide molecular evidence for the presence of characteristic inhibition patterns of these polyphenolic compounds to inhibit proinflammatory gene expression in IEC through the specific modulation of the NF-B, IRF and Akt signaling pathways.

KEY WORDS: • intestinal epithelial cell pathology • inflammation • NF-B signaling • flavonoids

Gastrointestinal infections, the genetic predisposition to dysregulated mucosal immune responses, and the concurrent prevalence of certain environmental triggers in developed countries (e.g., nutritional habits) are strong etiologic factors for the development of chronic intestinal inflammation including ulcerative colitis and Crohn's disease, the 2 distinct pathologies of inflammatory bowel disease (IBD)³ (1–3). Advancing knowledge about the molecular mechanisms of chronic intestinal inflammation has led to the development of specific biologic therapies (4); however, little is known about the anti-inflammatory effects of dietary components on disease pathology. Increased activity of the nuclear factor (NF)-B transcription factor system was documented recently in the intestinal epithelium of animal models for experimental colitis (5) and IBD patients (6–8); accordingly, pharmacologic blockade may become particularly important in the treatment of chronic intestinal inflammation (9). Intestinal epithelial cells (IEC) must adapt to a constantly changing environment by processing the combined biological information of luminal enteric bacteria/nutritional factors (10) as well as host-derived immune signals (11–13) to maintain gut homeostasis; thus, IEC become an excellent target cell type with which to assess the anti-inflammatory effects of dietary components on the host (14).

Tumor necrosis factor- (TNF) plays an important role in initiating and perpetuating NF-B signaling and chronic intestinal inflammation (15,16). Indeed, the treatment of a subset of IBD patients with monoclonal antibodies to TNF (infliximab) induced clinical remission of the inflammatory disease status (17). Additional experimental evidence for the importance of TNF signaling in triggering intestinal inflammation was demonstrated in TNF^ARE mice (18,19). These mice lack the translational repression of TNF due to the absence of TNF adenosine-uracil–rich elements in the 3'-untranslated region of the TNF mRNA transcripts; as a consequence, the mice develop experimental ileitis. At the cellular level, TNF activates the IB/NF-B transcription factor system through a TNF receptor 1 (TNFR1)-mediated cascade. Although controversial, the signal may converge on the NF-B–inducing kinase, which then activates the IB kinase (IKK) complex. The activation of IKK triggers NF-B RelA (Ser536) and IB (Ser32/34) phosphorylation as well as IB ubiquitination and proteasomal degradation. The activation of the IB/NF-B complex is then followed by the nuclear translocation of transcriptionally active NF-B subunits such as RelA and the induction of B-dependent gene expression (20,21). Interestingly, the full activation of TNF-induced NF-B activity and proinflammatory gene expression requires additional mechanisms including Akt serine-threonine kinase activation (22). Although TNF usually targets the cellular survival pathways in IEC through the activation of NF-B and Akt signal transduction, TNF-mediated inflammatory signals can also be diverted to induce proapoptotic mechanisms through the induction of the p38 MAPK pathway (23–25).

Dietary flavonoids, which comprise the most common group of plant polyphenols particularly abundant in fruits and vegetables, were shown to mediate anti-inflammatory, antioxidative, antiproliferative, and proapoptotic effects in various cell types (26). Although certain flavonoids affect stress/cytokine-induced NF-B signal transduction (27–29) and at least to some extent inhibit experimental colitis (30–32), the molecular mechanisms and the target specificity of these polyphenolic compounds in inhibiting epithelial cell activation is not well defined. We showed previously that under conditions of chronic experimental colitis, persistently activated NF-B signal transduction and gene expression of the T cell CXC chemoattractant IFN--inducible protein 10 (IP-10) in primary IEC was associated with the development of histopathologic changes in the colonic mucosa (5). In this study, we used preselected flavonoids that were shown to be highly efficient in inhibiting TNF-induced IP-10 expression in the murine IEC line Mode-K IEC. The aim of this study was to further characterize the molecular mechanisms of apigenin, luteolin, genistein, and 3'-hydroxy-flavone in inhibiting host factor-mediated activation of the intestinal epithelium.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
    Cell culture and treatments. The mouse IEC line Mode-K (passage 10–30) (a generous gift from Dr. Ingo B. Autenrieth, University of Tübingen, Germany) was grown in a humidified 5% CO₂ atmosphere at 37°C to confluence in 6-well tissue culture plates (Cell Star, Greiner bio-one) as previously described (10). Mode-K cells were stimulated with TNF (5 µg/L; R&D Systems), IL-1ß (5 µg/L; R&D Systems) in the absence or presence of apigenin, luteolin, 3'-hydroxy-flavone, flavone (all from Sigma-Aldrich), and genistein (Roth). Dose-response experiments were performed with the flavonoids in a concentration range of 1–200 µmol/L. The 50% effective inhibitory concentrations (EC₅₀) of these compounds were determined by calculating the inflection point of the inhibition curve. TNF-induced IP-10 protein concentrations were blotted against the flavonoid concentration. Where indicated, we used pharmacologic inhibitors including the p38 MAPK inhibitor SB203580 (20 µmol/L; Calbiochem, Merck Biosciences) and the proteasome inhibitor MG132 (20 µmol/L; BioMol). Cell viability was measured using MTT (thiazolyl blue tetrazolium bromide) (Sigma-Aldrich) and trypan blue exclusion.

    Western blot analysis. Mode K cells were pretreated with flavonoids (100 µmol/L) for 1 h followed by stimulation with IL-1ß and TNF. Cells were lysed in 1X Laemmli buffer and 20–50 µg of protein was subjected to electrophoresis on 10% SDS-PAGE gels. Anti-phospho-RelA (Ser536), anti-phospho-p38 (Thr180/Tyr182), anti-p38, anti-histone 3, anti-phospho-IB (Ser32), anti-phospho-Akt (Ser473), Akt, anti-phospho-glycogen synthase kinase (GSK)3/ß (Ser3/9), anti-cleaved caspase-3 (Asp175), anti-histone 3 (all from Cell Signaling), anti-interferon regulatory factor (IRF)-1, anti-IRF-3, anti-RelA, and anti-IB (all from Santa Cruz) were used to detect immunoreactive phospho-RelA, phospho-p38, p38, histone 3, phospho-IB, phospho-Akt, Akt, GSK-3/ß, IRF-1, IRF-3, RelA, and IB, respectively, using an enhanced chemiluminescence light-detecting kit (Amersham) as previously described (10).

    IKKß and Akt kinase assay. IKKß and Akt kinase assays were performed according to the protocol from Cell Signaling. Mode-K cells were lysed in cell lysis buffer (Cell Signaling) after treatment with TNF in the absence and presence of 100 µmol/L flavonoids for 30 min. Total cellular protein (200 µg) was incubated overnight with 0.01 µL/L anti-IKK (Cell Signaling) or anti-Akt (1G1, Cell Signaling) followed by incubation with 20 µL of A/G agarose beads (Santa Cruz) for an additional 2 h. The kinase reaction was performed in 40 µL of kinase buffer (Cell Signaling) supplemented with 200 µmol/L ATP and incubated in the presence of substrate including IB (1-317; Santa Cruz) or GSK-3 fusion proteins (Cell Signaling). Substrate protein was resolved by gel electrophoresis followed by the detection of phospho-IB and phospho-GSK-3/ß using immunoreactive antibodies.

    ELISA analysis. Mode K cells were pretreated with flavonoids (100 µmol/L) for 1 h followed by the stimulation with TNF and IL-1ß for an additional 24 h. Protein concentrations were determined in spent culture supernatants of IEC cultures. IL-6 and IP-10 production was determined by mouse-specific DuaSet Development ELISA assay kits, according to the manufacturer's instructions (R&D Systems).

    Nuclear extracts and NF-B RelA protein/DNA binding activity. Mode K cells were pretreated with flavonoids (100 µmol/L) for 1 h followed by the stimulation with TNF for an additional 30 min. Nuclear extracts were prepared according to the manufacturer's instructions (Active Motif). Extracts (5 µg) were used to determine nuclear RelA binding activity to the B-nucleotide consensus sequence 5'-GGGACTTTCC-3' using the TransAM ELISA-based NF-B transcription factor assay (Active Motif). Protein/oligonucleotide binding activity was quantified by colorimetric analysis using a MultiScan spectrophotometer.

    Cell transfection and selection. Mode K cells were grown to 80% confluence and then transfected with 2 µg of the NF-B–inducible reporter plasmid pNiFty-secreted alkaline phosphatase (SEAP) (InvivoGen) in the presence of 6µL FuGENE 6 Transfection Reagent (Roche Diagnostics). The pNiFty-SEAP reporter construct contains an engineered ELAM promoter with 5 NF-B binding sites (GGGGACTTTCC) and the SEAP as a reporter gene. Stable transfected cells were selected after the initial transfection (48 h) in the presence of the antibiotic zeocin (InvivoGen).

    Reporter (SEAP) gene assay for NF-B transcriptional activity. pNiFty-SEAP transfected Mode-K cells were pretreated with flavonoids (100 µmol/L) for 1 h followed by the stimulation with TNF for an additional 24 h. The spent culture supernatants (10 µL) were heated at 65°C for 5 min to eliminate the endogenous alkaline phosphatase activity. The SEAP secreted was measured according to the manufacturer's instructions (InvivoGen) at 405 nm in a MultiScan spectrophotometer.

    Statistical analysis. Data are expressed as means ± SD of triplicate stimulations from 3 independent experiments. Dose-dependent analysis for the calculation of EC₅₀ values was performed in duplicate stimulations. Statistical analysis was performed using 1-way ANOVA followed by Tukey's test. Differences were considered significant at P < 0.05.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
    Inhibition of IEC activation by flavonoids and their bacterial metabolites. To investigate anti-inflammatory mechanisms of flavonoids on IEC activation, we stimulated the murine noncarcinoma epithelial cell line Mode-K with the proinflammatory cytokine TNF for 24 h in the absence and presence of apigenin, luteolin, genistein, 3'-hydroxy-flavone, and flavone. Apigenin, luteolin, genistein, and 3'-hydroxy-flavone at a final concentration of 100 µmol/L completely blocked TNF-induced IP-10 protein secretion in Mode-K cells (Table 1). Dose-response analysis revealed EC₅₀ values for apigenin, luteolin, genistein and 3'-hydroxy-flavone in a concentration range of 20–27 µmol/L (Fig. 1). Of note, the maximal inhibition of TNF-induced IP-10 secretion was achieved for apigenin, luteolin, genistein, and 3'-hydroxy-flavone at a final concentration of 100 µmol/L without induction of significant cytotoxicity (<15%). Interestingly, nontoxic concentrations (1–200 µmol/L) of flavone also did not inhibit TNF-induced IP-10 secretion, suggesting that the inability of flavone to inhibit TNF-induced proinflammatory gene expression in IEC is not a concentration-dependent phenomenon.

View this table: [in this window] [in a new window]   TABLE 1 Differential effects of flavonoids on TNF-induced IP-10 expression, NF-B DNA binding activity, and NF-B reporter gene activity in Mode K cells1

View larger version (19K): [in this window] [in a new window]   FIGURE 1  Effective flavonoid concentrations in the inhibition of TNF-induced IP-10 expression in IEC. Mode-K cells were stimulated with TNF (5 µg/L) in the presence of apigenin, luteolin, genistein, and 3'-hydroxy-flavone in a concentration range of 1–200 µmol/L. TNF-induced IP-10 protein concentrations were blotted against the flavonoid concentration and the EC50 were determined. Medium alone and medium with vehicle dimethyl sulfoxide (DMSO) were used as controls. The results represent the mean of 2 independent experiments performed in duplicate 6-well cultures. IP-10 protein concentrations (µg/L) were measured in the spent culture supernatant after 24 h of stimulation using ELISA analysis.

View larger version (33K): [in this window] [in a new window]   FIGURE 2  3'-Hydroxy-flavone inhibits TNF-induced NF-B RelA and IB phosphorylation. Mode-K cells were stimulated with TNF (5 µg/L) in the presence of 100 µmol/L apigenin, luteolin, genistein, 3'-hydroxy-flavone, and flavone. Medium alone and medium with vehicle (DMSO) were use as controls; 20 µg of total protein was subjected to SDS-PAGE followed by (A) phospho-RelA, RelA, (B) phospho-IB, (C) IB and H3 immunoblotting. These results are representative of 3 independent experiments.

View larger version (29K): [in this window] [in a new window]   FIGURE 3  3'-Hydroxy-flavone inhibits IKK-activity. Mode-K cells were stimulated with TNF (5 µg/L) in the presence of 100 µmol/L 3'-hydroxy-flavone and flavone. The IB kinase reaction was performed using IB (1-317) fusion protein. Substrate protein was resolved by SDS-PAGE followed by the detection of phospho-IB and total IB using immunoreactive antibodies. These results are representative of 2 independent experiments.

    Luteolin inhibits NF-B RelA transcriptional activity.

    Luteolin, apigenin, and hydroxy-flavone inhibit Akt phosphorylation and induce IRF transcription factor degradation. We next measured the level of Akt phosphorylation/activity and IRF-1/3 transcription factor stability in TNF-stimulated Mode-K cells in the presence and absence of flavonoids. Although phospho-Akt was already present in unstimulated control cells, the presence of luteolin and apigenin dramatically diminished Akt phosphorylation in IEC after stimulation with TNF for 20 min (Fig. 4A). Interestingly, the level of phospho-Akt was restored to almost control levels in the presence of apigenin but not luteolin after 24 h of incubation (Fig. 4B), suggesting that luteolin had the most profound inhibitory effect on Akt phosphorylation. Consistent with the effects on phospho-Akt, apigenin and most particularly luteolin reduced Akt kinase activity in TNF-stimulated cells after 30 min of incubation (Fig. 5). The fact that 3-hydroxy-flavone did not affect the level of Akt kinase activity may further support the hypothesis for a characteristic inhibition pattern of flavonoids in TNF-activated IEC. In addition to the effects of these polyphenolic compounds on the survival pathway Akt, luteolin, and hydroxy-flavone selectively triggered IRF-1 but not IRF-3 transcription factor degradation in Mode-K cells after 24 h of stimulation (Fig. 6). Luteolin and hydroxy-flavone did not affect IRF-1 protein stability at the early stages (30 min) of signal transduction.

View larger version (22K): [in this window] [in a new window]   FIGURE 4  Luteolin and apigenin inhibit Akt phosphorylation/activity in TNF-stimulated IEC. Mode-K cells were stimulated with TNF (5 µg/L) in the presence of 100 µmol/L apigenin, luteolin, genistein, 3'-hydroxy-flavone, and flavone. Medium alone and medium with vehicle (DMSO) were used as controls; 20 µg of total protein was subjected to SDS-PAGE followed by phospho-Akt and total Akt immunoblotting for 20 min (A) or 24 h (B) These results are representative of 2 independent experiments.

View larger version (24K): [in this window] [in a new window]   FIGURE 5  Luteolin and apigenin inhibit Akt activity. The Akt kinase reaction was performed in the presence of GSK3/ß fusion protein. Substrate protein was resolved by SDS-PAGE followed by the detection of phospho- GSK3/ß and total Akt using immunoreactive antibodies. These results are representative of 2 independent experiments.

View larger version (23K): [in this window] [in a new window]   FIGURE 6  Luteolin and 3'-hydroxy-flavone trigger IRF-1 protein degradation. Mode-K cells were stimulated with TNF (5 µg/L) in the presence of 100 µmol/L apigenin, luteolin, genistein, 3'-hydroxy-flavone, and flavone. Medium alone and medium with vehicle (DMSO) were used as controls; 20 µg of total protein was subjected to SDS-PAGE followed by IRF-1 and IRF-3 immunoblotting. These results are representative of 2 independent experiments.

View larger version (28K): [in this window] [in a new window]   FIGURE 7  Induction of p38 phosphorylation in TNF-stimulated IEC. Mode-K cells were treated with 100 µmol/L apigenin, luteolin, genistein, 3'-hydroxy-flavone, and flavone in the presence of TNF (5 µg/L). Medium alone and medium with vehicle (DMSO) were used as controls; 20 µg of total protein was subjected to SDS-PAGE followed by phospho-p38 and p38 immunoblotting for 30 min (A) or 24 h (B). These results are representative of 2 independent experiments.

View larger version (22K): [in this window] [in a new window]   FIGURE 8  Caspase-3 cleavage in TNF-stimulated IEC. Mode-K cells were stimulated with TNF (5 µg/L) in the presence of 100 µmol/L apigenin, luteolin, genistein, 3'-hydroxy-flavone, and flavone. Medium alone and medium with vehicle (DMSO) were used as controls; 20 µg of total protein was subjected to SDS-PAGE followed by cleaved caspase-3 and H3 immunoblotting for 30 min (A) or 24 h (B). Positive controls for caspase-3 cleavage were generated from camptothecin-treated cells. These results are representative of 2 independent experiments.

View larger version (13K): [in this window] [in a new window]   FIGURE 9  Induction of caspase-3 cleavage and p38 phosphorylation by flavonoids. Mode-K cells were stimulated with TNF (5 µg/L) in the presence of 100 µmol/L apigenin, luteolin, genistein, 3'-hydroxy-flavone, and flavone. Medium alone and medium with vehicle (DMSO) were used as controls; 20 µg of total protein was subjected to SDS-PAGE followed by cleaved phospho-p38, p38, caspase-3, and H3 immunoblotting. These results are representative of 2 independent experiments.

    Differential effects of genistein in inhibiting IL-6 expression. To further evaluate whether the inhibitory effect of genistein is specific for TNF-induced IP-10 expression, we next measured the IL-1ß– and TNF-induced IL-6 expression in Mode-K cells after 24 h of stimulation in the presence and absence of 100 µmol/L genistein. Genistein did not inhibit TNF-induced IL-6 expression and, consistent with previously published results, genistein even increased IL-1ß–induced IL-6 expression (Table 2).

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

The 2 faces of TNF signaling and NF-B activation in the epithelium were shown in intestinal ischemia-reperfusion studies of IKKß–/– mice (34). The ablation of IKKß activity in enterocytes resulted in severe apoptotic damage to the reperfused intestinal mucosa but was protective in the development of systemic inflammation, demonstrating the dual function of NF-B inhibitors under conditions of acute inflammation. TNF mediates proinflammatory signals through the default survival pathways of the NF-B and Akt cascades (20,22), but at the same time, the signal can be diverted to induce proapoptotic mechanisms in IEC through induction of the p38 MAPK pathway (35). In Mode-K cells, TNF did not induce p38 phosphorylation and caspase-3 cleavage, suggesting that TNF signal transduction per se was biased in these cells toward the interrelated survival pathways NF-B and Akt. Although the inhibition of TNF-induced RelA and IB phosphorylation by 3'-hydroxy-flavone induced p38 phosphorylation, the inhibitory effect of 3'-hydroxy-flavone on NF-B transcriptional activity was not reversed in the presence of the pharmacologic p38 inhibitor SB203580. Consistent with the observation that SB203580 had only moderate effects on TNF-induced NF-B transcriptional activity and IP-10 expression, we concluded from these results that the inhibition of IKKß activity by 3'-hydroxy-flavone was independent of the p38 MAPK signaling pathway. In addition, the lack of significant nuclear fragmentation and cell death suggests that proapoptotic effects were not the primary mechanisms for the inhibition of NF-B transcriptional activity and IP-10 expression after 24 h of TNF stimulation. Nevertheless, the induction of late caspase-3 cleavage in the presence of 3'-hydroxy-flavone strongly indicates a late onset of proapoptotic effects in these cells. The pathological consequences of 3-hydroxy-flavone signaling to the epithelium under conditions of chronic inflammation remains to be determined.

Luteolin and apigenin inhibited TNF-induced IP-10 expression at the level of RelA nuclear translocation, NF-B DNA binding, and NF-B transcriptional activity. Although luteolin and apigenin differed in their ability to inhibit NF-B signaling at the various control points of this cascade, Akt was completely dephosphorylated by all 3 polyphenolic compounds, with luteolin having the most profound and long-lasting effects. The serine/threonine kinase Akt was shown to stimulate the NF-B signaling pathway at various levels including IKKß activity, NF-B DNA binding activity, and NF-B transcriptional activity (10,36–38). Because luteolin and apigenin did not block TNF-induced RelA and IB phosphorylation, the inhibition of the PI3-kinase/Akt pathway may not specifically interfere with the induction of IKK activity and the subsequent phosphorylation/degradation of the IB/NF-B complex. Mayo et al. (39) showed in prostate cells that the reintroduction of PTEN, which is a lipid phosphatase responsible for the deactivation of PI3K/Akt signaling, resulted in the inhibition of TNF-induced NF-B transcriptional activity by blocking the transactivation domain of the RelA/p65 subunit. Interestingly, the authors also showed that PTEN did not inhibit TNF-induced IKK activity, IB degradation, and NF-B RelA nuclear translocation. Although the molecular mechanisms for the inhibition of TNF-induced IP-10 expression through the Akt cascade remain controversial, the inhibition of Akt activity by flavonoids may interfere with the full recruitment of transcriptionally active NF-B RelA to the IP-10 promoter by partially inhibiting RelA nuclear translocation and NF-B transcriptional activity.

The IP-10 promoter is regulated in a complex way with interrelated roles of the transcription factor binding sites for NF-B and the family of IRF proteins. Promoter studies clearly indicated that the 2 NF-B and additional IRF binding sites interrelate to fully activate IP-10 gene expression (40,41). In addition, IRF-3 is acting as coactivator for IP-10 transactivation by recruiting IRF-3 protein to B-promoter sites where it can bind directly to NF-B RelA (42,43). Interestingly, we showed that luteolin and 3'-hydroxy-flavone but not apigenin selectively triggered IRF-1 protein degradation at late stages of TNF-induced IEC activation. Considering that the IRF inhibitor ribavarin completely blocked TNF-induced IP-10 expression, we may speculate about a specific role of IRF-1 in mediating TNF-induced IP-10 expression. It is interesting that 3'-hydroxy-flavone, apigenin, and luteolin differentially affect the IKK, IRF, and Akt pathways, but at the same time all of the compounds inhibited IP-10 expression at the various levels of the TNF signaling cascade.

Two previous studies showed that luteolin also blocked lipopolysaccharide (LPS)-induced NF-B signal transduction and proinflammatory gene expression at the level of IKKß activity and IB phosphorylation in nontransformed IEC (28) and macrophages (44). Interestingly, Kim and Jobin (28) showed in IEC that the blockade of LPS-induced IKKß activity by luteolin resulted in the inhibition of IB but not RelA phosphorylation. In addition and consistent with findings that LPS-induced IKK activity was linked to the activation of the PI3-kinase/Akt pathway (10), Xagorari et al. (44) showed in LPS-stimulated macrophages that luteolin indeed inhibited IB and Akt phosphorylation. In contrast, we showed that the inhibition of TNF-induced IKKß activity by 3'-hydroxy-flavone resulted in the blockade of both RelA (Ser536) and IB phosphorylation. In addition, luteolin completely failed to block TNF-induced activation of the NF-B cascade at the level of IKK and Akt activity. Signal-specific mechanisms may partially explain the discrepancy concerning the flavonoid-mediated inhibition of the NF-B signal transduction pathway.

In addition, genistein inhibited the NF-B signaling cascade through Akt-dependent mechanisms in PC3 prostate (45) and MDA-MB-231 breast (46) cancer cells. Interestingly, we showed that genistein completely failed to affect the NF-B and Akt signaling pathways but effectively inhibited TNF-induced IP-10 expression. These results may support the hypothesis that the inhibitory mechanisms of flavonoids are not only signal specific but also cell type dependent. Indeed, Gustin et al. (47) provided some molecular insights for cell type–dependent differences in the cross-talk between the NF-B and Akt signaling cascades. The authors showed that cells with a high proportion of IKK relative to IKKß were more sensitive to the inhibition of the PI3 kinase/Akt pathway; consequently, the transient overexpression of IKKß diminished the capacity of the PI3 kinase/Akt inhibitors to block NF-B DNA binding. The fact that genistein blocked cytokine-induced IP-10 expression but completely failed to inhibit IL-1ß/TNF-induced IL-6 expression further supports the hypothesis that the specific capability of flavonoids to interfere with distinct signaling pathways characteristically modulates the inhibition profile of target genes depending on their requirements for transcription and coactivation factors.

In conclusion, these results provide molecular evidence for characteristic inhibition patterns of flavonoids in the regulation of cytokine-induced NF-B/Akt signaling and IP-10 expression in noncarcinoma epithelial cells from the small intestine. Most interestingly, these polyphenolic compounds all exhibited inhibitory effects on proinflammatory cytokine expression but appeared to achieve their inhibitory effects by targeting distinct signaling pathways. The specific effects of the flavonoids on NF-B, IRF, and Akt signal transduction are summarized schematically in Figure 10. It seems important to fully understand the functional diversity of these polyphenolic compounds in targeting epithelial cell–specific signal transduction pathways and gene expression profiles to select effective anti-inflammatory compounds for adjunct nutritional therapy of chronic intestinal inflammation with little or no side effects for the epithelium. It is therefore important to evaluate the physiologic consequences of anti-inflammatory but potentially proapoptotic flavonoids for the development of chronic intestinal inflammation with respect to the various disease pathologies in human IBD. Future experiments in animal models of experimental colitis (e.g., IL-10^–/– mice) and ileitis (e.g., TNF^ARE) will add useful information about the therapeutic role of flavonoids in human IBD.

View larger version (26K): [in this window] [in a new window]   FIGURE 10  Schematic illustration for the inhibitory effects of flavonoids in IEC after stimulation with TNF. NF–B1 indicates NF-B transcriptional activity; black stop lines indicate the primary mechanism of inhibition.

	FOOTNOTES

³ Abbreviations used: DMSO, dimethyl sulfoxide; EC₅₀, 50 % effective inhibitory concentration; GSK, glycogen synthase kinase; IBD, inflammatory bowel diseases; IEC, intestinal epithelial cells; IKK, IB kinase; IP-10, interferon--inducible protein 10; IRF, interferon regulatory factor; LPS, lipopolysaccharide; MAPK, mitogen-activated protein kinase; NF-B, nuclear factor B; SEAP, secreted alkaline phosphatase; TNF, tumor necrosis factor-.

Manuscript received 16 August 2005. Initial review completed 28 September 2005. Revision accepted 13 December 2005.

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