Abstract. Sodium butyrate enhances TNF-a-induced complement C3 secretion but suppresses TNF-a-induced factor. B secretion in intestinal epithelial cells.
INTERNATIONAL JOURNAL OF MOLECULAR MEDICINE 6: 51-54, 2000
Modulation of complement component (C3 and factor B) biosynthesis by a histone deacetylase inhibitor in human intestinal epithelial cells AKIRA ANDOH, YOSHIHIDE FUJIYAMA, MITSUE SHIMADA and TADAO BAMBA Department of Internal Medicine, Shiga University of Medical Science, Seta-Tukinowa, Otsu 520-2192, Japan Received March 9, 2000; Accepted March 30, 2000
Abstract. Sodium butyrate enhances TNF-a-induced complement C3 secretion but suppresses TNF-a-induced factor B secretion in intestinal epithelial cells. To further evaluate the mechanism underlying these responses, we assessed the effects of trichostatin A, a compound structurally unrelated to butyrate and a potent inhibitor of histone deacetylase. The C3 and factor B secretion was evaluated by enzyme-linked immunosorbent assay (ELISA) and Northern blot, and the activation of transcription factor was assessed by an electro phoretic gel mobility shift assay (EMSA). Like sodium butyrate, trichostatin A enhanced TNF-a-induced C3 secretion, but suppressed TNF-a-induced factor B secretion. These effects were also observed at the level of mRNA. EMSAs indicated that trichostatin A weakly suppressed TNF-a-induced NF-KB and NF-IL6 activation. These observations differ from previous reports that sodium butyrate potently suppressed N F - K B activation but enhanced NF-IL6 activation. Trichostatin A modulated TNF-a-induced C3 and factor B secretion in a manner similar to that induced by sodium butyrate, suggesting that both sodium butyrate and trichostatin A exert certain counter-regulatory effects associated with histone hyperacetylation. However, it remains to be determined which factors other than histone acetylation are responsible for the counter-regulation of TNF-a-induced C3 and factor B gene expression. Introduction The complement system is a potent effector of normal immune and inflammatory responses (1). Complement activation occurs through three pathways: the classical, alternative and mannanbinding lectin (MBL)-initiated pathways (1,2). The C3
Correspondence to: Dr Akira Andoh, Department of Internal Medicine, Shiga University of Medical Science, Seta-Tukinowa, Otsu 520-2192, Japan Abbreviation: IL, interleukin; TNF, tumor necrosis factor; DMEM, Dulbecco's modified Eagle's medium; ELISA, enzymelinked immunosorbent assay; EMSA, electrophoretic gel mobility shift assay; NF-KB, nuclear factor-KB Key words: trichostatin A, histone acetylation, TNF-a, NF-KB
convertase of the alternative pathway, C3b,Bb, is a complex of C3 and factor B, and the generation of this complex is the rate-limiting step in the activation of the alternative pathway (1). Interestingly, we have found C3 and factor B mRNA expression in human colonic epithelial cells by in situ hybridization technique (3). Studies in several cell lines have indicated that complement secretion by intestinal epithelial cells is regulated by various pro-inflammatory cytokines (4,5). Thus, the complement system may participate in local immune protection in combination with the secretory-IgA system in the intestinal mucosa. Butyrate is one of the short-chain fatty acids which is physiologically generated by anaerobic bacterial fermentation of dietary fiber in the colon (6,7). Recently, we reported that sodium butyrate differentially modulates tumor necrosis factor (TNF)-a-induced C3 and factor B secretion in intestinal epithelial cells (8). Sodium butyrate up-regulated TNF-ainduced C3 secretion, but down-regulated TNF-a-induced factor B secretion. The down-regulation of factor B secretion induces suppression of alternative pathway activation at the initial step of the complement cascade and results in the inhibition of complement-mediated inflammatory responses in the mucosa. Sodium butyrate appears to be important as a physiological anti-inflammatory mechanism in the colon. Chromatin structure is an important component of gene expression. Recent studies have demonstrated that core histone acetylation facilitates gene expression through several mechanisms (9). One hypothesis is that the changes in core histone structure induced by histone acetylation facilitate access and binding of transcription factors and subsequently evoke gene transcription (9). Treatment with sodium butyrate induces the accumulation of hyperacetylated core-particle histones, notably histones H3 and H4, due to the inhibition of activity of histone deacetylase (10,11). In previous studies, the biological activity of sodium butyrate has been explained in association with its ability to induce histone hyperacetylation (10,11). In this study, to define the mechanisms mediating counterregulation of TNF-a-induced complement C3 and factor B secretion by sodium butyrate, we tested the effects of trichostatin A, a compound structurally unrelated to butyrate, which has been shown to be a potent and specific inhibitor of histone deacetylase (12). The findings in this study suggest that the regulation of complement secretion by sodium butyrate may be partially mediated by histone hyperacetylation in intestinal epithelial cells.
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Figure 1. Effects of trichostatin A on TNF-a-induced C3 secretion in HT-29 cells. Cells were stimulated with medium alone, TNF-a (25 ng/ml), or TNF-a (25 ng/ml) plus increasing concentrations of trichostatin A for 24 h. Values were expressed as mean ± SD (n=4).
Figure 2. Effects of trichostatin A on TNF-a-induced factor B secretion in HT-29 cells. Cells were stimulated with medium alone, TNF-a (25 ng/ml), or TNF-a (25 ng/ml) plus increasing concentrations of trichostatin A for 24 h. Values were expressed as mean ± SD (n=4).
Materials and methods
method of Dignam and Roeder (14). Consensus oligo
Reagents and cells. Recombinant human TNF-a (specific activity, 2.5xl06 U/mg) was kindly provided from Dainippon Pharmaceutical Co. (Osaka, Japan). All other reagents were purchased from Sigma Chemical Co. (St. Louis, MO). Human colonic adenocarcinoma cell lines HT-29 and T84 were obtained from the American Type Culture Collection (ATCC; Rockville, MD). The HT-29 and T84 cells were maintained in RPMI-1640 (Gibco Labs, Grand Island, NY) and a 1:1 mixture of Dulbecco's modified Eagle's medium (DMEM; Gibco) and Ham's F-12 medium (Gibco), respectively. Each medium contains 10% fetal bovine serum (Gibco), 50 U/ml penicillin and 50 u.g/ml streptomycin.
AGC C) (15) and NF-IL6 (TGC AGA JTG ÇGÇ AAT CTG CA) (16) were used. The consensus sequence for binding of transcription factor is underlined. Oligonucleotides were 5' end-labeled with T4 polynucleotide kinase (Promega, Madison, WI) and [y-32P]ATP (Amersham). Binding reactions were performed by preincubating 7.5 |ig of nuclear proteins in HEPES (20 mM; pH 7.9), KCl (60 mM), MgCl2 (1 mM), EDTA (0.1 M), glycerol (10%), dithiothreitol (0.5 mM), and poly (dl-dC; 2 |Xg) on ice for 10 min, followed by addition of the 32P-labeled oligonucleotide and a second incubation at room temperature for 20 min. Samples were loaded directly on non-denaturing 4% Polyacrylamide gels prepared in Trisglycin-EDTA buffer (pH 8.5). The gels were dried and exposed to Kodak XRP film with intensifying screen.
nucleotides of NF-KB (5': AGT TGA GGG GAC TTT CCC
Quantification of human C3 and factor B. The levels of antigenic C3 and factor B in the samples were determined by sandwich ELISA established in our laboratory (4). Northern blot analysis. Total cellular RNA was isolated by the acid guanidinium thiocyanate-phenol-chloroform (AGPC) method (13). Samples of RNA (20 (ig) were denatured in a loading buffer for 15 min at 65°C. The RNA was then electrophoresed through a 1.0% agarose-formaldehyde gel and transferred to a nylon membrane (Gene Screen Plus; New England Nuclear Research Products, Boston, MA) in a 10X SSPE. After the transfer, the membrane was washed and baked at 80°C for 2 h. The prehybridization was performed for 4 h at 42°C in a solution containing 50% formamide, 5X SSPE, IX Denhardt's solution, 0.2% sodium dodecyl sulfate, and 100 |xg/ ml denatured salmon sperm DNA. The hybridization was performed with 32P-labeled human C3 and factor B probes (3), generated by a random primed DNA labeling kit (Amersham, Arlington Heights, IL), and evaluated by autoradiography. Nuclear extracts and electrophoretic gel mobility shift assays. Nuclear extracts were prepared from HT-29 cells exposed to TNF-a (25 ng/ml) and trichostatin A (5 |xM) for 1.5 h by the
Results Effects of trichostatin A on the secretion of complement C3 and factor B in HT-29 cells. HT-29 cells were incubated for 24 h with medium alone, TNF-a (25 ng/ml), or TNF-a plus increasing concentrations of trichostatin A. As shown in Fig. 1, TNF-a induced C3 secretion, and trichostatin A dosedependently enhanced TNF-a-induced C3 secretion. As demonstrated in Fig. 2, TNF-a induced a marked increase in factor B secretion. In contrast to effects on C3 secretion, trichostatin A dose-dependently inhibited TNF-a-induced factor B secretion. These effects of trichostatin A were observed at concentrations as low as 1 (xM and reached a plateau at a concentration of 5.0 )uM. The addition of trichostatin A (5 uM) enhanced TNF-a-induced C3 secretion by 5.0-fold, but suppressed TNF-a-induced factor B secretion by 78%. Similar responses were also observed in another intestinal epithelial cell line, T84 (Fig. 3). These observations in HT-29 and T84 cells were compatible with the effects of sodium butyrate on TNF-a-induced C3 and factor B secretion.
INTERNATIONAL JOURNAL OF MOLECULAR MEDICINE 6: 51-54, 2000
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Figure 5. Electrophoretic gel mobility shift assays (EMSA) for NF-KB and NF-IL6 DNA-binding activities. HT-29 cells were incubated with medium alone, trichostatin A (5 Ug/ml), TNF-a (25 ng/ml), or TNF-a (25 ng/ml) plus trichostatin A (5 u.g/ml) for 1.5 h, and the nuclear extracts prepared. Figure 3. Effects of trichostatin A on TNF-a-induced C3 and factor B secretion in T84 cells. Cells were stimulated with medium alone, TNF-a (25 ng/ml) alone, or TNF-a (25 ng/ml) plus increasing concentrations of trichostatin A for 24 h. Values were expressed as mean ± SD (n=4).
Modulation of transcription factor activation by trichostatin A. TNF-a induces the activation of NF-KB (p50/p65) and NF-IL6 in HT-29 cells (8). Previously, we observed that sodium butyrate differentially modulates TNF-a-induced activation of transcription factors in HT-29 cells. Sodium butyrate suppressed TNF-a-induced NF-KB activation, while TNF-ainduced NF-IL6 activation was enhanced. As shown in Fig. 5, the addition of trichostatin A suppressed TNF-a-induced NF-KB and NF-IL6 activation. The inhibitory effects of trichostatin A on NF-KB activation were very weak as compared to those induced by sodium butyrate (8). Discussion
Figure 4. Northern blot analysis of C3 and factor B mRNA expression in HT-29 cells. Cells were stimulated with medium alone, TNF-a (25 ng/ml) alone, trichostatin A (5 U.M) or TNF-a (25 ng/ml) plus trichostatin A (5 U.M) for 6 h, and then total cellular RNA was extracted. These were also evaluated in the presence of cycloheximide (5 u.g/ml).
Trichostatin A modulates C3 and factor B mRNA abundance. We evaluated the effects of trichostatin A on the abundance of C3 and factor B mRNA in HT-29 cells (Fig. 4). The cells were cultured for 6 h with medium alone, TNF-a (25 ng/ml), or trichostatin A (5 |^M) plus TNF-a (25 ng/ml). Unstimulated HT-29 cells weakly expressed the factor B gene. The addition of TNF-a induced a marked increase in the abundance of C3 (5.2 kb) and factor B (2.4 kb) mRNA. The addition of trichostatin A alone exerted no effect, but enhanced TNF-ainduced C3 mRNA abundance by 5.6-fold and inhibited TNFa-induced factor B mRNA abundance by 75%, respectively. Furthermore, the presence of the protein synthesis inhibitor, cycloheximide, did not affect these responses.
The secretion of complement proteins C3 and factor B by intestinal epithelial cells is thought to play an important role in the local immunologic protection system of the intestinal mucosa. Among many complement proteins C3 and factor B have been regarded as acute phase proteins, and the induction of C3 and factor B secretion is coupled in various cell types (17). For example, both C3 and factor B secretion are induced by pro-inflammatory cytokines including interleukin (IL)-lß, JX-6, and TNF-a in hepatocytes, fibroblasts, and epithelial cells (17-19). Furthermore, the promoter analyses of the C3 and factor B genes have indicated that transcription factors NF-KB and NF-IL6 drive the expression of these genes (20,21). Recently, we found that sodium butyrate has a novel effect on C3 and factor B secretion in intestinal epithelial cells (8). In HT-29, Caco-2 and T84 cells, TNF-a induces C3 and factor B secretion, and sodium butyrate differentially modulates TNF-ainduced C3 and factor B secretion; sodium butyrate markedly enhanced TNF-a-induced C3 secretion, but potently suppressed TNF-a-induced factor B secretion (8). Such a counterregulatory effect on C3 and factor B secretion has not been reported previously. In this study, to assess the role of histone acetylation in the counter-regulation of C3 and factor B secretion by sodium butyrate, we tested the effects of trichostatin A, a compound structurally unrelated to butyrate, which has been shown to be a potent and specific inhibitor of histone deacetylase and to induce histone hyperacetylation (12). As demonstrated in
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Figs. 1 and 2, trichostatin A exerted counter-regulatory effects on TNF-ct-induced C3 and factor B secretion in HT-29 cells; trichostatin A dose-dependently enhanced TNF-a-induced C3 secretion, but dose-dependently decreased TNF-a-induced factor B secretion. These effects were also confirmed in T84 cells. Trichostatin A, however, exerted its effects at much lower concentrations than sodium butyrate; the effects of sodium butyrate were observed at mM order but the effects of trichostatin A were observed at \xM order. Northern blot analysis demonstrated that trichostatin A regulated C3 and factor B expression at the mRNA level. The addition of the protein synthesis inhibitor, cycloheximide, did not alter the effects of trichostatin A, indicating that new protein synthesis was not required for the effects of trichostatin A. This mimicry of effect between sodium butyrate and trichostatin A suggests that certain aspects of counter-regulation of TNF-a-induced C3 and factor B secretion may be associated with histone hyperacetylation. Histone hyperacetylation is believed to have a positive role in facilitating transcription factor access to promoter sequences, thus inducing transcription (9). However, it has been also recognized that high levels of histone hyperacetylation are not sufficient for active transcription of all genes (9,11) because the selective induction of gene transcriptions by either sodium butyrate or trichostatin A have been reported (11). These observations prompted us to assess the effects of trichostatin A on transcription factor activation. In our recent study, we found that sodium butyrate differentially modulates TNF-a-induced transcription factor activation in HT-29 cells (8). Sodium butyrate potently down-regulated TNF-a-induced N F - K B activation, but slightly enhanced TNF-a-induced NF-IL6 activation. However, as demonstrated in Fig. 4, trichostatin A weakly inhibited TNF-a-induced N F - K B and NF-IL6 activation. When comparing trichostatin A with sodium butyrate, the inhibitory effect of trichostatin A on N F - K B activation was modest as compared with that induced by sodium butyrate. The changes in transcription factor activation were very weak as compared to those in C3 and factor B mRNA expression. These observations suggest a possibility that the modulation of transcription factors does not actively participate in the regulation of counter-regulation of TNF-a-induced C3 and factor B secretion by trichostatin A. Recently, several hypotheses for the mechanisms by which protein acetylation affects gene transcription have been postulated (9). For example, acetylation of non-histone proteins including t r a n s c r i p t i o n factor may play an important role in transcriptional regulation. In conclusion, we demonstrated that trichostatin A, as well as sodium butyrate, exerted counter-regulatory effects on TNF-a-induced C3 and factor B secretion in intestinal epithelial cells. However, the modulations of N F - K B and NF-IL6 activation by trichostatin A differed from those induced by sodium butyrate. In the future, it should be determined how protein acetylation modulates gene transcription. Acknowledgements This study was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, and Culture, Japan (9470135 and 9670541).
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