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• W2008431663 abstract "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-α (TNF-α)-induced nuclear translocation of the proinflammatory transcription factor NF-κB. Butyrate inhibited NF-κB DNA binding within 30 min of TNF-α stimulation, consistent with an inhibition of nuclear translocation. IκB·NF-κB complexes extracted from butyrate-treated cells were relatively resistant to in vitro dissociation by deoxycholate, suggesting a change in cellular IκB composition. Butyrate treatment increased p100 expression, an IκB that was not degraded upon TNF-α treatment. Butyrate also reduced the extent of TNF-α-induced IκB-α degradation and enhanced the presence of ubiquitin-conjugated IκB-α. The suppression of IκB-α 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 IκB-α 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. 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-α (TNF-α)-induced nuclear translocation of the proinflammatory transcription factor NF-κB. Butyrate inhibited NF-κB DNA binding within 30 min of TNF-α stimulation, consistent with an inhibition of nuclear translocation. IκB·NF-κB complexes extracted from butyrate-treated cells were relatively resistant to in vitro dissociation by deoxycholate, suggesting a change in cellular IκB composition. Butyrate treatment increased p100 expression, an IκB that was not degraded upon TNF-α treatment. Butyrate also reduced the extent of TNF-α-induced IκB-α degradation and enhanced the presence of ubiquitin-conjugated IκB-α. The suppression of IκB-α 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 IκB-α 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. trichostatin A tumor necrosis factor-α nuclear factor-κB inhibitor-κB electrophoretic mobility shift assay 1,4-piperazinediethanesulfonic acid N-succinyl-Leu-Leu-Val-Tyr-7-amido-4-methylcoumarin 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 (1Velazquez O.C. Rombeau J.L. Adv. Exp. Med. Biol. 1997; 427: 169-181Crossref PubMed Scopus (31) Google Scholar, 2Hague A. Butt A.J. Paraskeva C. Proc. Nutr. Soc. 1996; 55: 937-943Crossref PubMed Scopus (113) Google Scholar, 3Hodin R. Gastroenterology. 2000; 118: 798-801Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar, 4Velazquez O.C. Lederer H.M. Rombeau J.L. Adv. Exp. Med. Biol. 1997; 427: 123-134Crossref PubMed Scopus (174) Google Scholar, 5Velazquez O.C. Lederer H.M. Rombeau J.L. Dig. Dis. Sci. 1996; 41: 727-739Crossref PubMed Scopus (148) Google Scholar). Colonocytes utilize short chain fatty acids as their primary energy source, and evidence has been obtained that they undergo apoptosis in its absence (2Hague A. Butt A.J. Paraskeva C. Proc. Nutr. Soc. 1996; 55: 937-943Crossref PubMed Scopus (113) Google Scholar, 5Velazquez O.C. Lederer H.M. Rombeau J.L. Dig. Dis. Sci. 1996; 41: 727-739Crossref PubMed Scopus (148) Google Scholar, 6Hassig C.A. Tong J.K. Schreiber S.L. Chem. Biol. 1997; 4: 783-789Abstract Full Text PDF PubMed Scopus (82) Google Scholar, 7Luciano L. Hass R. Busche R. von Engelhardt W. Reale E. Cell Tissue Res. 1996; 286: 81-92Crossref PubMed Scopus (41) Google Scholar). Interestingly, short chain fatty acids, particularly butyrate, have the opposite effect on transformed cells in culture, inducing rather than suppressing apoptosis (2Hague A. Butt A.J. Paraskeva C. Proc. Nutr. Soc. 1996; 55: 937-943Crossref PubMed Scopus (113) Google Scholar,8Avivi-Green C. Polak-Charcon S. Madar Z. Schwartz B. Oncol. Res. 2000; 12: 83-95Crossref PubMed Scopus (95) Google Scholar, 9Hague A. Manning A.M. Hanlon K.A. Huschtscha L.I. Hart D. Paraskeva C. Int. J. Cancer. 1993; 55: 498-505Crossref PubMed Scopus (529) Google Scholar, 10Thomas G.L. Henley A. Rowland T.C. Sahai A. Griffin M. Birckbichler P.J. In Vitro Cell Dev. Biol. Anim. 1996; 32: 505-513Crossref PubMed Scopus (15) Google Scholar). The ability of butyrate to induce cancer cell apoptosis may contribute to the cancer preventive activities of dietary fiber (1Velazquez O.C. Rombeau J.L. Adv. Exp. Med. Biol. 1997; 427: 169-181Crossref PubMed Scopus (31) Google Scholar, 5Velazquez O.C. Lederer H.M. Rombeau J.L. Dig. Dis. Sci. 1996; 41: 727-739Crossref PubMed Scopus (148) Google Scholar,6Hassig C.A. Tong J.K. Schreiber S.L. Chem. Biol. 1997; 4: 783-789Abstract Full Text PDF PubMed Scopus (82) Google Scholar, 11Whiteley L.O. Klurfeld D.M. Nutr. Cancer. 2000; 36: 131-149Crossref PubMed Scopus (29) Google Scholar, 12Csordas A. Eur. J. Cancer Prev. 1996; 5: 221-231Crossref PubMed Scopus (69) Google Scholar). Short chain fatty acids can also suppress intestinal inflammation: butyrate is effective for treating selected inflammatory conditions of the distal alimentary tract (13Kanauchi O. Iwanaga T. Mitsuyama K. Saiki T. Tsuruta O. Noguchi K. Toyonaga A. J. Gastroenterol. Hepatol. 1999; 14: 880-888Crossref PubMed Scopus (51) Google Scholar, 14Scheppach W. Sommer H. Kirchner T. Paganelli G.M. Bartram P. Christl S. Richter F. Dusel G. Kasper H. Gastroenterology. 1992; 103: 51-56Abstract Full Text PDF PubMed Google Scholar, 15Wachtershauser A. Stein J. Eur. J. Nutr. 2000; 39: 164-171Crossref PubMed Scopus (202) Google Scholar). 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 (16Boffa L.C. Vidali G. Mann R.S. Allfrey V.G. J. Biol. Chem. 1978; 253: 3364-3366Abstract Full Text PDF PubMed Google Scholar, 17Sealy L. Chalkley R. Cell. 1978; 14: 115-121Abstract Full Text PDF PubMed Scopus (559) Google Scholar). 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) (18Liu L. Scolnick D.M. Trievel R.C. Zhang H.B. Marmorstein R. Halazonetis T.D. Berger S.L. Mol. Cell. Biol. 1999; 19: 1202-1209Crossref PubMed Scopus (655) Google Scholar, 19Gu W. Roeder R.G. Cell. 1997; 90: 595-606Abstract Full Text Full Text PDF PubMed Scopus (2189) Google Scholar, 20Boyes J. Byfield P. Nakatani Y. Ogryzko V. Nature. 1998; 396: 594-598Crossref PubMed Scopus (633) Google Scholar, 21Luo J. Su F. Chen D. Shiloh A. Gu W. Nature. 2000; 408: 377-381Crossref PubMed Scopus (693) Google Scholar). We and others have reported that butyrate can modulate the activity of the transcription factor NF-κB in a number of different cell types including colon cancer cell lines, cells isolated from the lamina propria of the colon, and macrophages (22Chakravortty D. Koide N. Kato Y. Sugiyama T. Mu M.M. Yoshida T. Yokochi T. J. Endotoxin Res. 2000; 6: 243-247Crossref PubMed Scopus (66) Google Scholar, 23Segain J.P. Raingeard de la Bletiere D. Bourreille A. Leray V. Gervois N. Rosales C. Ferrier L. Bonnet C. Blottiere H.M. Galmiche J.P. Gut. 2000; 47: 397-403Crossref PubMed Scopus (964) Google Scholar, 24Wu G.D. Huang N. Wen X. Keilbaugh S.A. Yang H. J. Leukoc. Biol. 1999; 66: 1049-1056Crossref PubMed Scopus (33) Google Scholar, 25Inan M.S. Rasoulpour R.J. Yin L. Hubbard A.K. Rosenberg D.W. Giardina C. Gastroenterology. 2000; 118: 724-734Abstract Full Text Full Text PDF PubMed Scopus (382) Google Scholar). The ability of butyrate to modulate NF-κB 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-κB activity and that propionate, a weaker deacetylase inhibitor, is less effective than butyrate in altering NF-κB activity (25Inan M.S. Rasoulpour R.J. Yin L. Hubbard A.K. Rosenberg D.W. Giardina C. Gastroenterology. 2000; 118: 724-734Abstract Full Text Full Text PDF PubMed Scopus (382) Google Scholar). The ability of butyrate to modulate NF-κB activity resonates with its proposed cancer chemopreventive and anti-inflammatory activities since NF-κB 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-κB 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-κB activation in the human colon-derived HT-29 cell line. NF-κB is regulated through the binding of inhibitory molecules referred to collectively as IκBs (26Baeuerle P.A. Cell. 1998; 95: 729-731Abstract Full Text Full Text PDF PubMed Scopus (418) Google Scholar, 27Gilmore T.D. Koedood M. Piffat K.A. White D.W. Oncogene. 1996; 13: 1367-1378PubMed Google Scholar, 28Gerondakis S. Grossmann M. Nakamura Y. Pohl T. Grumont R. Oncogene. 1999; 18: 6888-6895Crossref PubMed Scopus (273) Google Scholar). There are a number of IκBs, including IκB-α, IκB-β, IκB-ε, p105, and p100. NF-κB activation usually requires the phosphorylation-dependent ubiquitination and subsequent proteasome degradation of one or more IκB molecules (29Hatakeyama S. Kitagawa M. Nakayama K. Shirane M. Matsumoto M. Hattori K. Higashi H. Nakano H. Okumura K. Onoe K. Good R.A. Nakayama K. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3859-3863Crossref PubMed Scopus (182) Google Scholar, 30Crinelli R. Bianchi M. Gentilini L. Magnani M. Hiscott J. Eur. J. Biochem. 1999; 263: 202-211Crossref PubMed Scopus (5) Google Scholar, 31Krappmann D. Wulczyn F.G. Scheidereit C. EMBO J. 1996; 15: 6716-6726Crossref PubMed Scopus (180) Google Scholar, 32Miyamoto S. Seufzer B.J. Shumway S.D. Mol. Cell. Biol. 1998; 18: 19-29Crossref PubMed Google Scholar). Here we provide evidence that butyrate prevents the entrance of NF-κB into the nucleus triggered by TNF-α 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 (33Adams J. Palombella V.J. Elliott P.J. Investig. New Drugs. 2000; 18: 109-121Crossref PubMed Scopus (240) Google Scholar, 34Hallahan D.E. Teng M. Int. J. Radiat. Oncol. Biol. Phys. 2000; 47: 859-860Abstract Full Text Full Text PDF PubMed Scopus (6) Google Scholar, 35Meng L. Mohan R. Kwok B.H. Elofsson M. Sin N. Crews C.M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 10403-10408Crossref PubMed Scopus (827) Google Scholar, 36Phillips J.B. Williams A.J. Adams J. Elliott P.J. Tortella F.C. Stroke. 2000; 31: 1686-1693Crossref PubMed Scopus (176) Google Scholar, 37Ciechanover A. Orian A. Schwartz A.L. J. Cell. Biochem. Suppl. 2000; 34: 40-51Crossref PubMed Scopus (246) Google Scholar). 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-α 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. Nuclear extracts were prepared based on a previously reported protocol with minor modifications as described in Inan et al. (25Inan M.S. Rasoulpour R.J. Yin L. Hubbard A.K. Rosenberg D.W. Giardina C. Gastroenterology. 2000; 118: 724-734Abstract Full Text Full Text PDF PubMed Scopus (382) Google Scholar). For the DNA binding assays, double strand NF-κB DNA oligonucleotide (Promega, Madison, WI) were end-labeled with [γ-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-κB subunits do not supershift the TNF-α-activated NF-κB complexes from HT-29 cells, providing evidence that p65 and p50 are the predominant NF-κB subunits active in these cells (25Inan M.S. Rasoulpour R.J. Yin L. Hubbard A.K. Rosenberg D.W. Giardina C. Gastroenterology. 2000; 118: 724-734Abstract Full Text Full Text PDF PubMed Scopus (382) Google Scholar). For deoxycholate activation (38Baeuerle P.A. Baltimore D. Science. 1988; 242: 540-546Crossref PubMed Scopus (1689) Google Scholar, 39Baeuerle P.A. Baltimore D. Genes Dev. 1989; 3: 1689-1698Crossref PubMed Scopus (368) Google Scholar), 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. 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 IκB-α (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. 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 mmdithiothreitol, 2 mm MgCl2, 0.5 mmphenylmethylsulfonyl fluoride, 0.5 m sucrose, 2 μg/ml leupeptin, 10 μm clasto-lactacystin β-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 IκB-β (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 IκB-α antibody (C-21, Santa Cruz Biotechnology). Proteasome activity in cytosolic extracts was quantified using the fluorogenic proteasome substrate Suc-LLVY-AMC (Calbiochem) (40Rock K.L. Gramm C. Rothstein L. Clark K. Stein R. Dick L. Hwang D. Goldberg A.L. Cell. 1994; 78: 761-771Abstract Full Text PDF PubMed Scopus (2206) Google Scholar). 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 inhibitorclasto-lactacystin β-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. Previous reports have indicated that the treatment of HT-29 cells with butyrate for 24 h dampens their ability to activate NF-κB in response to TNF-α stimulation (25Inan M.S. Rasoulpour R.J. Yin L. Hubbard A.K. Rosenberg D.W. Giardina C. Gastroenterology. 2000; 118: 724-734Abstract Full Text Full Text PDF PubMed Scopus (382) Google Scholar). NF-κB 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-κB p65 subunit was determined by immunocytochemistry and confocal microscopy. As shown in Fig.1 A, treatment of HT-29 cells with TNF-α 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-κB remains in the cytoplasm (41Jobin C. Haskill S. Mayer L. Panja A. Sartor R.B. J. Immunol. 1997; 158: 226-234PubMed Google Scholar). Treatment of the HT-29 cells with 4 mm butyrate for 24 h prior to TNF-α stimulation prevented much of the TNF-α-induced nuclear translocation of the p65 subunit (Fig. 1 A). To confirm these data, a cell fractionation study was performed (Fig.1 B). In unstimulated cells, most of the cellular p65 was found in the cytosolic fraction of HT-29 cells. After TNF-α 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 IκB-α or IκB-β, two proteins that can also shuttle between the cytoplasm and nucleus (Ref. 25Inan M.S. Rasoulpour R.J. Yin L. Hubbard A.K. Rosenberg D.W. Giardina C. Gastroenterology. 2000; 118: 724-734Abstract Full Text Full Text PDF PubMed Scopus (382) Google Scholar and data not shown). These data indicate that butyrate inhibits a step in NF-κB activation at or prior to nuclear translocation. Fig. 2 A shows the influence of butyrate on the NF-κB DNA binding activity induced by TNF-α. The butyrate inhibition of NF-κB 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.2 A revealed a consistent level of inhibition, consistent with a model in which butyrate suppresses the movement of NF-κB from the cytoplasm to the nucleus (as opposed to an increase in the rate of NF-κB nuclear export). Also shown in Fig. 2 B is a DNA competition analysis, which demonstrated that the protein·DNA complex inhibited by butyrate is a specific NF-κB complex. The butyrate suppression of NF-κB DNA binding activity is reflected in a reduced level of NF-κB-regulated gene expression (24Wu G.D. Huang N. Wen X. Keilbaugh S.A. Yang H. J. Leukoc. Biol. 1999; 66: 1049-1056Crossref PubMed Scopus (33) Google Scholar, 25Inan M.S. Rasoulpour R.J. Yin L. Hubbard A.K. Rosenberg D.W. Giardina C. Gastroenterology. 2000; 118: 724-734Abstract Full Text Full Text PDF PubMed Scopus (382) Google Scholar). A number of different IκB molecules control NF-κB activity in HT-29 cells, and it is possible that butyrate is suppressing NF-κB nuclear translocation by altering the IκB composition of the cytoplasm. To determine whether this was the case, we first characterized the IκB·NF-κB complexes extracted from control and butyrate-treated cells. The IκB·NF-κB complexes can be disrupted in vitro by incubation of cytosolic extracts with deoxycholate (38Baeuerle P.A. Baltimore D. Science. 1988; 242: 540-546Crossref PubMed Scopus (1689) Google Scholar,39Baeuerle P.A. Baltimore D. Genes Dev. 1989; 3: 1689-1698Crossref PubMed Scopus (368) Google Scholar). The level of NF-κB DNA binding activity activated in vitro by deoxycholate was consistently lower in extracts from butyrate-treated cells (Fig.3 A). The identity of the deoxycholate-activated DNA binding activity as NF-κB was confirmed by a supershift reaction using antibodies that have previously been shown to specifically recognize the p65 and p50 NF-κB subunits (Fig.3 A). Fig. 3 B 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 IκB·NF-κB complexes in butyrate-treated cells with butyrate inducing the formation of complexes that are relatively resistant to deoxycholate dissociation. To determine how IκB·NF-κB complexes might be altered in butyrate-treated HT-29 cells, we determined the expression levels of a number of known IκBs by immunoblotting. We were unable to detect increased expression of IκB-β, IκB-ε, or p105 (data not shown). The p100 IκB, however, was increased in HT-29 cells treated with butyrate, and it was not degraded upon TNF-α treatment (Fig.4 A). The resistance of p100 to TNF-α-induced degradation has been reported in other cell types as well (42Dejardin E. Deregowski V. Chapelier M. Jacobs N. Gielen J. Merville M.P. Bours V. Oncogene. 1999; 18: 2567-2577Crossref PubMed Scopus (96) Google Scholar). IκB-α was also influenced by butyrate treatment. In control cells, IκB-α was completely degraded in response to TNF-α (at 15 min), whereas its degradation was dampened in butyrate-treated cells (Fig. 4 A). In addition, butyrate treatment of HT-29 cells caused a number of larger proteins to appear on the IκB-α immunoblot (marked with an asterisk in Fig. 4 A). One possibility is that some of these larger bands are ubiquitinated forms of IκB-α, 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 IκB-α after butyrate treatment was obtained by performing an immunoprecipitation experiment (Fig. 4 B). An anti-ubiquitin antibody precipitated more IκB-α from butyrate-treated cells than from control cells (relative to the amount of unconjugated IκB-α in the extract, Fig. 4 B). The finding that an antibody against IκB-β could not precipitate IκB-α-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 IκB-α. 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 (40Rock K.L. Gramm C. Rothstein L. Clark K. Stein R. Dick L. Hwang D. Goldberg A.L. Cell. 1994; 78: 761-771Abstract Full Text PDF PubMed Scopus (2206) Google Scholar). As shown in Fig.5 A, 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. 5 B). 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. Since NF-κB activation in the absence of IκB degradation has been reported (41Jobin C. Haskill S. Mayer L. Panja A. Sartor R.B. J. Immunol. 1997; 158: 226-234PubMed Google Scholar, 43Wilson L. Szabo C. Salzman A.L. Gastroenterology. 1999; 117: 106-114Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar), we determined whether proteasome activity was required for NF-κB activation by TNF-α in HT-29 cells. As shown in Fig. 5 C, the MG-132 proteasome inhibitor was able to suppress both TNF-α-induced NF-κB activation (Fig. 5 C) and IκB-α degradation (Fig. 4 A). The ability of butyrate to suppress proteasome activity could therefore contribute to the inhibition of NF-κB activation by TNF-α. Butyrate influences a number of cellular activities, including ion transport, glutathione synthesis, reactive oxygen generation, and protein acetylation (16Boffa L.C. Vidali G. Mann R.S. Allfrey V.G. J. Biol. Chem. 1978; 253: 3364-3366Abstract Full Text PDF PubMed Google Scholar, 46Umesaki Y. Yajima T. Yokokura T. Mutai M. Pfluegers Arch. 1979; 379: 43-47Crossref PubMed Scopus (101) Google Scholar, 47Edmonds C.J. Philos. Trans. R Soc. Lond. B Biol. Sci. 1982; 299: 575-584Crossref PubMed Scopus (5) Google Scholar, 48Binder H.J. Mehta P. Gastroenterology. 1989; 96: 989-996Abstract Full Text PDF PubMed Google Scholar, 49Benard O. Balasubramanian K.A. Mol. Cell. Biochem. 1997; 170: 109-114Crossref PubMed Scopus (24) Google Scholar, 50Giardina C. Inan M.S. Biochim. Biophys. Acta. 1998; 1401: 277-288Crossref PubMed Scopus (65) Google Scholar). We have reported previously that the influence of butyrate on NF-κB activation arises from its ability to inhibit histone deacetylases (25Inan M.S. Rasoulpour R.J. Yin L. Hubbard A.K. Rosenberg D.W. Giardina C. Gastroenterology. 2000; 118: 724-734Abstract Full Text Full Text PDF PubMed Scopus (382) Google Scholar). We therefore determined whether the effects of butyrate on p100 expression, IκB-α 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. 6 A), suppress TNF-α-induced IκB-α degradation (Fig. 6 B), and increase p100 expression (Fig. 6 B). These data are consistent with a model in which the influence of butyrate on NF-κB 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. Cells of the gastrointestinal tract constantly interact with the contents of the lumen. These conte" @default.
• W2008431663 created "2016-06-24" @default.
• W2008431663 creator A5029257492 @default.
• W2008431663 creator A5044713388 @default.
• W2008431663 creator A5079553213 @default.
• W2008431663 date "2001-11-01" @default.
• W2008431663 modified "2023-10-14" @default.
• W2008431663 title "Butyrate Suppression of Colonocyte NF-κB Activation and Cellular Proteasome Activity" @default.
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