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MEMBRANE TRANSPORTERS, ION CHANNELS, AND PUMPS
Departments of Pediatrics and Physiology, Steele Children's Research Center, University of Arizona Health Sciences Center, Tucson, Arizona
Submitted 19 March 2006 ; accepted in final form 27 February 2007
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Slc9a3; Sp1; Sp3; phosphorylation; acetylation; small interfering RNA
2 liters of electrolyte-rich fluid. Of this amount, 1.5–1.9 liters is absorbed in the colon, although the maximal capacity of the human large intestine to absorb fluids may be as high as 5–6 l/day (14). Therefore, there is a large margin within which a healthy colon can compensate for increased ileocecal flow ensuing from defective small intestinal absorption. Exceeding the maximal capacity will result in diarrhea. On the other hand, in colonic disease relatively small changes in water and electrolyte absorption will produce a significant increase in stool water output, emphasizing the relevance of fine-tuning of colonic transport processes. The bulk colonic electrolyte absorption occurs via electroneutral NaCl transport and takes place in both crypts and surface epithelium of proximal and distal colon. The remaining electrolyte absorption is mediated by electrogenic sodium channels (ENaC) and by transcellular and/or paracellular absorption of Cl–. The electroneutral component is generally believed to represent coupled apical Na+/H+ and Cl–/HCO3– exchange and is greatly enhanced by short-chain fatty acids (SCFAs). Of all the SCFAs produced in large amounts in the colonic lumen by bacterial fermentation of carbohydrates, butyrate seems to exert the most significant influence on colonocyte biology. In addition to being a preferred fuel for the colonic epithelial cells (37) and a potentially beneficial factor in colon cancer prevention (16) and in colonic inflammation (2), butyrate has profound effects on colonic fluid and electrolyte absorption. In fact, the use of amylase-resistant starch as an additive to oral rehydration solution proved effective in reducing diarrheal stool output in cholera patients (35), thus showing that SCFAs can be potent antidiarrheal agents. Indeed, SCFAs have been demonstrated many times as potent stimuli of sodium and water absorption in the colon (4, 12, 34, 40), with butyrate being the most effective (23). Although the precise molecular mechanism of butyrate on Na+ absorption in the gut is unclear, it is thought that SCFA-mediated increase in Na+ absorption is due to two coupled exchange mechanisms, Na+/H+ and SCFA–/Cl– exchange.
Na+/H+ exchanger (NHE)3 is one of nine Na+/H+ exchangers cloned to date (50). NHE3 protein is expressed on apical membranes of intestinal epithelial cells (7, 15), where it is thought to be the major absorptive Na+/H+ exchanger, as shown by gene targeting experiments in mice. Lack of the exchanger results in diarrhea and distension of all intestinal segments due to fluid accumulation, and NHE3-knockout mice are considered a model of chronic secretory diarrhea (43). Experiments in dogs demonstrate that NHE3 accounts for all basal ileal Na+ absorption and the neurohormonally induced increase in ileal Na+ absorption that occurs after meals (26, 49). Furthermore, SCFAs have been demonstrated to stimulate NHE3 activity and protein and mRNA expression both in vivo (rat colon) and in vitro (Caco-2 cells) (31). Our previously published studies (19) demonstrate that in transient transfection NHE3 gene promoter is potently stimulated by sodium butyrate (NaB) and that the mechanism of this regulation appears different from the effects of the specific histone deacetylase (HDAC) inhibitor trichostatin A (TSA).
Many published reports on transcriptional regulation by NaB point to multiple mechanisms of action by this SCFA going beyond the traditionally attributed reversible inhibition of HDACs (36). Among other effects on chromatin, NaB has been reported to inhibit phosphorylation of histones H1 and H2 (5, 24), to increase phosphorylation of histone H3 (48), to decrease global histone methylation (5), and to induce hypermethylation of cytosine residues in DNA (6). Indeed, microarray analyses of colonic epithelial gene expression identified a significantly larger fraction of cellular genes whose expression was modulated by butyrate than by TSA (28). Many gene promoters have been described as butyrate responsive, and although the involvement of Sp family transcription factors has been implicated by several studies, the detailed molecular basis of gene-specific effects of NaB is generally poorly defined. It is also conceivable that these mechanisms will differ among genes, dependent on the chromatin context and spatial and functional organization of relevant cis-elements within the promoter.
The transcriptional response to NaB has been perhaps most extensively studied for p21Waf1, a cyclin-dependent kinase inhibitor responsible for NaB-induced cell cycle arrest in cancer cell lines. In this model gene, hyperacetylation of histones by NaB was not sufficient to increase p21Waf1 mRNA levels but required recruitment of several transcription factors including Sp1 and ZBP-89 (21). Merchant et al. (30) proposed a model in which Sp1 and ZBP-89 act in concert by binding to the GC-rich elements within the proximal promoter of p21Waf1 and recruiting HDAC1 and p300, respectively, thereby permitting a local increase in acetylase activity. Because of the high relevance of NaB-mediated transcription of NHE3 in colonic physiology and pathophysiology, we further pursued the molecular mechanism of this regulation. Here we describe a novel gene-specific effect of this SCFA on NHE3 gene promoter activity, which is mediated by a combination of two events: a switch in DNA binding of Sp1 and Sp3 resulting from their posttranslational modifications (phosphorylation and acetylation, respectively) to favor the more potent trans-activator Sp3 and recruitment of an unknown modulator to an upstream cis-element.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Plasmids. Rat NHE3 gene promoter constructs were created in phGR-b vector (Promega) containing a synthetic humanized Renilla firefly cDNA as a reporter gene. Constructs were made in this vector by moving respective promoter fragments by restriction digestion from previously reported constructs in pGL3-basic vector (19, 20). Hybrid promoters were created containing regions of NHE3 gene amplified by PCR and cloned upstream of a heterologous transcription start site [TATA box of thymidine kinase promoter from pTA-Luc vector (Clontech, Mountain View, CA)]. pSp-rLuc plasmid was created by subcloning an oligonucleotide (5'-CGCGTGGGCGGAACTGGGCGGAGTTAGGGGCGGGA-3') containing three SV40 promoter-derived consensus Sp binding sites into the SmaI site of phRG-b vector. Sp1 and Sp3 expression vectors driven by Drosophila actin 5 gene promoter were kindly provided by Drs. Robert Tjian (University of California at Berkeley, Berkeley, CA) and Guntram Suske (Philipps-University, Marburg, Germany), respectively. Hemagglutinin (HA) epitope tag was engineered on the NH2 terminus of respective Sp protein (pPacSpx-HA) for comparative Western blot detection. The Drosophila expression plasmid with human ZBP-89 cDNA was provided by Dr. Juanita Merchant, (University of Michigan, Ann Arbour, MI). For stable transfection of Caco-2 cells with a reporter construct containing rat NHE3 promoter, a blasticidin-resistance cassette was removed from pCMV/Bsd (Invitrogen, Carlsbad, CA) with BglII and BamHI and subcloned into the BamHI site of phRG-b vector containing the –320/+58 nt fragment of NHE3 promoter to create a construct phRG-320/+58Bsd. All new constructs were sequenced to confirm fidelity.
Cell culture and transfections.
Human colonic adenocarcinoma (Caco-2) cells and Drosophila SL2 cells were obtained from the American Type Culture Collection (Manassas, VA). Caco-2 cells were maintained in high-glucose DMEM supplemented with 2 mM glutamine, 1 mM sodium pyruvate, 0.1 mM nonessential amino acids, 100 µg/ml streptomycin and 100 U/ml penicillin G, and 10% (vol/vol) fetal bovine serum at 37°C in a 5% CO2-95% air incubator. Caco-2 cells were transfected in 24-well plates at 80% confluence with 200 ng of NHE3 promoter constructs, using Effectene (Qiagen). Clonally selected Caco-2 cells stably transfected with phRG-320/+58Bsd plasmid were grown in the presence of 2 µg/ml Blasticidin S (Invitrogen). Drosophila SL2 cells were maintained in Schneider's insect medium supplemented with 10% (vol/vol) fetal bovine serum and antibiotics and were grown at 25°C without CO2. SL2 cells were seeded at 500,000 cells/well in 24-well plates 24 h before transfection with 200 ng of p
Gal-320/+58 and various amounts of Sp1 or Sp3 plasmids with Effectene. All reporter gene activities were normalized to protein concentration. Cells were treated with control medium or medium supplemented with 5 mM NaB for 24 h. Since NaB stimulated reporter humanized Renilla luciferase (hrLuc) gene activity in cells transfected with promoterless vector by 30–50%, for clarity purposes values obtained from these transfections were subtracted from the activity of NHE3 promoter in respective experiments.
Gel mobility shift assays. An Sp1/3 kit (NuShift, Active Motif) was utilized for gel mobility shift assay (GMSA) analysis of Sp transcription factor binding with a 32P-labeled probe spanning the three Sp binding sites as described previously (20), with all three Sp sites intact or with sites A and C mutated to study binding to the functionally most relevant site, B (for probe sequences, see Fig. 3). Because of close spacing, SpA, -B, and -C sequences used as competitors had to be slightly modified at the 5'- and 3'-ends to prevent overlap and to provide sufficient length for binding (see Fig. 3). GMSA with –196/–175 nt probe was performed in binding buffer containing 4% glycerol, 1 mM MgCl2, 0.5 mM EDTA, 0.5 mM DTT, 50 mM NaCl, 10 mM Tris·HCl (pH 7.5), and 0.5 mg/ml poly(dI-dC)·poly(dI-dC). Binding reactions were resolved on 6% DNA retardation gels (Invitrogen). Gels were exposed to a K screen (Kodak) and imaged with Molecular Imager FX laser scanning densitometer and Quantity One software version 4.5 (both from Bio-Rad, Hercules, CA). Binding reactions were also performed with nuclear extracts from control or NaB-treated Caco-2 cells blocked by preincubation with excess antibodies against ZBP-89 (negative control) or neutralizing IgG against Sp1 or Sp3 (5 µg/reaction). Both anti-Sp1 and anti-Sp3 antibodies were verified to eliminate binding to DNA and not to cause a supershift.
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One-dimensional and two-dimensional Western blotting. Antibodies for Sp1, Sp3, and ZBP-89 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Monoclonal antibodies for HA epitope were obtained from Sigma. Nuclear protein or whole cell lysates were separated by 8% denaturing PAGE, blotted to nitrocellulose membranes, and reacted with respective antibodies according to standard protocols. One-dimensional Western blotting, with standard SDS-PAGE protocol, was utilized with total cell lysate (cells lysed with boiling Laemmli sample buffer to preserve sumoylated species of Sp3) or with isolated nuclear protein. For two-dimensional Western blotting, nuclear protein from control or NaB-treated Caco-2 cells was acetone precipitated and resuspended in sample/rehydration buffer containing 8 M urea, 2% CHAPS, 50 mM DTT, 0.2% Bio-Lyte 3/10 ampholyte, and 0.001% bromophenol blue. Protein samples (50 µg) were loaded into linear 3–10 pH range immobilized pH gradient (IPG) strips for isoelectric focusing (IEF) in a Protean IEF cell (all from Bio-Rad). To solubilize focused proteins and allow SDS binding, IPG strips were equilibrated before two-dimensional electrophoresis with 375 mM Tris·HCl, pH 8.8, 6 M urea, 2% SDS, 2% DTT for 10 min to reduce sulfhydryl groups, followed by 375 mM Tris·HCl, pH 8.8, 6 M urea, 2% SDS, 2% iodoacetamide for an additional 10-min period to alkylate sulfhydryl groups. Two-dimensional electrophoresis was performed in Criterion precast 10% acrylamide gels (Bio-Rad). Gels were transferred to nitrocellulose membrane and processed for immunoblotting with anti-Sp1 or anti-Sp3 antibodies (Santa Cruz Biotechnology) with SuperSignal West Pico (Pierce) as a chemiluminescent horseradish peroxidase substrate.
Ser/Thr phosphoprotein enrichment. Nuclear proteins from control and NaB-treated Caco-2 cells (5 mM, 24 h) were used to purify Ser/Thr phosphorylated proteins with a Pro-Q Diamond Phosphoprotein Enrichment Kit (Invitrogen) under denaturing conditions according to the manufacturer's instructions. Concentration of the phosphoprotein-enriched samples was measured by bicinchoninic acid assay, and 12.5 µg was loaded onto 10% SDS-PAGE gels, transferred to nitrocellulose, and processed for immunoblotting with anti-Sp1 or anti-Sp3 antibodies (Santa Cruz Biotechnology), as above.
Detection of Sp3 acetylation. Control and NaB-treated Caco-2 cells (5 mM, 24 h) were lysed with RIPA buffer with protease inhibitor cocktail (HALT, Pierce), and 1 mg of protein was immunoprecipitated with rabbit anti-Sp3 antibodies or control IgG. Immunoprecipitated protein was loaded onto 10% SDS-PAGE gels, blotted to nitrocellulose, blocked with 5% bovine serum albumin (BSA) and reacted with pan-acetyl antibodies (Santa Cruz Biotechnology). The blots were subsequently stripped (Restore Western Blot Stripping Buffer, Pierce) and reprobed with anti-Sp3 antibodies to confirm even loading.
Small interfering RNA transfection studies. SMARTpool small interfering RNA (siRNA) for Sp1 was purchased from Dharmacon. Lamin A siRNA (Qiagen) was used as a positive control. The respective siRNA at 50 nM was transfected with Dharmafect-4 reagent (Dharmacon) into clonally selected Caco-2 cells stably transfected with phRG-320/+58Bsd promoter construct (under Blasticidin selection). Ninety-six hours after transfection cells were treated with 5 mM NaB for 24 h. Cells were then harvested for reporter gene assay, Western blot, and real-time PCR to determine the extent of knockdown and its effects on NaB stimulation of NHE3 gene promoter.
Southwestern blotting.
Fifty micrograms of Caco-2 nuclear extract was fractionated on a 4–20% SDS-polyacrylamide gel and transferred to a nitrocellulose membrane in a buffer containing 25 mM Tris and 190 mM glycine for 30 min at 100 V. The membrane was then incubated with blocking buffer [2% nonfat dry milk, 1% BSA, 50 mM HEPES (pH 7.9), 75 mM MgCl2, 40 mM KCl, 0.05 mM EDTA, 5% glycerol, 140 mM -mercaptoethanol, and 16 µg/ml sonicated salmon sperm DNA] for 2 h and then incubated with binding buffer (same as blocking buffer but with 0.2% nonfat dry milk) containing 32P-labeled double-stranded probe (–196/–173 nt NHE3 promoter fragment; 106 cpm/ml) for 16 h, washed, and subjected to autoradiography. An identical binding reaction was performed with 100-fold excess of unlabeled probe as a negative control.
Statistical analysis. The results were statistically analyzed by ANOVA followed by Fisher protected least significant difference (PLSD) test or Student's t-test as indicated in Figs. 1, 2, 4, 7, and 8 (StatView 4.0; SAS Institute, Cary, NC). P < 0.05 was considered statistically significant.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Sp binding site is indispensable but not sufficient for maximal stimulation by NaB. A series of reporter plasmids containing various lengths of the NHE3 5'-flanking regions from nt –1360 to nt –118 (all with 3'-end at nt +58) upstream of the hrLuc gene were transiently transfected into Caco-2 cells. The levels of hrLuc activity were normalized to protein concentration, because all tested internal control reporter plasmids were stimulated by NaB treatment. As depicted in Fig. 1, promoter fragments spanning between –193 and –1360 nt were stimulated by 5 mM NaB approximately threefold, consistent with the 281% increase in NHE3 mRNA abundance reported in the colon of pectin-fed rats (31). Constructs ranging from –175 to –118 nt were marginally, yet significantly, induced by NaB (Fig. 1). These results suggested two regions within NHE3 promoter participating in the transcriptional response to NaB, located between –175 and –193 nt and downstream of –118 nt.
To further narrow the search, three hybrid promoter constructs were created with fragments spanning –320/–79 nt, –320/–33 nt, and –118/–33 nt cloned upstream of a heterologous transcription initiation site provided by the TATA box of thymidine kinase promoter. As depicted in Fig. 2, the construct containing –320/–33 nt of NHE3 promoter was maximally stimulated with NaB, whereas the –118/–33 nt construct tended toward a less significant response to SCFA treatment, consistent with data presented in Fig. 1. Surprisingly, the construct spanning –320/–79 nt was not significantly stimulated with NaB treatment, suggesting that the region –79/–33 nt (depicted as a gray overlapping bar in Fig. 2A) contained an element(s) indispensable for NaB-mediated transcriptional response.
Since we have previously described (20) dependence of NHE3 gene transcription on a cluster of three Sp binding sites located in this region (sites SpA, SpB, and SpC at –77/–36 nt), and Sp1 and Sp3 have been implicated in the mechanism of NaB action in several gene promoters, we next investigated the effects of NaB on the activity of NHE3 promoter with individually mutated Sp sites A, B, and C. As illustrated in Fig. 1, mutation of sites A and C was without effect, while mutation of site B completely eliminated the transcriptional response of NHE3 promoter to NaB. Mutation of sites A and C was also without effect on NaB-stimulated hrLuc activity in stably transfected Caco-2 cells (data not shown). Importantly, site SpB has been also shown to be important in basal transcription of NHE3 gene, although its mutation alone did not completely eliminate basal promoter activity (20). To further validate the role of Sp transcription factors in the response to NaB, we transiently transfected the Sp-driven reporter vector pSp-hrLuc into Caco-2 cells, which when treated with 5 mM NaB exhibited marked upregulation of luciferase activity (Fig. 2B).
ZBP-89 is unlikely to be involved in NaB-mediated NHE3 gene transcription. Expression of ZBP-89, a Krüppel-type zinc finger protein that participates in the activation of p21Waf1 gene transcription by NaB by binding to proximal GC-rich elements within the human promoter has been shown previously (30). Detectable expression of ZBP-89 in Caco-2 cells has been documented by others (17, 27), and it was induced by 5 mM NaB treatment in Caco-2 as evidenced by real-time PCR (our observations; data not shown). In SL2 cells cotransfected with equivalent amounts of Drosophila expression plasmid containing ZBP-89 cDNA, only a moderate increase in NHE3 promoter activity was observed (3.5 ± 0.13-fold, P < 0.05; data not shown) compared with the much more dramatic effects of overexpression of Sp1 or Sp3 (see Fig. 6 and Ref. 20). More importantly, ZBP-89 was not detected in supershift assays utilizing the –77/–36 nt region of NHE3 promoter as a probe and nuclear protein from either control or NaB-treated Caco-2 cells (data not shown). For this reason, anti-ZBP-89 antibody was used as a negative control in subsequent GMSAs.
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Expression and posttranslational modifications of Sp1 and Sp3 in response to NaB. We next evaluated the effects of NaB on Sp1 and Sp3 protein expression in Caco-2 cells. As demonstrated in Fig. 5A, expression of neither Sp transcription factor was affected. Also, we did not observe changes in mobility of Sp3 species, which could suggest major posttranslation modifications, such as sumoylation (41) (Fig. 5A). Comparison of Sp1 and Sp3 protein expression in total cell lysates (presented in Fig. 5A) and in purified nuclear fraction (data not shown) yielded similar results, suggesting that nuclear translocation of Sp1 or Sp3 was also not affected by NaB treatment. Since less extensive modifications (such as phosphorylation or acetylation) restricted to few residues in large-molecular-weight proteins such as Sp1 and Sp3 could go unnoticed based on their mobility in SDS-PAGE, two-dimensional separation was used, followed by immunodetection of Sp1 and Sp3 proteins. As demonstrated in Fig. 5B, NaB treatment resulted in a significant shift in the isoelectric point (pI) of both Sp1 and Sp3, confirming that both proteins undergo posttranslational modifications in NaB-treated cells. Since our previous observations (19) demonstrated that the effects of NaB on NHE3 promoter activity were abrogated by inhibition of Ser/Thr kinases, and Sp1 has been previously demonstrated to be regulated by phosphorylation (13), we studied expression of Sp1 and Sp3 in Ser/Thr phosphoprotein-enriched pools of nuclear protein. In purified nuclear phosphoprotein fraction Sp3 was undetectable (both from control and NaB-treated cells), suggesting that Sp3 is not phosphorylated (data not shown). However, a significantly higher amount of Sp1 was detected in the phosphorylated pool of nuclear protein purified from NaB-treated cells (Fig. 5C). This observation indicates that in response to NaB Sp1 is phosphorylated on Ser/Thr residues and that this phenomenon may participate in the induction of NHE3 promoter activity by SCFA.
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Sp3 is a more potent transactivator of NHE3 gene transcription than Sp1. Although our earlier studies (20) suggested that Sp3 is a more potent transactivator of NHE3 gene transcription than Sp1, the effects of plausible differences in expression of Sp1 and Sp3 proteins in transfected SL2 cells could not be addressed unless both proteins could be detected with the same antibody. For this reason, we tagged Sp1 and Sp3 cDNA in respective Drosophila expression vectors with HA epitope to facilitate quantitative comparison of expression by Western blotting. This was performed from the same cell lysates as those used for reporter gene assays. Figure 6 demonstrates expression of Sp1 and Sp3 in SL2 cells cotransfected with NHE3 reporter plasmid and increasing amounts of HA-tagged Sp expression vectors. At 200 ng of cotransfected Sp vectors, despite lower expression of Sp3 protein (Fig. 6A), NHE3 promoter was activated significantly more strongly by Sp3 than by Sp1 (89.1 ± 11.6-fold vs. 20.9 ± 3.5-fold, P < 0.001; Fig. 6B). These results support the hypothesis that NaB-initiated switch in binding of Sp1 and Sp3 to the proximal NHE3 gene promoter would translate into a higher transcription rate.
Partial knockdown of Sp1 by siRNA further stimulates response to NaB in stably transfected Caco-2 cells.
While siRNA transfection effectively knocked down Sp1 mRNA expression within 24 after transfection by 90% (data not shown), protein expression was not significantly affected until 96 h (64% knockdown). Therefore, Caco-2 cells stably transfected with phRG-320/+58Bsd reporter plasmid were treated with NaB between 96 and 120 h after transfection with the Sp1 siRNA pool. In Fig. 7A, we demonstrate that siRNA effectively reduced Sp1 protein expression without affecting Sp3 protein levels. Reporter gene assays indicated that reduced expression of Sp1 led to further, statistically significant increase in response of NHE3 promoter activity to NaB (Fig. 7B).
NaB inversely regulates Sp1- and Sp3-stimulated NHE3 promoter activity in SL2 cells. Since binding of Sp3 to site SpB increased in response to NaB treatment without a concomitant change in Sp1 or Sp3 protein expression in Caco-2 cells, we utilized Drosophila SL2 cells, naturally devoid of Sp-like transcription factors, as a reconstitution model to study the effects of NaB on NHE3 promoter activity in the absence or presence of individually expressed Sp1 or Sp3. Consistent with the results obtained with siRNA in stably transfected Caco-2 cells and with our overall forming hypothesis, the effects of NaB treatment on NHE3 promoter activity were reduced when Sp1 was overexpressed and significantly enhanced in Sp3-expressing SL2 cells (Fig. 8).
Identity of upstream modulator of NaB-mediated NHE3 gene transcription.
As demonstrated in Fig. 1, a cis-element located within –196/–175 nt was necessary for maximal induction of NHE3 promoter by NaB. The promoter fragment containing this element without the downstream cluster of Sp sites was not, however, sufficient to promote induction by NaB (Fig. 2). Moreover, mutation of site SpB in the –320/+58 nt construct completely abolished the effect of NaB on NHE3 promoter, thus suggesting that binding of Sp3 site may secondarily affect a trans-factor binding upstream at –196/–175 nt. Consistent with this notion, GMSA analysis with a probe containing only this sequence in isolation from downstream elements did not demonstrate any difference in protein-DNA binding (Fig. 9A). Prediction analysis of this sequence under low-stringency conditions with Match software (18) utilizing the TRANSFAC database of transcription factor binding sites (29) identified three putative proteins binding to an overlapping core TCCAG (Fig. 9, inset). These include CP2, CDP, and Ets-1 transcription factors. The formation of protein-DNA complex was abolished by excess unlabeled probe, but not by a probe with mutated core nucleotides (Fig. 9B). Southwestern blotting performed with nuclear protein isolated from Caco-2 cells and radiolabeled –196/–175 nt probe demonstrated binding to two proteins, 35 and 120 kDa in size. Binding was significantly attenuated by preincubation with excess of unlabeled probe (Fig. 9C). Neither of these two detected DNA-protein complexes had molecular mass consistent with the TRANSFAC-predicted trans-factors.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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In our previous report (19), we utilized a pGL-3 reporter vector with firefly luciferase as a reporter gene (Promega). In these studies, promoterless vector was stimulated by NaB 3- to 5-fold and maximal stimulation of NHE3 promoter activity was as high as 25-fold, a stimulation inconsistent with reported changes in NHE3 mRNA levels in vivo and in vitro. To avoid potential synergistic interactions between NHE3 promoter and cryptic cis-elements within pGL3-basic, we have tested a number of reporter plasmids and selected phRG-b containing a version of Renilla luciferase with improved codon usage and with a drastically reduced number of potential trans-factor binding sites. In this vector NHE3 gene promoter was stimulated approximately threefold, consistent with the data reported for the endogenous gene.
NaB is widely accepted as a chromatin remodeling agent, acting primarily through posttranslational modifications of histone proteins to increase accessibility of promoter and enhancer sequences for relevant transcription factors. It was therefore important to verify whether stimulation of NHE3 gene promoter by NaB was dependent on the chromatin context. Replicating episomal DNA, such as several eukaryotic viruses (bovine papilloma virus type 1, Epstein-Barr virus, Kaposi sarcoma-associated human herpesvirus 8), and even replicating plasmid transiently transfected into COS-7 cells (45) are known to assume nucleosomal structures and form minichromosomes. However, there are no data available to date that would demonstrate similar assembly of histones on nonreplicating plasmids in transient transfection. One may therefore postulate that while the effects of NaB on endogenous gene expression have a chromatin structure-dependent component, in transiently transfected cell lines these changes may not be related to histone modifications and rather represent changes in transcription factor binding and ensuing trans-activation or trans-repression. Indirectly, an identical response of the NHE3 promoter to NaB in transiently transfected cells, and in a number of clonally selected stably transfected Caco-2 cells, seems to support this hypothesis.
Our studies identified two cis-elements involved in the transcriptional response of NHE3 gene to NaB, the SpB site at nt –58/–55, and a binding site for an unknown factor in the region located at nt –196/–173. The effect of butyrate mapping to a single Sp site present in the proximal gene promoter binding Sp1 and Sp3 proteins was also described earlier by Camarero et al. (9). We took this observation further to describe that the principal component of the transcriptional response of NHE3 promoter to NaB appears to involve a switch in binding between Sp1 and Sp3 in favor of the latter, to site SpB (with the core binding sequence located on the reverse complementary strand as opposed to sites SpA and SpC). Moreover, we have demonstrated that the Sp3/Sp1 switch would have profound functional consequences, because Sp3 is a significantly more potent transactivator of NHE3 promoter. Partial siRNA-mediated knockdown of Sp1 in Caco-2 cells stably transfected with NHE3 reporter vector resulted in increased stimulation of reporter gene activity by NaB, confirming our hypothesis on the effects of NaB on the dynamic balance of Sp1 vs. Sp3. This equilibrium of Sp protein binding within the proximal promoter seems to be further validated by the fact that in rat IEC-6 small intestinal epithelial cells, Sp3 is the predominant Sp protein binding, while in Caco-2 cells binding to this region of NHE3 promoter is dominated by Sp1 (20). Consistent with this, such a switch in Sp1/Sp3 balance would be less consequential in IEC-6 cells. Indeed, our earlier observations (19) demonstrated that NaB treatment is much less effective in IEC-6 cells than in Caco-2 cells. A similar observation on NaB-induced balance in Sp1/Sp3 binding in colonic epithelial cells was recently described for the proapoptotic BAK gene (11) and postulated to participate in the mechanism of NaB-induced apoptosis in colon cancer.
In our experimental settings, neither total cellular expression of Sp1 or Sp3 nor their nuclear localization was affected by NaB. It was therefore conceivable that posttranslational modifications or protein-protein interactions are involved in this switch. Data obtained from GMSA and DAPA assays presented in Figs. 3 and 4 suggest that NaB affects both Sp1 and Sp3, albeit in a reciprocal mode. The mobility of respective Sp proteins by Western blotting in cells lysed with SDS buffer (to prevent protein degradation) was not altered. It is conceivable, however, that relatively small changes in mobility such as phosphorylation or acetylation could have gone unnoticed because of the relatively high molecular masses of both proteins. Indeed, two-dimensional immunoblotting demonstrated clearly that both Sp1 and Sp3 are postranslationally modified in NaB-treated cells, in which a significant shift in the proteins’ pI was documented (Fig. 5B).
Our previously published data (19) showed that the effect of NaB on NHE3 transcription was sensitive to H-7, a general inhibitor of Ser/Thr kinases, as well as PKA inhibitors and the dominant-negative form of the catalytic subunit of PKA, thus making a change in phosphorylation status of either Sp protein a plausible explanation. Sp1 was overrepresented in a pool of Ser/Thr nuclear phosphoproteins isolated from NaB-treated cells compared with controls (Fig. 5C), which strongly indicates that Sp1 was phosphorylated in response to NaB, consistent with our previously published data (19). Sp3 was not detectable in the phosphorylated nuclear protein fraction, confirming that this transcription factor is not significantly modified by Ser/Thr kinases. Sp1 has been shown to be phosphorylated through multiple kinase pathways, resulting in either decrease or increase in transcriptional activation, depending on the cell model and gene investigated (13). Interestingly, PKA-mediated phosphorylation has been reported to activate Sp1 in vivo and in vitro (38), a result contrasting with our observations. In previous studies, 10 µM H-89 was needed to inhibit NaB-induced NHE3 promoter activity (19), a concentration that could also have inhibited protein kinase CK2 (formerly casein kinase II). CK2 has been demonstrated to reduce Sp1 affinity for its consensus binding site by threonine phosphorylation of the zinc finger domain (3). In addition, there exists an emerging link between phosphorylation status of Sp1 and cell cycle, although at the current state of knowledge it would be premature to discuss a potential link between NaB-induced cell cycle arrest and Sp1 phosphorylation.
Sp3-mediated transcription is controlled translationally and posttranslationally via a complex set of modifications. The complete Sp3 coding region contains four in-frame start codons at positions 1, 37, 856, and 907, all of which are utilized to translate an array of proteins not only of different molecular weight but also of varying activities (41). Recent reports demonstrated that sumoylation of Sp3 can have profound effects on transcriptional activation and subnuclear localization of the transcription factors (39, 41, 42, 44). Our studies, however, showed not only that total or nuclear expression of Sp3 did not change but also that Sp3 species presumably representing different translation and sumoylation products were not affected by 24-h NaB treatment (Fig. 5), thus making sumoylation, or differential use of initiation codons, an unlikely mechanism of NaB effect. Another modification of Sp3 potentially involved in the mechanism of NHE3 promoter activation by NaB is acetylation. Acetylation of Sp3 was recently reported to be stimulated by HDAC inhibitor (1), although depending on experimental settings and the gene promoter studied the consequences of this modification varied from transcriptional activation (1) to transcriptional repression (8). Acetylation of Sp3 has also been demonstrated as a mechanism leading to a decrease in transcription of IGF-binding protein (IGFBP)-3 (47). This finding is, however, uncertain because other groups showed induction of IGFBP-3 by NaB (32, 46). Here we demonstrate by immunoprecipitation and acetyl-Lys detection that Sp3 is constitutively acetylated and that NaB treatment increased the acetylating status of this transcription factor (Fig. 5D).
Data presented in Figs. 1 and 2 suggest that while an upstream element located between nt –193/–175 is indispensable for maximal induction of NHE3 gene promoter by NaB, it is not sufficient without downstream site SpB, because mutation of the latter completely abrogated the response to butyrate. This finding implies that increased binding of Sp3 to site SpB is a preceding event and likely leads to Sp3-mediated recruitment of an unknown transcription factor to the aforementioned upstream cis-element. Consistent with this recruitment hypothesis, GMSA did not detect changes in binding to this site with NaB treatment. If such recruitment occurred, it could be verified by in vivo assays such as chromatin immunoprecipitation. Unfortunately, the identity of the transcription factor binding could not be confirmed at this stage since the two proteins detected to interact with this region in Southwestern blotting have molecular masses disparate from the transcription factors suggested by prediction analysis (Fig. 9). Future studies will be needed to identify this protein and its participation in NaB-induced colonic NHE3 expression.
In conclusion, our study demonstrates a novel mechanism underlying stimulation of NHE3 gene promoter by butyrate that involves a change of balance between Sp1 and Sp3 binding to the proximal NHE3 promoter, which favors transcriptionally more active Sp3. This event involves phosphorylation of Sp1 and acetylation of Sp3 and is followed by a recruitment of a yet unidentified transcription factor to its upstream binding site, thus bringing about a maximal stimulation of NHE3 gene transcription. Our observations, while posing many new questions about the mechanisms and pleiotropy of the effects of butyrate on gene expression in intestinal epithelial cells, provide important insight into the molecular mechanism of proabsorptive and antidiarrheal effects of this SCFA in the mammalian colon.
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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.
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TTTAG; 100x mut). C: Southwestern blotting with nuclear protein from Caco-2 cells and radiolabeled probe –196/–173 nt in absence and presence of excess unlabeled competitor. Inset depicts the sequence of the used probe with cis-element and putative binding proteins as predicted by Match analysis. + or –, DNA strand where binding site is located; molecular masses of the predicted proteins according to SDS-PAGE analysis are in parentheses.