The regulation of gene expression by specific nutrients exerts a major influence on the health of a child. Several dietary factors are implicated as specific modifiers of gene expression. These include glucose, fatty acids, amino acids, ions, and vitamins (1,2). The targets of these nutrients include transacting factor nuclear proteins and cis-acting elements, which regulate gene transcription, splicing, and editing of nuclear RNA and cytosolic proteins that alter RNA stability and translation. Dietary factors play an important role in the cross-talk between bacteria and human intestinal epithelium. One such nutrient, the pleiotropic byproduct of bacterial fermentation, butyrate, plays an active role as a modulator of gene expression (3) and initiator of cell differentiation (4,5) and regulates binding protein expression within the insulin-like growth factor (IGF) axis (6). It has long been established that members of this axis affect intestinal growth and may modulate the immune system (7). Early childhood is a time of great change in butyrate concentrations in the gastrointestinal tract. Butyrate is undetectable at birth and remains low in the infant until weaning (8). By 2 years its concentration has reached adult levels. Bottle-feeding greatly enhances colonic butyrate concentration, which increases rapidly during the neonatal period (8). Intestinal diseases are also associated with changes in butyrate concentrations, with increases occurring during necrotizing enterocolitis (9) and Crohn's disease (10).
Traditionally, IGF binding proteins (IGFBPs) were thought to serve primarily as circulating carrier proteins that prolong the plasma half-life of the IGFs and limit the bioavailability of active IGF-I or IGF-II for mitogenic actions (11). IGFBP-3 has a 20-fold greater affinity for IGF-I than does IGFBP-II (12). Consequently, circulating IGFBP-3 binds to IGF-I as part of the soluble 150 kDa complex that serves as a reservoir for circulating IGF-I in the blood. Reports have also suggested that IGF binding proteins have direct antiproliferative effects independent of IGF in colonic epithelium (13). Furthermore, IGFBP-3 has been shown to induce apoptosis in an IGF-independent manner and is a mediator of transforming growth factor-β induced apoptosis in prostate and breast cancer cells (14-16).
Butyrate affects gene expression in two ways; first, it alters the binding of nuclear proteins at the 5’ promoter region of the gene (17), and second, it alters electrostatic interactions between histones and DNA in chromatin by altering histone acetylation (18-20). There is a growing family of proteins with histone-acetyltransferase activity and a similarly diverse family that possess histone deacetylase activity (HDAC) (21,22). Butyrate is a noncompetitive inhibitor of HDAC activity (17) and as such is known to mediate alterations in gene expression ultimately through effects on the expansion of the nucleosome through the acetylation of histones. Histone acetylation increases access of transcription factors to the DNA and generally correlates with transcriptional activation. However, the expansion of the nucleosome does not easily explain how butyrate may inhibit gene expression.
We have previously reported that sodium butyrate (NaB) up-regulates the constitutive transcription and secretion of IGFBP-2 by intestinal epithelial cells and that this correlates with an increase in histone acetylation (6). However, NaB also down-regulates IGFBP-3 in intestinal epithelial cells (6), and it is difficult to explain how loosening of the nucleosome results in gene repression. In this study, we examined whether NaB acts in an indirect manner (e.g., through the upregulation of a repressor) or in a direct manner at the IGFBP-3 promoter. We hypothesized that NaB may regulate the acetylation of transcription factor that inhibits IGFBP-3 expression.
MATERIALS AND METHODS
Caco-2 cells, obtained from the American Type Tissue Culture, Manassas, VA, were cultured as previously described (6). Cells were used between the 26th and 32nd passages. Caco-2 cells were seeded in complete medium (10% fetal bovine serum [FBS]) at 5 × 104 cells cm2 into T75 tissue culture flasks. At 90% confluence, cells were washed twice with minimal medium and transferred to minimal medium for a further 24 hours. Cells were then exposed to 5 mmol/L NaB or controls to 1× phosphate-buffered saline (PBS) in minimal medium for an additional 24 hours. Cells were maintained in complete media, Dulbecco's Modified Eagles Medium (DMEM) supplemented with 10% FBS, 2 mmol/L L-Glutamine, 1 × nonessential amino acids, 10 U/mL penicillin, and 10 μg/mL streptomycin (Invitrogen, Paisley, United Kingdom). Cells were incubated at 37°C in 5% CO2/95% air.
Reverse-Transcriptase Polymerase Chain Reaction
Total RNA was extracted using TRIzol (Invitrogen), according to the manufacturer's instructions. Oligo (dT)-primed cDNA was prepared by reverse transcriptase. PCR was performed with 200 μmol/L of dNTPs and 1U of Taq polymerase. Cycles within the range of liner expansion of amplicon product were determined experimentally. Normally, 30 cycles were used. β-actin transcripts were amplified using described primers. Human (h)IGFBP-3 transcripts were prepared using primers hIGFBP-3 UP1 (-11-gCCTCCACATTCAgAggCAT-30-) and hIGFBP-3 DN1(antisense) (-501-CCTgACTTTgCCAgACCTTC-520-). β-actin primers used were UP (CgAggCCCAgAgCAAgAgA) and DOWN (CACAgCTTCTCCTTAATgTCACg). Reverse-transcriptase polymerase chain reaction (RT-PCR) was performed as a minimum in triplicate.
Inhibition of Protein Synthesis by Cycloheximide
Caco-2 cell lines were cultured in complete medium and resuspended in minimal media 24 hours before fresh minimal media and the addition of 5 mmol/L NaB in the presence or absence of 10 μmol/L cycloheximide (Chx) (Sigma, Poole, United Kingdom) for 6, 12, 24, 36, and 48 hours. hIGFBP-3 mRNA transcripts were analyzed by Southern blotting of RT-PCR amplicons, whereas hIGFBP-3 protein was assessed by Western immunoblotting using an anti-IGFBP-3 antibody (Upstate Biotechnology, Dundee, United Kingdom). Fifty micrograms of total protein was added to each well as determined by the Bradford Assay (Biorad, Hemel Hempstead, United Kingdom). SQRT PCR were performed in triplicate and experiments, as a minimum, in triplicate.
Electro-Mobility Shift Assay
NP-40 mediated nuclear extracts (NE) were prepared by lysis of 106 cells with Dignam buffer A (10 mmol/L Hepes [pH 7.9 at +4°C], 1.5 mmol/L MgCl2, 10 mmol/L KCl, 0.5 mmol/L dithiothreitol) using 0.1% NP-40. After isolation of nuclei by centrifugation, proteins were extracted at 4°C for 60 minutes with Dignam buffer C (20 mmol/L Hepes [pH 7.9 at +4°C], 25% [w/v] glycerol, 0.42 mol/L NaCl, 1.5 mmol/L MgCl2, 0.2 mmol/L EDTA, 0.5 mmol/L DTT) in the presence of protease inhibitors (0.5 mmol/L phenylmethylsulphonylfluorid, 1.0 mmol/L benzamidine, 30 mg/mL leupeptin, 5 mg/mL aprotinin, 5 mg/mL pepstatin). Starting with 25 ng DNA and 25 μCi (α32P) dCTP (6,000 Ci/mmol) (Amersham), Large Klenow fragment incorporated 32P into oligonucleotides over 5 minutes at room temperature to make probes. Probes were gel purified on a 15% nondenaturing polyacrylamide two-dimensional (2D) gel. NE were incubated for 15 minutes at 37°C with 32P-labelled oligonucleotides. For supershift assays, NE were pre-incubated, on ice, with antibody for 1 hour before incubation with oligonucleotide. DNA-protein complexes were size fractionated by electrophoresis through a 5% nondenaturing polyacrylamide 2D prerun gel and bands visualized through exposure of film. Oligonucleotide sequences were Sp1/3 5’OH hIGFBP-3 WT (-237gCCTgCgCCgACCCgCCCCCCTCCCAACCC-221), Sp3 5’OH hIGFBP-3 MUT (-237gCCTgCgCCgACCCgCCCCTCTAgAAACCC-221), and Sp1 5’OH hIGFBP-3 MUT (-237gCCTgCgCCCATgggCCCCCCTCCCAACCC-221).
Protein Transfer (Western) Immunoblots
For the detection of hIGFBP-3, 125 μg total protein from serum-free conditioned media from Caco-2 cultures treated with 5 mmol/L NaB or controls with 1× PBS for 36 hours was used. For the detection of acetyl-lysine and Sp3 proteins, 25 μg NE after 6 hours of 5 mmol/L NaB treatment and 1× PBS control treatment was used. Samples were electrophoresed through 12.5% reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) overnight at constant voltage, electroblotted onto Hybond P (Amersham, Chalfont, United Kingdom), blocked with 5% nonfat dry milk in Tris-buffered saline, probed with specific primary antibodies, and detected using an electrogenerated chemiluminescence (ECL) detection system (Amersham). To reprobe the membranes with a different primary antibody, probes were stripped from the membranes by washing twice in excess of 0.1% TBS-T for 10 minutess followed by 2% SDS, 100 mmol/L mercaptoethanol, 62.5 mmol/L pH 6.8 TrisHCl at 65°C for 30 minutes. The membrane was washed twice more for 10 minutes in excess of 0.1% TBS-T before continuing with the new primary antibody.
Cells were lysed with RIPA buffer (1%Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 0.15 mol/L NaCl, 0.01 mol/L sodium phosphate pH 7.2, 1% Trasylol). IPs were performed by incubating 55 μg of precleared total protein with 0.3 μg of polyclonal anti-Sp3 IP antibody in S. aureus Cowan I (SAC I) buffer. Protein A Sepharose beads were used (Sigma) in the incubation, and precipitates were washed four times with SAC I buffer and resolved on a 7.5% SDS-PAGE gel under reducing conditions. Immunoprecipitated proteins were examined using anti-Sp3 antibody to check for consistent loading. The Hybond P (Amersham) membrane was stripped as per manufacturer's instructions and re-examined by immunoblotting using an anti-acetyl lysine antibody. ECL (Amersham) chemiluminescence detection reagents were used to detect immunoreactive proteins.
Anti-Sp3 antibody used according to manufacturer's recommendation (Upstate, Dundee, UK). Anti-E1A antibody used according to manufacturer's recommendation (Upstate). Anti-acetyl-lysine antibody used according to manufacturer's recommendation (Upstate).
Probe manufacture and hybridization were performed according to manufacturer's instructions (Rpn3000 Alkphos Direct Labeling Kit, Amersham). Hybond N+ membrane and ECL detection reagents were used as per manufacturers instruction.
Deletional Mutations of IGFBP-3 Promoter
Deletional mutations linked to the luciferase reporter gene were a generous gift of Dr. Youngman Oh (University of Oregon) (23). They were individually transfected into Caco-2 cells using Fugene (Roche, Lewes, United Kingdom), as previously described (24).
NaB Acts Directly on hIGFBP-3 Gene in Caco-2 Cells When Downregulating Its Transcription
NaB has been shown to increase histone acetylation in cultured cells through the inhibition of HDAC activity (25-27). We investigated whether the downregulation of hIGFBP-3 in Caco-2 cells was indirect (i.e., casued by the induction of a repressor of hIGFBP-3) through hyperacetylation of histones at the repressor's promoter. Because repressors are proteins, we studied whether the downregulation by NaB of hIGFBP-3 mRNA in Caco-2 cells was interrupted by cycloheximide, an inhibitor of protein synthesis. RT-PCR analysis of cycloheximide treated and untreated Caco-2 cells showed a reduction in hIGFBP3 mRNA levels in Caco-2 cells treated with 5 mmol/L NaB (Fig. 1) over those cells that were not treated with NaB. hIGFBP-3 mRNA accumulation was greater in the absence of NaB and was unaffected by the presence of 10 μmol/L cycloheximide. Cells subjected to 5 mmol/L NaB showed a reduction in mRNA levels, even when the cells had been pre-treated with 10 μmol/L cycloheximide. The same cells showed equivalent expression of β-actin transcripts (Fig. 1). These experiments demonstrate that protein synthesis was not necessary for NaB to inhibit hIGFBP-3 mRNA. Therefore, NaB does not act by increasing the transcription and translation of a repressor.
To determine whether the observed decrease in hIGFBP-3 mRNA transcripts by NaB in the Caco-2 cell was caused by a decrease in mRNA stability, mRNA decay studies were performed (Fig. 2). Caco-2 cells pre-incubated for 24 hours in minimal media and then for a further 6 hours in minimal media with or without 5 mmol/L NaB were treated with 25 μg/mL 5,6dichlororibofuranosylbenzimidazole (DRB), a potent inhibitor of transcription. Cells were harvested at various time-points from 0 to 37 hours and mRNA transcript levels measured by densitometry of Southern blots. Figure 2 shows the decay of hIGFBP-3 mRNA transcripts over time in the presence and absence of NaB. The gradient of the graphs plotted in Figure 2 indicate that exposure to 5 mmol/L NaB did not alter the decay of the hIGFBP-3 transcripts. Because the inhibitory action of NaB occurs in the absence of de novo protein synthesis without affecting mRNA stability, we concluded that it acts directly on the hIGFBP-3 gene.
Identification of a NaB Response Element Within hIGFBP-3 Promoter in Caco-2 Cells
Promoter sequence analysis revealed putative binding sites for various transcription factors 5’ to the hIGFBP-3 gene including AP2, p300, Ying Yang1 (YY1), p53, and Sp1/Sp3 (Fig. 3). A characterization study, through transient transfection of attenuating promoter regions, revealed a NaB responsive element in the IGFBP-3 promoter of Caco-2 cells that maps to a region approximately 50 bp 5’ to the initiation codon, encompassing the p300 and Sp1/Sp3 binding motifs (Fig. 3). As well as functioning as a transcription factor, p300 is known to have intrinsic acetyltransferase activity.
Acetyltransferase Activity of p300 Is Involved in NaB Mediated Downregulation of hIGFBP-3
We investigated the acetyltransferase involvement of p300 by transfecting Caco-2 cells with a plasmid encoding the adenoviral oncogene protein E1A. E1A abrogates the acetyltransferase activity of p300 (28). Transfection of NaB treated Caco-2 cells with E1A restored the expression of the hIGFBP-3 protein to nonbutyrate treated levels, as demonstrated by Western blot analysis and RT-PCR (Fig. 4). A mutant, E1AmCBP, lacking the ability to bind and block the acetyltransferase domain of p300, did not affect the downregulation of hIGFBP-3 by NaB.
p300 has been shown to possess the ability to acetylate both histones and transcription factors. p53 is a known target for this factor-acetyltransferase activity by p300 (29). However, acetylation of p53 has been reported to enable it to act as a stronger activator of transcription (30), and in WiDr p53-negative cells, NaB has been shown to activate the WAF1/Cip1 gene promoter (31). Furthermore, the butyrate response element in the hIGFBP-3 promoter did not include a putative p53 binding site. However, Sp3 may act as an activator or repressor of gene transcription, and one molecular event that controls this dual function is acetylation (32,33). We hypothesized that the repression of hIGFBP-3 may involve modulation by NaB of the acetylation status of Sp3 by p300.
Sp3 Is Acetylated in NaB Treated Caco-2 Cells and Binds NaB Responsive Element
Sp1 and Sp3 compete for overlapping binding sites on promoter DNA. Generally, Sp1 binds preferentially and acts as a strong activator. Sp3 binds weakly and has been shown to act as a repressor of Sp1 activity (34-36). We performed electromobility shift assays on NE prepared from NaB treated and nontreated Caco-2 cells using either an oligo of the wild-type Sp1/Sp3 sequence (WTSp1/WTSp3), a mutated-Sp1/wt-Sp3 sequence (MUTSp1/WTSp3), or a wt-Sp1/Sp3-mutated sequence (WTSp1/MUTSp3) as a probe. The MUTSp1/WTSp3 oligomer generated an extra retarded bandshift in the NaB treated extract that was not present in the nontreated extract, (Fig. 5A), which indicated a posttranslational modification of the bound Sp3. Sp1 binding to its wild-type promoter was very strong and not affected by butyrate (Fig. 5B).
We suspected that this bandshift was Sp3-Ac, which was preferentially binding in the absence of steric hindrance from Sp1. Pre-incubation of the nuclear extracts with an anti-acetyl-lysine antibody produced a band corresponding to a supershift whose intensity was stronger in the NaB treated versus nontreated samples (Fig. 6), suggesting that this was Sp3-Ac.
An anti-acetyl-lysine antibody was used to probe nuclear proteins transferred to Western blots from Caco-2 cells treated with 5 mmol/L NaB compared with controls. An acetylated protein was found in the treated cells, which was not present in the nontreated extract (Fig. 7A). Stripping and reprobing the transfer blot with the Sp3 specific antibody demonstrated a band at the same position (Fig. 7B). Furthermore, immunoprecipitation assays of total protein derived from NaB treated Caco-2 cells demonstrated that the amount of Sp3-Ac was increased compared with that derived from nontreated cells (Fig. 8). Total Sp3 protein levels were unchanged in treated cells when compared with nontreated cells.
The present study demonstrates that a bacterial product in the intestine can regulate gene expression in epithelial cells by inducing acetylation of a transcription factor. In particular, the short chain fatty acid, NaB, down-regulates hIGFBP-3 expression by the acetylation of Sp3 in Caco-2 cells. The Caco-2 cell line is an excellent model in which to examine signal transduction because the Caco-2 cell IGFBP profile is equivalent to other human intestinal epithelial cells, including nonmalignant epithelial cells (37). Furthermore, the growth and yield of nuclear proteins from Caco-2 cells permits examination of nuclear events that would not be feasible in other intestinal epithelial cell types.
IGFBPs inhibit the binding of IGFs to IGF receptors in the intestine (38,39), thereby altering cell proliferation and differentiation. IGF receptors are present both on epithelial cells (40,41) and on activated lymphocytes (42). Three IGF binding proteins have been identified in the conditioned media of Caco-2 cells (43), and each has a distinct affinity for IGF-I and -II (12). For example, IGFBP-3 binds to IGF-I with an affinity manifold greater than that of IGFBP-2 (12,44). The specificity of the effect of different binding proteins on the concentration of free IGFs is exaggerated by the presence of proteases, which are IGFBP specific (23). IGFBP-3 is particularly susceptible to the action of such proteases. Together, these data indicate that altering the profile of IGFBPs has profound effects on the bioavailability of IGF-I and -II.
We have previously suggested (6) that IGFBP expression can be changed by three possible mechanisms. They include (1) an alteration in binding of nuclear proteins to IGFBP gene promoters; (2) changes in chromatin structure; and (3) alterations in enterocytic differentiation. We demonstrated that up-regulation of IFGBP-2 acted through the second of these pathways. NaB is known as a reversible inhibitor of HDAC activity, and exposure is correlated with an increase in levels of acetylated-histones (45). However, the expansion of the nucleosome caused by histone acetylation does not easily explain the down-regulation of genes by NaB. To answer whether exposure to NaB increased the up-regulation of a specific repressor of IGFBP-3 in Caco-2 cells, we exposed NaB treated and nontreated Caco-2 cells to cycloheximide, a potent inhibitor of eukaryotic protein translation. Had the induction of a repressor been necessary to mediate the effect of NaB, exposure to cycloheximide would have abrogated the down-regulation of IGFBP-3 in Caco-2 cells. However, the effect of NaB persisted in the presence of cycloheximide. Through promoter deletion studies, we identified a NaB responsive element in the proximal promoter of hIGFBP-3 similar to that previously reported, which encompassed binding sites for Sp1/Sp3 and p300. Electromobility shift assays demonstrated that there was an increase in Sp3-Ac bound to its putative binding site within its response element in NaB treated NE. Sp3 has been reported as a target of acetylation by p300 (32) and is recognized as a repressor of transcription at several gene promoters (33-36). Because of the presence of the p300 putative binding site within the NaB responsive element, we hypothesized that Sp3 was acetylated in situ by p300. In agreement, transfection with a plasmid encoding the adenoviral protein E1A, known to block the acetyltransferase activity of p300, into Caco-2 cells abrogated the effect of NaB on hIGFBP-3.
Our experiments therefore support the following mechanism for the downregulation of IGFBP-3 in Caco-2 cells by NaB: p300 acetylates Sp3, increasing its binding to the IGFBP-3 promoter. This in turn represses IGFBP-3 transcription by blocking the binding of the active transcription factor, Sp1, through steric hindrance. Normally, Sp3-Ac would be rapidly de-acetylated. However, NaB increases acetylation by inhibiting Sp3 deacetylase in manner analogous to its action of inhibiting histone deacetylase. Preventing the p300 acetylation of Sp3 with E1A interrupted the inhibitory effect of NaB by depriving the NaB-sensitive deacetylase of Sp3-Ac substrate.
This study has wider implications than the molecular pathway from NaB through Sp3 to IGFBP-3. Sp3 is present in the promoter of many other genes. These include interleukin-10 (46), ENA-78 (47), and MUC2 and MUC5a (48). Thus, a mechanism exists where regulatory proteins modify the relationship of the enterocyte with its surrounding cells and substrates on sensing the environment of the intestinal lumen. Because that environment is influenced by diet, the cell is continually modifying its signaling profile according to food intake.
In summary, NaB acts by inhibiting the de-acetylation of Sp3 bound within a NaB response element at the hIGFBP-3 promoter in Caco-2 cells. We have shown, for the first time to our knowledge, that NaB can modulate the acetylation status of nonhistone DNA binding proteins.
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Keywords:© 2006 Lippincott Williams & Wilkins, Inc.
Butyrate; Intestinal epithelium; IGF; Sp3; Acetylation.