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Originally published In Press as doi:10.1074/jbc.M105170200 on September 25, 2001

J. Biol. Chem., Vol. 276, Issue 48, 44641-44646, November 30, 2001
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Butyrate Suppression of Colonocyte NF-kappa B Activation and Cellular Proteasome Activity*

Lei Yin, Gary Laevsky, and Charles GiardinaDagger

From the Department of Molecular and Cellular Biology, University of Connecticut, Storrs, Connecticut 06269

Received for publication, June 6, 2001, and in revised form, August 21, 2001


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Butyrate is derived from the microbial metabolism of dietary fiber in the colon where it plays an important role in linking colonocyte turnover and differentiation to luminal content. In addition, butyrate appears to have both anti-inflammatory and cancer chemopreventive activities. Using confocal microscopy and cell fractionation studies, butyrate pretreatment of a human colon cell line (HT-29 cells) inhibited the tumor necrosis factor-alpha (TNF-alpha )-induced nuclear translocation of the proinflammatory transcription factor NF-kappa B. Butyrate inhibited NF-kappa B DNA binding within 30 min of TNF-alpha stimulation, consistent with an inhibition of nuclear translocation. Ikappa B·NF-kappa B complexes extracted from butyrate-treated cells were relatively resistant to in vitro dissociation by deoxycholate, suggesting a change in cellular Ikappa B composition. Butyrate treatment increased p100 expression, an Ikappa B that was not degraded upon TNF-alpha treatment. Butyrate also reduced the extent of TNF-alpha -induced Ikappa B-alpha degradation and enhanced the presence of ubiquitin-conjugated Ikappa B-alpha . The suppression of Ikappa B-alpha degradation corresponded with a reduction in cellular proteasome activity as determined by in vitro proteasome assays and the increased presence of ubiquitin-conjugated proteins. The butyrate suppression of Ikappa B-alpha degradation and proteasome activity may derive from its ability to inhibit histone deacetylases since the specific deacetylase inhibitor trichostatin A had similar effects. These results suggest a potential mechanism for the anti-inflammatory activity of butyrate and demonstrate the interplay between short chain fatty acids and cellular proteasome activity.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Butyrate and other short chain fatty acids are generated in the intestine by the bacterial metabolism of dietary fiber. Short chain fatty acids benefit the colonic mucosa in a number of ways (1-5). Colonocytes utilize short chain fatty acids as their primary energy source, and evidence has been obtained that they undergo apoptosis in its absence (2, 5-7). Interestingly, short chain fatty acids, particularly butyrate, have the opposite effect on transformed cells in culture, inducing rather than suppressing apoptosis (2, 8-10). The ability of butyrate to induce cancer cell apoptosis may contribute to the cancer preventive activities of dietary fiber (1, 5, 6, 11, 12). Short chain fatty acids can also suppress intestinal inflammation: butyrate is effective for treating selected inflammatory conditions of the distal alimentary tract (13-15). Many aspects of intestinal health and function clearly depend on the appropriate levels of short chain fatty acids in the lumen. Understanding the effects of short chain fatty acids on normal and transformed colonocytes could provide insight into the mechanisms that maintain intestinal health and function.

It is not clear how all the beneficial aspects of short chain fatty acids are achieved. Considerable attention has focused on the influence of butyrate on cellular gene expression, derived in part from its ability to inhibit histone deacetylases (16, 17). Histone acetylation plays a central role in regulating gene expression, and cellular treatment with butyrate stabilizes histones in their acetylated state. In addition, a number of transcription factors are directly regulated by acetylation, and butyrate can enhance their activity (including GATA-1 and p53) (18-21). We and others have reported that butyrate can modulate the activity of the transcription factor NF-kappa B in a number of different cell types including colon cancer cell lines, cells isolated from the lamina propria of the colon, and macrophages (22-25). The ability of butyrate to modulate NF-kappa B activity may arise from its ability to inhibit protein deacetylases. This conclusion is based on the finding that the specific deacetylase inhibitor trichostatin A (TSA)1 also modulates NF-kappa B activity and that propionate, a weaker deacetylase inhibitor, is less effective than butyrate in altering NF-kappa B activity (25). The ability of butyrate to modulate NF-kappa B activity resonates with its proposed cancer chemopreventive and anti-inflammatory activities since NF-kappa B regulates genes involved in controlling cell proliferation, cell death, immune response, and inflammatory responses. We have been particularly interested in the mechanism by which butyrate influences NF-kappa B activation since understanding this mechanism could reveal how colonocyte growth and inflammatory responses are designed to sense luminal content. For this reason we have been studying the mechanism by which butyrate influences NF-kappa B activation in the human colon-derived HT-29 cell line.

NF-kappa B is regulated through the binding of inhibitory molecules referred to collectively as Ikappa Bs (26-28). There are a number of Ikappa Bs, including Ikappa B-alpha , Ikappa B-beta , Ikappa B-epsilon , p105, and p100. NF-kappa B activation usually requires the phosphorylation-dependent ubiquitination and subsequent proteasome degradation of one or more Ikappa B molecules (29-32). Here we provide evidence that butyrate prevents the entrance of NF-kappa B into the nucleus triggered by TNF-alpha in part by suppressing cellular proteasome activity. Butyrate therefore influences the cell in a manner similar to the relatively new class of cancer chemotherapeutic and anti-inflammatory agents, the proteasome inhibitors (33-37).

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cell Culture and Treatments-- HT-29 cells were purchased from American Type Culture Collection (Manassas, VA) and were propagated in McCoy's 5A medium supplemented with 10% fetal bovine serum, nonessential amino acids, streptomycin (50 µg/ml), and penicillin (50 units/ml). All media were purchased from Life Technologies, Inc. TNF-alpha was purchased from R&D Systems (Minneapolis, MN) and used at a final concentration of 100 ng/ml. MG-132 was purchased from Calbiochem and used at a final concentration of 60 µM. Sodium butyrate was used at a final concentration of 4 mM. All butyrate treatments were performed for ~24 h prior to further manipulation of the cells. TSA, purchased from Calbiochem, was used at a 10 µM concentration with TSA preincubations also performed for ~24 h.

Electrophoretic Mobility Shift Assay (EMSA)-- Nuclear extracts were prepared based on a previously reported protocol with minor modifications as described in Inan et al. (25). For the DNA binding assays, double strand NF-kappa B DNA oligonucleotide (Promega, Madison, WI) were end-labeled with [gamma -32P] adenosine triphosphate (3000 Ci/mmol at 10 mCi/ml, Amersham Pharmacia Biotech) using T4 polynucleotide kinase. Binding reactions were performed by mixing 7.5 µg of nuclear extract (in 7.5 µl) with 2.5 µg of poly(dIdC) and 1 µg of bovine serum albumin to give a final volume of 14 µl. After a 15-min incubation on ice, 40 fmol of labeled oligonucleotide (1 µl) was added to each reaction mixture. Reaction mixtures were then transferred to room temperature for an additional 15 min. Reaction products were separated on a 4% polyacrylamide/Tris borate/EDTA gel and analyzed by autoradiography. EMSA and immunoblot images were scanned and quantified using NIH Image software. For supershift experiments, 1 µl (0.2 µg) of antibody was preincubated with cellular extract on ice for 30 min. After this preincubation, the binding reaction was performed as usual. The p65 C-20 and p50 N-19 supershifting antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Evidence for the specificity of the supershifting antibodies comes from the observation that they do not supershift nonspecific protein·DNA complexes observed on the EMSA gel, and they do not bind to nonspecific proteins on an immunoblot. In addition, antibodies against the other three NF-kappa B subunits do not supershift the TNF-alpha -activated NF-kappa B complexes from HT-29 cells, providing evidence that p65 and p50 are the predominant NF-kappa B subunits active in these cells (25). For deoxycholate activation (38, 39), 10 µg of cytosolic extract was first incubated with deoxycholate at a final concentration of 0.6% for 10 min at 4 °C. One microliter of 10% Nonidet P-40 was then added, and the incubation continued for another 10 min at 4 °C. After this pretreatment, the supershift assay or regular binding reaction was performed as usual.

Immunoblotting and Immunofluorescence-- For immunoblotting studies, 25 µg of cytoplasmic or nuclear protein (quantified by the Bio-Rad protein assay) was denatured under reducing conditions, separated on 10% SDS-polyacrylamide gels, and transferred to nitrocellulose by voltage gradient transfer. The resulting blots were blocked with 5% nonfat dry milk. Specific proteins were detected with appropriate antibodies using enhanced chemiluminescence detection (Santa Cruz Biotechnology) as recommended by the manufacturer. Immunoblotting antibodies against p52/p100 (K-27) and Ikappa B-alpha (C-21) were obtained from Santa Cruz Biotechnology. For immunofluorescence, HT-29 cells were grown on glass coverslips to 50-70% confluence. Following appropriate treatments, the cells were washed with cold phosphate-buffered saline and fixed with 100% methanol for 30 min at -20 °C. The methanol was removed, and permeabilizing reagent (0.3% Triton X-100, 2 mM EGTA, and 5 mM Pipes) was added to the surface of cells for 5 min at 4 °C. Goat serum (10%) diluted in phosphate-buffered saline was used to block nonspecific binding for 30 min following permeabilization. After blocking, the cells were incubated with a rabbit anti-p65 antibody (C-20, Santa Cruz Biotechnology) at a 1:200 dilution followed by incubation with a fluorescein isothiocyanate-conjugated anti-rabbit immunoglobulin (Jackson ImmunoResearch Laboratories, Inc.). Confocal imaging of cells was performed using a Bio-Rad MRC 600 microscope equipped with a 25-milliwatt krypton-argon laser.

Immunoprecipitation-- Cytosolic extract was prepared from control or butyrate-treated cells using a Nonidet P-40-containing lysis buffer (0.1% Nonidet P-40, 10 mM HEPES-KOH, pH 7.9, 10 mM KCl, 0.1 mM EDTA, 0.5 mM dithiothreitol, 2 mM MgCl2, 0.5 mM phenylmethylsulfonyl fluoride, 0.5 M sucrose, 2 µg/ml leupeptin, 10 µM clasto-lactacystin beta -lactone (Calbiochem)). Cytosolic extract (200 µl) was precleared with 10 µl of protein A-Sepharose (Amersham Pharmacia Biotech) and then incubated with 3 µl of an anti-ubiquitin rabbit antibody (Calbiochem) overnight on ice. To demonstrate that the anti-ubiquitin immunoprecipitation was specific, control immunoprecipitation reactions were performed with a rabbit antibody raised against Ikappa B-beta (N-20, Santa Cruz Biotechnology). Precipitation was performed by incubating extracts with 30 µl of protein A-Sepharose (3 h at 4 °C), centrifuging for 1 min at 12,000 × g, and then washing the pellet three times with ice cold lysis buffer. The pellets were then suspended in 60 µl of SDS gel loading buffer and analyzed by immunoblotting using an Ikappa B-alpha antibody (C-21, Santa Cruz Biotechnology).

Proteasome Activity Assay-- Proteasome activity in cytosolic extracts was quantified using the fluorogenic proteasome substrate Suc-LLVY-AMC (Calbiochem) (40). Cytosolic extract (10 µg of protein in 5 µl) was incubated in a 100-µl reaction containing 20 mM Tris-HCl (pH 7.8), 0.5 mM EDTA, 0.035% SDS, and 70 µM Suc-LLVY-AMC at room temperature for 30 min. Fluorescence was measured in a microtiter plate fluorometer (excitation, 360 nm; emission, 460 nm). Proteasome-dependent activity was determined by performing the assay in the presence of the proteasome inhibitor clasto-lactacystin beta -lactone (final concentration, 10 µM) (Calbiochem). Proteasome activity values shown were derived by subtracting the fluorescence obtained in the presence of this inhibitor from the values obtained in its absence. Assays were performed in triplicate, and statistical significance was determined with a paired Student's t test.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Butyrate Treatment Inhibits the TNF-alpha -induced Nuclear Translocation of NF-kappa B-- Previous reports have indicated that the treatment of HT-29 cells with butyrate for 24 h dampens their ability to activate NF-kappa B in response to TNF-alpha stimulation (25). NF-kappa B activation can be regulated at numerous steps, including its translocation into the nucleus. To determine whether nuclear translocation was being suppressed in butyrate-treated cells, the cellular localization of the NF-kappa B p65 subunit was determined by immunocytochemistry and confocal microscopy. As shown in Fig. 1A, treatment of HT-29 cells with TNF-alpha alone resulted in the translocation of some of the cellular p65 into the nucleus. As has been described by other groups, p65 translocation into the nucleus is only partial in stimulated HT-29 cells and a large fraction of NF-kappa B remains in the cytoplasm (41). Treatment of the HT-29 cells with 4 mM butyrate for 24 h prior to TNF-alpha stimulation prevented much of the TNF-alpha -induced nuclear translocation of the p65 subunit (Fig. 1A). To confirm these data, a cell fractionation study was performed (Fig. 1B). In unstimulated cells, most of the cellular p65 was found in the cytosolic fraction of HT-29 cells. After TNF-alpha stimulation of HT-29 cells, a small fraction of the total cellular p65 appeared in the nuclear fraction. Consistent with the immunocytochemical analysis, pretreatment of cells with butyrate reduced the nuclear translocation of p65 into the nucleus by ~50%. The inhibition of nuclear translocation is not observed for Ikappa B-alpha or Ikappa B-beta , two proteins that can also shuttle between the cytoplasm and nucleus (Ref. 25 and data not shown). These data indicate that butyrate inhibits a step in NF-kappa B activation at or prior to nuclear translocation.


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  		Fig. 1.   A, subcellular localization of the NF-kappa B p65 subunit in HT-29 cells as determined by immunocytochemistry and confocal microscopy. HT-29 cells were preincubated with 4 mM butyrate for ~24 h and stimulated with TNF-alpha (TNF) for 30 min as indicated. Cell treatments were performed as described under "Experimental Procedures." In all panels a 400 × magnification is shown. B, subcellular localization of the NF-kappa B p65 subunit in HT-29 cells as determined by cellular fractionation. HT-29 cells were treated as described in A. Cellular proteins (25 µg) were then fractionated into cytoplasmic and nuclear fractions, which were then analyzed by immunoblotting. Proteins from control cells (C), TNF-alpha -treated cells (T), butyrate-treated cells (B), or cells treated with TNF-alpha and butyrate (T/B) are shown.

Fig. 2A shows the influence of butyrate on the NF-kappa B DNA binding activity induced by TNF-alpha . The butyrate inhibition of NF-kappa B nuclear translocation (shown in Fig. 1) corresponded with a decrease in its DNA binding activity (with an estimated inhibition of 75%). The time course analysis shown in Fig. 2A revealed a consistent level of inhibition, consistent with a model in which butyrate suppresses the movement of NF-kappa B from the cytoplasm to the nucleus (as opposed to an increase in the rate of NF-kappa B nuclear export). Also shown in Fig. 2B is a DNA competition analysis, which demonstrated that the protein·DNA complex inhibited by butyrate is a specific NF-kappa B complex. The butyrate suppression of NF-kappa B DNA binding activity is reflected in a reduced level of NF-kappa B-regulated gene expression (24, 25).


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  		Fig. 2.   A, butyrate suppresses NF-kappa B activation at early and late time points after TNF-alpha treatment. HT-29 cells were pretreated with or without butyrate (4 mM) as described under "Experimental Procedures" and then stimulated by TNF-alpha for the indicated lengths of time. EMSA analysis was then performed to measure NF-kappa B DNA binding activity. The two NF-kappa B complexes formed in HT-29 cells (p65·p50 and p50·p50) are indicated. B, a DNA competition reaction to show that the NF-kappa B DNA binding activity induced by TNF-alpha is specific. Nuclear extract prepared from TNF-alpha -stimulated HT-29 cells was incubated with a 50-fold excess of cold competitor DNA containing an NF-kappa B or AP-1 binding site (as indicated). Competition for the radioactive NF-kappa B oligonucleotide binding is observed only with the cold NF-kappa B oligonucleotide.

Butyrate Influences Cellular Ikappa B Composition-- A number of different Ikappa B molecules control NF-kappa B activity in HT-29 cells, and it is possible that butyrate is suppressing NF-kappa B nuclear translocation by altering the Ikappa B composition of the cytoplasm. To determine whether this was the case, we first characterized the Ikappa B·NF-kappa B complexes extracted from control and butyrate-treated cells. The Ikappa B·NF-kappa B complexes can be disrupted in vitro by incubation of cytosolic extracts with deoxycholate (38, 39). The level of NF-kappa B DNA binding activity activated in vitro by deoxycholate was consistently lower in extracts from butyrate-treated cells (Fig. 3A). The identity of the deoxycholate-activated DNA binding activity as NF-kappa B was confirmed by a supershift reaction using antibodies that have previously been shown to specifically recognize the p65 and p50 NF-kappa B subunits (Fig. 3A). Fig. 3B shows that the level of p65 was the same in butyrate-treated and control cells. These data suggest that there is an alteration in the composition of Ikappa B·NF-kappa B complexes in butyrate-treated cells with butyrate inducing the formation of complexes that are relatively resistant to deoxycholate dissociation.


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  		Fig. 3.   Top panel, characterization of NF-kappa B·Ikappa B complexes in the cytoplasm of control and butyrate-treated HT-29 cells by in vitro deoxycholate activation. Cytosolic extracts were activated with 1% deoxycholate and then tested for DNA binding activity by EMSA. Extracts were prepared from butyrate or control cells as indicated. Anti-p65 or anti-p50 antibodies were included in the binding reaction for supershift analysis (as indicated). Bottom panel, immunoblotting assay shows the level of the p65 subunit found in the cytoplasm of the control and butyrate-treated HT-29 cells used for deoxycholate activation.

To determine how Ikappa B·NF-kappa B complexes might be altered in butyrate-treated HT-29 cells, we determined the expression levels of a number of known Ikappa Bs by immunoblotting. We were unable to detect increased expression of Ikappa B-beta , Ikappa B-epsilon , or p105 (data not shown). The p100 Ikappa B, however, was increased in HT-29 cells treated with butyrate, and it was not degraded upon TNF-alpha treatment (Fig. 4A). The resistance of p100 to TNF-alpha -induced degradation has been reported in other cell types as well (42). Ikappa B-alpha was also influenced by butyrate treatment. In control cells, Ikappa B-alpha was completely degraded in response to TNF-alpha (at 15 min), whereas its degradation was dampened in butyrate-treated cells (Fig. 4A). In addition, butyrate treatment of HT-29 cells caused a number of larger proteins to appear on the Ikappa B-alpha immunoblot (marked with an asterisk in Fig. 4A). One possibility is that some of these larger bands are ubiquitinated forms of Ikappa B-alpha , which could appear as a result of decreased proteasome activity. An overlapping set of bands also appeared in cells treated with the MG-132 proteasome inhibitor (although intensities of the individual bands differ). Additional evidence for the accumulation of ubiquitinated Ikappa B-alpha after butyrate treatment was obtained by performing an immunoprecipitation experiment (Fig. 4B). An anti-ubiquitin antibody precipitated more Ikappa B-alpha from butyrate-treated cells than from control cells (relative to the amount of unconjugated Ikappa B-alpha in the extract, Fig. 4B). The finding that an antibody against Ikappa B-beta could not precipitate Ikappa B-alpha -reactive proteins under the same immunoprecipitation conditions supports the specificity of the anti-ubiquitin immunoprecipitation. These results suggested that butyrate may be interfering with proteasomal degradation of Ikappa B-alpha .


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  		Fig. 4.   A, the butyrate influence on p100 and Ikappa B-alpha levels in HT-29 cells. Cells were pretreated with 4 mM butyrate and then stimulated by TNF-alpha for the indicated lengths of time. Cytosolic proteins were then analyzed by immunoblotting for p100 and Ikappa B-alpha as indicated. Also shown are the results of an experiment in which HT-29 cells were pretreated with the proteasome inhibitor MG-132 prior to TNF-alpha treatment. The asterisk indicates larger proteins that bind to the Ikappa B-alpha antibody in butyrate- and MG-132-treated cells. B, immunoprecipitation to determine the levels of ubiquitinated Ikappa B-alpha in control and butyrate-treated cells. The top panel is an immunoblot that shows the Ikappa B-alpha levels in the cytosolic extracts of control and butyrate-treated cells. These extracts were immunoprecipitated with an anti-ubiquitin antibody (+ anti-Ub). The immunoprecipitates were then analyzed by immunoblotting with an anti-Ikappa B-alpha antibody (bottom panel). The right-most panel of B shows the results of a control immunoprecipitation reaction performed with an anti-Ikappa B-beta antibody. The Ikappa B-beta antibody does not precipitate Ikappa B-alpha -reactive proteins, indicating that the precipitation reaction performed with the anti-ubiquitin antibody is specific.

Butyrate Suppresses Proteasome Activity in the Cell-- To determine whether butyrate was affecting proteasome activity in the cell, cytosolic extracts prepared from control and butyrate-treated HT-29 cells were tested for proteasome activity using the synthetic substrate Suc-LLVY-AMC (40). As shown in Fig. 5A, butyrate treatment decreased proteasome activity in the extract by ~40%. Since a decrease in proteasome activity may generally increase the presence of polyubiquitinated proteins in the cell, a blot of cytosolic proteins from control and butyrate-treated cells was probed with an anti-ubiquitin antibody (Fig. 5B). Exposure of the HT-29 cells to butyrate resulted in the accumulation of ubiquitin-conjugated proteins. These data indicate butyrate suppresses proteasome activity in HT-29 cells.


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  		Fig. 5.   A, proteasome activity was suppressed in HT-29 cells after exposure to butyrate. Cell lysates were prepared from untreated (0), butyrate-treated (BA), or TNF-alpha -treated (TNF) HT-29 cells and analyzed for proteasome activity. The butyrate reduction of proteasome activity was significant (**, p < 0.01). B, ubiquitinated proteins found in the extracts of control, butyrate (BA)- and TNF-alpha (TNF)-treated cells. The level of protein ubiquitination in these extracts was determined by immunoblotting with an anti-ubiquitin antibody. Duplicate samples were analyzed. C, MG-132 suppressed NF-kappa B activation induced by TNF-alpha in HT-29 cells. The cells were incubated with MG-132 followed by treatment with TNF-alpha for 30 min (as indicated). EMSA was then performed to measure NF-kappa B DNA binding activity. Duplicate samples were analyzed.

Since NF-kappa B activation in the absence of Ikappa B degradation has been reported (41, 43), we determined whether proteasome activity was required for NF-kappa B activation by TNF-alpha in HT-29 cells. As shown in Fig. 5C, the MG-132 proteasome inhibitor was able to suppress both TNF-alpha -induced NF-kappa B activation (Fig. 5C) and Ikappa B-alpha degradation (Fig. 4A). The ability of butyrate to suppress proteasome activity could therefore contribute to the inhibition of NF-kappa B activation by TNF-alpha .

Deacetylase Inhibition and NF-kappa B Activation-- Butyrate influences a number of cellular activities, including ion transport, glutathione synthesis, reactive oxygen generation, and protein acetylation (16, 46-50). We have reported previously that the influence of butyrate on NF-kappa B activation arises from its ability to inhibit histone deacetylases (25). We therefore determined whether the effects of butyrate on p100 expression, Ikappa B-alpha degradation, and proteasome activity were also obtained with the deacetylase inhibitor TSA. As shown in Fig. 6, TSA was able to inhibit proteasome activity (Fig. 6A), suppress TNF-alpha -induced Ikappa B-alpha degradation (Fig. 6B), and increase p100 expression (Fig. 6B). These data are consistent with a model in which the influence of butyrate on NF-kappa B activation and proteasome activity derives from its ability to inhibit histone deacetylases. It should be noted, however, that butyrate and TSA might be accomplishing these outcomes through different molecular mechanisms.


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  		Fig. 6.   A, the influence of the histone deacetylase inhibitor TSA and butyrate (BA) on the proteasome activity in HT-29 cells. Cells were pretreated with TSA or butyrate for ~24 h. Proteasome activity in cell extracts was then determined. The reduction of proteasome activity by TSA and butyrate was found to be significant (**, p < 0.01). B, immunoblotting shows that TSA also increased p100 levels (top panel) and suppressed Ikappa B-alpha degradation and enhanced the formation of larger Ikappa B-alpha forms (bottom panel). The bracket on the bottom panel indicates the position of the larger Ikappa B-alpha forms.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cells of the gastrointestinal tract constantly interact with the contents of the lumen. These contents range from food and bacterial antigens to pathogenic and nonpathogenic bacteria. Bacteria in the gut also produce a number of metabolites and products that can be either beneficial or harmful, such as vitamin K and lipopolysaccharides. For the proper functioning of the gastrointestinal tract, cells must sense and respond appropriately to the contents of the lumen. One molecule found at relatively high concentrations in the colonic lumen is butyrate. Butyrate is an energy molecule for colonic epithelial cells, serving as a substrate for mitochondrial beta -oxidation pathways. Evidence is also accumulating that colonic epithelial cells sense butyrate directly and adjust gene expression and signal transduction pathways in response to its presence (51-58). These changes in signal transduction and gene expression impact colonocyte proliferation, differentiation, and death. Evidence has also been obtained that the influence of butyrate on gene expression and signal transduction serves to suppress inflammation in the colon. For example, epithelial cells in inflamed colonic tissue are often found to be deficient for butyrate metabolism (59-61). Moreover, direct application of butyrate to inflamed tissue by irrigation can be an effective means of suppressing intestinal inflammation (14, 15). Determining how butyrate influences inflammatory signal transduction pathways and gene expression may therefore provide insight into how inflammatory responses are regulated in the colon.

We have been studying how the NF-kappa B signal transduction pathway is influenced by butyrate in the HT-29 cell culture model. Our analysis indicates that butyrate influences NF-kappa B in part by preventing the complete degradation of Ikappa B-alpha by reducing proteasome activity in the cell. Whether this is the only mechanism by which butyrate influences NF-kappa B activation is not clear. We also found that the levels of the p100 Ikappa B are increased upon butyrate treatment, but it is not clear whether the butyrate reduction in proteasome activity is solely responsible for the accumulation of p100. Our findings suggest that alterations in the cellular proteasome activity may influence the sensitivity of the colon to inflammatory stimuli.

Although this report is the first to demonstrate the influence of butyrate on cellular proteasome activity, other groups have reported that butyrate stabilizes Ikappa B-alpha in macrophages and nonepithelial cells isolated from colonic lamina propria (22, 23). The increase in Ikappa B-alpha stability reported by other groups may also be the result of a lower proteasome activity in butyrate-treated cells. Interestingly, NF-kappa B inhibition by butyrate has been observed in Caco-2 cells without any apparent change in Ikappa B-alpha degradation (24). Butyrate does, however, increase Ikappa B-beta levels in Caco-2 cells without affecting Ikappa B-beta mRNA levels (24). This response may be due in part to an enhanced stabilization of the Ikappa B-beta protein through a decrease in cellular proteasome activity. In a number of instances, NF-kappa B activation has been found to occur in the absence of Ikappa B-alpha degradation by the proteasome. These proteasome-independent pathways for NF-kappa B activation have been observed in some colon cell lines (43), and we predict that these pathways would not be inhibited by butyrate.

There are a number of potential mechanisms by which butyrate could influence the proteasome. The core mammalian 20 S proteasome has four stacked rings, each containing seven subunits (62). The two inner rings are each composed of seven distinct alpha  subunits, and the outer rings are each composed of seven distinct beta  subunits. In addition, there are genes that encode three nonessential beta  subunits, two of which are interferon-gamma -inducible. Butyrate may suppress proteasome activity by reducing the expression of one or more of these 17 core subunits (63, 64). This possibility seems feasible given that the actions of butyrate appear to be mediated through its ability to inhibit deacetylases: histone deacetylation can have a multifaceted impact on cellular gene expression. However, the post-transcriptional modulation of proteasome activity is also possible. Regardless of the exact mechanism of the activity of butyrate, the identification of the proteasome as a potential target of butyrate is interesting from a pharmacological viewpoint since proteasome inhibitors represent a promising new class of anti-inflammatory and anticancer drugs (33, 65, 67). These activities have long been associated with butyrate (14, 15) and butyrogenic dietary fiber (5, 6, 68-71).

Although our studies have focused of NF-kappa B regulation, the decrease in proteasome activity we describe here could impact a number of colonocyte and colon cancer cell activities. For example, many proteins that regulate the cell cycle (e.g. cyclins and beta -catenin) (72-76) and cell death (e.g. p53) are regulated by the ubiquitin-proteasome pathway (72, 77-79, 81-83). The butyrate suppression of proteasome activity might therefore contribute to the alterations in colonocyte proliferation observed in animals fed a high fiber diet (66, 68, 69, 80, 84, 85) as well as the ability of butyrate to induce colon cancer cell apoptosis (9, 45). The proteasome is also involved in antigen processing, so the suppression of cellular proteasome activity by butyrate might impact the antigen-presenting function of colonic epithelial cells (44). Exactly how extensive these and other processes are impacted by a decreased proteasome activity remains to be determined.

    FOOTNOTES

* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: Dept. of Molecular and Cell Biology, 75 North Eagleville Rd., U-125, University of Connecticut, Storrs, CT 06269. Tel.: 860-486-0454; Fax: 860-486-4331; E-mail: Giardina@uconnvm.uconn.edu.

Published, JBC Papers in Press, September 25, 2001, DOI 10.1074/jbc.M105170200

    ABBREVIATIONS

The abbreviations used are: TSA, trichostatin A; TNF-alpha , tumor necrosis factor-alpha ; NF-kappa B, nuclear factor-kappa B; Ikappa B, inhibitor-kappa B; EMSA, electrophoretic mobility shift assay; Pipes, 1,4-piperazinediethanesulfonic acid; Suc-LLVY-AMC, N-succinyl-Leu-Leu-Val-Tyr-7-amido-4-methylcoumarin.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1. Velazquez, O. C., and Rombeau, J. L. (1997) Adv. Exp. Med. Biol. 427, 169-181
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