Departments of Pediatrics and Physiology, Steele Memorial Children's Research Center, University of Arizona Health Sciences Center, Tucson, Arizona 85724
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ABSTRACT |
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Short-chain fatty acids, and especially butyrate (NaB),
stimulate sodium and water absorption by inducing colonic
Na+/H+ exchange (NHE). NaB induces NHE3
activity and protein and mRNA expression both in vivo and in vitro.
NaB, as a histone deacetylase (HDAC) inhibitor, regulates gene
transcription. We therefore studied whether NaB regulates transcription
of the rat NHE3 promoter in transiently transfected Caco-2 cells. NaB
(5 mM) strongly stimulated reporter gene activity, and this stimulation
was prevented with actinomycin D, indicating transcriptional
activation. NaB effects on the NHE3 promoter depended on the activity
of Ser/Thr kinases, in particular, protein kinase A (PKA). However, PKA
stimulation alone did not have an effect on promoter activity, and it
did not act synergistically with NaB. Another HDAC inhibitor,
Trichostatin A (TSA), stimulated NHE3 promoter in a Ser/Thr
kinase-independent fashion. The putative NaB-responsive elements were
localized within 320/
34 bp of the NHE3 promoter. These findings
suggest that PKA mediates NaB effects on NHE3 gene transcription and
that the mechanism of NaB action is different from that of TSA.
sodium-hydrogen exchanger; short-chain fatty acids; signal transduction; serine/threonine kinases; protein kinase A
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INTRODUCTION |
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SHORT-CHAIN FATTY ACIDS
(SCFAs) are produced in large amounts in the colonic lumen by
bacterial fermentation of carbohydrates that have escaped digestion in
the proximal gut (13). They play an important role in
homeostasis of the colonic mucosa by inducing pathways of cell
maturation, including cell cycle arrest, differentiation, and
apoptosis (20, 21). Of all SCFAs, butyrate (NaB)
seems to exert the most significant influence on colonocyte biology. Butyrate is the preferred fuel for the distal colonic epithelial cells
(39) and is considered as essential for maintaining
colonic health. A lack of SCFAs can lead to colonic inflammation, such as in diversion colitis (19), and reduced luminal
availability and/or impaired intracellular oxidation of butyrate have
been implicated in the pathogenesis of colonic disorders such as
ulcerative colitis (47) and pseudomembranous colitis
(18). Furthermore, inhibition of SCFA-stimulated
Na+/H+ exchange (NHE) and SCFA absorption also
contribute to diarrheal fluid losses observed in acute colitis
(7). Additionally, SCFAs are potent stimuli of sodium and
water absorption in the colon (3, 9, 24, 36, 41), with
butyrate being the most effective (24). The precise
molecular mechanism of SCFA influence on sodium absorption in the gut
is unclear. It is speculated that the SCFA-mediated increase in
Na+ absorption is due to the coupling of two exchange
mechanisms, Na+/H+ and
SCFA/Cl
exchange (4). The use
of amylase-resistant starch as an additive to oral rehydration solution
proved effective in reducing diarrheal stool output in cholera patients
(37), thus showing that SCFAs can be potent antidiarrheal agents.
Butyrate is known to modulate expression of an array of genes, and its action has traditionally been attributed to reversible inhibition of histone deacetylases (HDAC) (38). Despite the extensive knowledge about histone acetylation, the relationship between butyrate and transcriptional activation remains relatively unclear. In microarray analyses of colonic epithelial gene expression (8,063 sequences), butyrate modulated expression of a significantly larger fraction of cellular genes than a specific HDAC inhibitor, Trichostatin A (TSA) (28). These observations suggest that butyrate has multiple mechanisms of action involving more than just the inhibition of histone deacetylation traditionally ascribed to it. Because stimulation of histone H10 gene expression by butyrate could take place in the absence of de novo protein synthesis (11), it was hypothesized that one mode of sodium butyrate action involves posttranslational modification of a factor involved in the transcription process. This modification, however, as well as the target protein(s) has not yet been characterized.
NHE3 is one of six NHEs cloned to date (1, 10, 32-34). NHE3 protein is expressed on apical membranes of intestinal epithelial cells (5, 22) where it is thought to be the major absorptive NHE, 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 (43). Experiments in dogs demonstrated that NHE3 accounts for all basal ileal Na+ absorption and the neurohormonally induced increase in ileal Na+ absorption that occurs after meals (27, 48). 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) (30).
The involvement of SCFA, with butyrate in particular, in the absorption of electrolytes by the large intestine and in preventing certain types of diarrhea, increasing therapeutic potential of butyrate, as well as limited understanding of the multiple mechanisms of action of NaB on colonocyte biology and gene expression prompted us to investigate the effects of SCFAs on NHE3 gene promoter. In the present study, we demonstrated the effectiveness of NaB as an activator of NHE3 gene transcription. We also showed that activation of the NHE3 gene promoter by NaB depends on the activity of protein kinase A (PKA) and that this mechanism is different from that of a specific HDAC inhibitor, TSA. We also provide preliminary mapping of the cis element involved in SCFA regulation of transcriptional initiation of the NHE3 gene promoter.
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MATERIALS AND METHODS |
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Reagents. SCFAs were obtained from Sigma-Aldrich (St. Louis, MO). All protein kinase and phosphatase inhibitors as well as actinomycin D were purchased from Calbiochem-Novabiochem (San Diego, CA). Hydrogen peroxide-activated sodium orthovanadate was used as a broad-spectrum inhibitor of protein tyrosine phosphatases. Okadaic acid (from Proroncentrum concavum) is a potent inhibitor of Ser/Thr protein phosphatases PP1 and PP2A. Genistein (4',5,7-trihydroxyisoflavone) was used as an inhibitor of protein tyrosine kinases. H-7 {[1-(5-isoquinolinesulfonyl)-2-methylpiperazine, 2HCl]} was used as a broad-based Ser/Thr kinase inhibitor. Bisindolylmaleimide I {Gö 6850, 2-[1-(3-dimethylaminopropyl)-1H-indol-2-yl]-3-(1H-indol-3-yl)-maleimide} was used as a selective inhibitor of protein kinase C (PKC). H-89 dihydrochloride (N-[2-((p-bromocinnamyl)amino)ethyl]-5-isoquinolinesulfonamide, 2HCl), and Rp-cAMPS (adenosine 3',5'-cyclic monophosphorothioate, Rp-Isomer, triethylammonium salt) were used as selective inhibitors of PKA. Forskolin (an activator of adenylate cyclase and cAMP synthesis) and 8-Bromo-cAMP (adenosine 3',5'-cyclic monophopshate, 8-Bromo-, sodium salt; a cell permeable cAMP analog resistant to phosphodiesterases) were used to stimulate the activity of cAMP-dependent protein kinase (PKA). TSA (4,6-dimethyl-7-[p-dimethylaminophenyl]-7-oxahepta-2,4-dienohydroxaminic acid) was used as a potent and reversible HDAC inhibitor.
Reporter vector pGL3-basic, luciferase assay reagent, and all restriction enzymes were obtained from Promega (Madison, WI). Pfu proofreading DNA polymerase was purchased from Promega or Stratagene (LaJolla, CA). All other reagents were obtained from Sigma-Aldrich or Fisher Scientific (Pittsburgh, PA).Reporter construct preparation.
On the basis of the published sequence (8, 23), a 1360-
to +58-nt fragment of the 5'-flanking region of NHE3 gene was PCR
amplified from rat genomic DNA using primers with adapters containing
Sac I and Nhe I restriction enzyme sites (primers
1 and 2; Table 1). This fragment was
directionally cloned into the pGL3-basic vector with firefly
(Photinus pyralis) luciferase as a reporter gene and
confirmed by sequencing. A construct containing
1195/+58 bp of the
NHE3 promoter was prepared by digesting the above plasmid with
Sac I, blunting the 3' end with Klenow fragment of DNA
polymerase I, and subsequently digesting with Pvu II and religating. Constructs (
715/+58,
450/+58,
320/+58,
118/+58) were prepared by PCR amplification using the
1360/+58-bp construct as
a template, using a vector-specific reverse primer (primer 8; Table 1)
and primers 3, 4, 5, and 6, respectively (Table 1). For these
construsts, PCR amplicons were digested with Bgl II and
subcloned into pGL3-basic vector, which was cut with Bgl II and dephosphorylated. A construct containing
34/+58 bp of NHE3 promoter was prepared by PCR using primers 7 and 8 (Table 1). The
amplicon was digested with Bgl II and subcloned between
Sma I and Bgl II sites in the pGL3-basic vector.
A fragment of the NHE3 promoter encompassing
118/
19 bp was removed
from the
118/+58 construct by digesting it with Sma I. This fragment was subcloned in both orientations into the
Sma I site in the pTA-Luc plasmid (Clontech, Palo Alto, CA).
This vector contains the firefly luciferase reporter gene under control
of a minimal promoter (TATA-box only) of the herpes simplex virus
thymidine kinase gene, which provides the transcriptional start site.
The
118/
19-bp fragment of the NHE3 promoter was located 19 bp
upstream from the TATATAA sequence in pTA-Luc.
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Cell culture, transfection, and treatments. The HTB-37 clone of the human colonic adenocarcinoma cell line, Caco-2 (ATCC; Manassas, VA) and rat small intestinal cells IEC-6 (ATCC) were cultured in DMEM supplemented with 10% fetal bovine serum, 2 mM glutamine, 0.1 mM nonessential amino acids, 1 mM sodium pyruvate, 100 U/ml penicillin G, and 100 µg/ml streptomycin (all from Life Technologies; Rockville, MD or from Irvine Scientific; Irvine, CA). Cells were transiently transfected with the indicated plasmid at 70-80% confluency in 24-well plates using Lipofectamine (Life Technologies) according to manufacturer's protocol. Twenty-four hours after transfection, cells were washed with PBS (pH 7.4), and the medium was replaced with medium containing various SCFAs at the indicated concentration. For all studies with actinomycin D and protein kinase and phosphatase inhibitors, cells were pretreated for 1 h with the inhibitor and then treated with 5 mM sodium butyrate in the presence of the inhibitor for an additional 6 h, unless indicated otherwise in the figure legend.
Luciferase assays. Cells were washed with PBS and lysed in 100 µl of passive lysis buffer (Promega). Ten microliters of cell lysate were used for firefly luciferase assays with luciferase assay reagent (Promega) with a tube luminometer (FB12; Zylux, Maryville, TN). Relative light unit (RLU) values obtained from the assay were normalized by the amount of protein used as determined by bicinchoninic acid (BCA) protein assay (Pierce; Rockford, IL) and are expressed as RLU per microgram of protein.
PKA assay. PepTag assay for nonradioactive detection of cAMP-dependent protein kinase (Promega) was used with whole cell lysates or with nuclear protein prepared from Caco-2 cells treated with 5 mM NaB for 6 or 24 h. The assay was performed according to the manufacturer's protocol. PKA activity was quantitated by scanning densitometry of phosphorylated peptide substrate resolved in 0.8% Tris · HCl agarose (pH 8.0).
To further test the role of PKA activity in the effects of NaB on gene expression, we employed two independent reporter systems to study the PKA-mediated signaling pathway, both of them based on the premise that transactivating effects of cAMP response element (CRE) binding protein (CREB) are dependent upon PKA-mediated phosphorylation. Two sets of reporter constructs were used in this study. The first system included luciferase driven by a minimal promoter of the herpes simplex thymidine kinase gene with or without an upstream CRE enhancer (1×CRE; Mercury Pathway Profiling System, Clontech). The second system included luciferase reporter gene driven by a TATA box only or with an upstream enhancer consisting of four CRE elements (4×CRE, PathDetect CRE cis-reporting system, Stratagene). These plasmids were transiently transfected into Caco-2 cells. Cells were treated with control medium and medium supplemented with 5 mM NaB, and luciferase assays were performed as described above.Statistical analyses. The results were statistically analyzed by ANOVA followed by Fisher's protected least-significant difference test or Student's t-test as indicated in figure legends (StatView 4.0; SAS Institute, Cary, NC). P < 0.05 was considered statistically significant.
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RESULTS |
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Butyrate is a potent activator of the rat NHE3 promoter.
We first examined the effects of sodium salts of six SCFAs normally
found in the colonic lumen: acetate, propionate, butyrate, valerate,
caproate and isobutyrate. Of these six, butyrate (NaB) was the most
potent inducer of NHE3 promoter activity in transient transfections.
Acetate and isobutyrate did not affect NHE3 promoter activity (Fig.
1). The induction of the NHE3 promoter by
NaB was dose dependent, with maximal fold increase observed at
5-10 mM (Fig. 2A), which
represents concentrations within the physiological range observed in
the rodent large intestine. Statistically significant activation was
observed after 6 h of treatment with 5 mM NaB, and the induction
was further increased up to 24 h. Interestingly, we observed that
the activity of luciferase in Caco-2 cells transfected with the
promoterless vector pGL3-basic was also moderately induced by 5 mM NaB
(Fig. 3; see also Fig. 9). This
phenomenon may have been related to cryptic regulatory sites in the
luciferase gene, because similar effects were observed with pGL2-basic
(a precursor plasmid of pGL3-basic lacking a Kozak sequence and minor
modifications in the luciferase cDNA) and pTA-Luc plasmids (the latter
with a different plasmid backbone) but not with pGal-basic vector (Clontech; with
-galactosidase as a reporter gene). The induction of
luciferase expression from the promoterless pGL3-basic vector was much
slower and less pronounced than that of the NHE3 promoter constructs.
Cells transfected with pGL3-basic demonstrated no change after 6-h NaB
treatment, and there was about a three- to fivefold increase in
luciferase activity after 24 h of exposure to NaB. However, pGL3
vector with the
1360/+58-bp fragment of the NHE3 promoter was induced
by nearly 5- and 30-fold, respectively.
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Role of protein phosphatases in activation of the NHE3 promoter by
NaB.
Previous reports on the effects of NaB on gene promoter activity and
cell differentiation indicated a role for Ser/Thr protein phosphatases
(11, 14) as well as protein tyrosine phosphatases (PTP)
(42). To verify the involvement of protein phosphatase activity in the mechanism of the regulation of NHE3 gene transcription by SCFA, we studied the effect of NaB on NHE3 gene promoter activity in
the presence of okadaic acid, an inhibitor of PP1 and PP2A Ser/Thr
protein phosphatases, or sodium orthovanadate, a broad-spectrum inhibitor of PTP. Inhibition of PTP with sodium orthovanadate did not
significantly influence either unstimulated or NaB-stimulated activity
of the NHE3 promoter (Fig. 4), indicating
that tyrosine phosphatase activity is not involved in the NaB mechanism
of action. Furthermore, inhibition of PP1 and PP2A Ser/Thr protein
phosphatases with okadaic acid, which profoundly reduced both basal and
NaB-stimulated activity of NHE3 promoter, did not affect the fold
increase in NHE3 promoter activity after NaB treatment (Fig. 4).
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Protein tyrosine kinases are not involved in the activation of the NHE3 promoter by NaB. Modulation of tyrosine kinase activity has been postulated to be an important signal-transduction event in the mechanism of butyrate action in Caco-2 cells (2). The authors of the cited publication, however, used a very high dose of genistein (75 µg/ml; equivalent to 277 µM) to inhibit NaB-mediated induction of alkaline phosphatase activity. At this concentration, genistein may no longer be specific for tyrosine kinases (49). We therefore tested increasing concentrations of genistein, ranging from 10 to 277 µM on NaB-induced increase of the NHE3 promoter activity. Concentrations of genistein up to 100 µM did not affect the stimulation of NHE3 promoter by NaB. Only at 277 µM did genistein effectively inhibit this activation (data not shown). We thus concluded that this phenomenon represented nonspecific inhibition of kinase activity other than that of protein tyrosine kinases.
Protein Ser/Thr Kinases are involved in activation of the NHE3
promoter by NaB but not by TSA.
Ser/Thr kinases have been previously implicated in signal-transduction
pathways leading to activation of gene transcription by NaB
(14). Inhibition of this kinase family with 40 µM H-7 completely abolished activation of the NHE3 promoter by 5 mM NaB (Fig.
5A). This indicated that the
activity of Ser/Thr protein kinases is indispensable for NaB-mediated
induction of NHE3 promoter activity. Additionally, the NHE3 promoter
was also strongly activated by another specific inhibitor of HDAC, TSA.
Surprisingly, this activation was not reduced by H-7 either at 6 (Fig.
5A) or 24 h (not shown) exposure to 1 µM TSA. NaB and
TSA administered in combination did not have a synergistic effect on
NHE3 promoter activity (Fig. 5B).
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PKA plays a permissive role in NHE3 promoter activation by NaB.
We further demonstrated that the PKC group of Ser/Thr protein kinases
is unlikely to be involved in the mechanism of NaB action on NHE3
promoter activity, because inhibition of PKC activity with 0.1-2
µM bisindolylmaleimide I (Gö 6850) did not affect NaB-induced
activity of the NHE3 promoter (data not shown). These concentrations
are sufficient to inhibit most of the PKC isoforms (29)
except for PKC (IC50 = 5.8 µM) (29).
Further increase in Gö 6850 concentration to 20 µM resulted in
reduction of the effects of NaB on NHE3 promoter activity (data not
shown). However, at this concentration, this inhibitor may also have an
effect on PKA (IC50 = 2 µM) (44).
Consistently, inhibition of PKA activity with two specific inhibitors,
Rp-cAMPS and H-89, repressed this induction in a dose-dependent fashion
(Fig. 6A). Cotransfection of
Caco-2 cells with NHE3 reporter construct along with an expression vector encoding a dominant-negative mutant form of the regulatory subunit of PKA [MT-REV(AB); a kind gift from Dr. G. S. McKnight] resulted in a complete inhibition of NaB-mediated
induction of the NHE3 promoter (Fig. 6B). These experiments
clearly indicate that PKA activity is required for NaB action on the
NHE3 promoter. PKA activity assays in whole cell extracts of Caco-2
cells treated with 5 mM NaB for increasing amounts of time did not
demonstrate any changes in PKA-dependent phosphorylation (not shown).
PKA assays with nuclear protein extract purified from control and NaB-treated cells indicated a small, 9-10% increase in
PKA activity (Fig.
7A). In vivo
kinase assay using CRE-driven reporter vectors indicated, however, that
these small changes in PKA activity were capable of inducing
significant increases in CREB-dependent gene transcription. In these
experiments, expression of the reporter gene (luciferase) driven by a
TATA box or a minimal promoter of herpes simplex thymidine kinase (TK)
gene fused with upstream CRE enhancer (4×CRE and 1×CRE, respectively)
was significantly induced by 24-h exposure of transfected cells to 5 mM
NaB (Fig. 7, A and B). In this system, the CREB
acts as a transcriptional activator when phosphorylated by PKA
(26). Therefore, it appears that strong activation of PKA
is not a prerequisite for NaB action. We further tested whether
activation of PKA with forskolin, an activator of adenylate cyclase, or
8-Bromo-cAMP, a cell permeable, phosphodiesterase-resistant analog of
cAMP, would reproduce the effects of NaB on NHE3 promoter activity. We
demonstrated that none of these PKA activators influenced NHE3 promoter
activity (Fig. 8). These studies also did
not demonstrate a synergistic effect when coadministered with 5 mM NaB
(not shown). The efficiency of PKA stimulation in Caco-2 cells was
confirmed by forskolin treatment of cells transfected with pCRE-Luc
(Clontech), a reporter vector controlled by a minimal promoter of
herpes simplex virus thymidine kinase gene fused with an upstream CRE
(Fig. 8).
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Deletion analysis of the NHE3 promoter in Caco-2 cells.
To provide initial mapping of the putative cis elements and
to facilitate identification of transcription factor(s) involved in the
action of NaB on the NHE3 promoter, we created a series of reporter
constructs containing progressive deletions of the NHE3
promoter. Constructs containing 1360 to
320 bp of the NHE3 promoter at the 5' end [all numbers relative to the transcription start site identified by Kandasamy and Orlowski (23)]
were similarly stimulated by 5 mM NaB (Fig.
9). However, deletion of a fragment located between
320 and
118 bp reduced the responsiveness of the
promoter to NaB by ~40%, and further deletion of the fragment from
118 to
34 bp reduced NaB activation of the reporter gene to
background levels. Furthermore, a fragment encompassing
118 to
18
bp (Sma I restriction fragment) subcloned into the pTA-Luc reporter vector ~19 bp upstream of a TATA box promoted the activation of this hybrid promoter in the same fashion as observed for the
118/+58-bp construct. This transactivation was independent of the
orientation of the
118/
18-bp fragment (Fig. 9).
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DISCUSSION |
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In this report, we demonstrated that SCFAs induce transcriptional
initiation of the rat NHE3 promoter. Of the three most abundant colonic
SCFAs (acetate, propionate, and butyrate), butyrate was the most
effective, and acetate had no influence on NHE3 promoter activity. We
also showed that Ser/Thr and Tyr protein phosphatase activities,
although involved in regulation of basal activity of NHE3 promoter, do
not participate in the mechanism of NaB-mediated induction of NHE3
promoter activity. We further provide evidence that NaB action on the
NHE3 gene promoter depends on the activity of PKA. Preliminary mapping
of cis elements involved in transcriptional activation by
NaB indicated sequences located between 320 and
34 bp relative to
the transcription initiation site of the NHE3 gene promoter.
Mounting evidence indicates that SCFAs, and butyrate in particular, have a great impact on the physiology of the colonic epithelium. Beneficial effects of butyrate in pathological states such as colonic neoplasia, ulcerative colitis, diversion colitis, and colonic injury are increasingly acknowledged (46). Additionally, SCFAs provide compensatory and conservatory mechanisms in some diarrheal disorders. These effects are mediated in part by stimulating sodium, chloride, and water absorption in the colon (6). Although the mechanisms by which SCFAs regulate fluid and electrolyte fluxes are not fully understood, it is believed that stimulation of NHE is critical (3, 4, 24). Furthermore, SCFAs have been recently demonstrated to stimulate NHE3 activity and protein and mRNA expression in rat colon and in Caco-2 cells (30).
Butyrate is the preferred oxidizable fuel for colonocytes. Over 70% of butyrate is metabolized to CO2 and ketones in the colonic epithelium (12). In our studies on the rat NHE3 gene promoter in Caco-2 cells, butyrate, in physiological concentrations, was the most potent activator of promoter activity, and this activation was more prominent in colonic epithelial cells (Caco-2) than in the small intestinal epithelial cell line (IEC-6). Other tested SCFAs had less influence on NHE3 promoter, and acetate and isobutyrate were ineffective. This contrasts recently published observations by Musch et al. (30), who showed that NaB, acetate, propionate, and isobutyrate were equally effective in stimulating NHE3 activity and protein expression in Caco-2 cells. This discrepancy may result from different SCFA concentrations and incubation time (10 mM for 48 h in the above-cited study vs. 5 mM for 24 h in our report). It is also conceivable that the mechanism of action of SCFAs other than NaB on NHE3 expression and activity does not involve changes in gene transcription. The effects of acetate, propionate, and isobutyrate on NHE3 mRNA have not been reported.
Butyrate has been demonstrated to increase transfection efficiency (17). The differences observed in reporter gene activity in control and NaB-treated Caco-2 cells could not, however, be attributed to changes in transfection efficiency, because inhibition of RNA synthesis by actinomycin D abolished the effects of NaB. Also, extending the time between transfection and treatment from 24 to 48 h did not affect the fold induction of reporter gene activity by NaB (Fig. 3). These results, therefore, confirm and emphasize the importance of butyrate as a dietary factor regulating intestinal gene transcription.
Despite extensive studies, the mechanisms of NaB-mediated changes in gene transcription are still not fully understood. It has been proposed that NaB inhibits HDAC, resulting in hyperacetylation of histone proteins, modification of chromatin structure, and consequently in increased accessibility of transcription factors to their DNA recognition motifs. This hypothesis, however, does not explain why the effects of NaB on ubiquitous nuclear histone proteins and global modifications of chromatin structure affect the expression of only a very restricted set of cellular genes. In human lymphoid cells, for example, a specific inhibitor of HDAC, TSA, affects expression of only ~2% of cellular genes (8 genes of ~340 examined) (45). In a microarray analysis of 8,063 sequences, treatment of colonic epithelial cells (SW620 and Caco-2) with butyrate resulted in elevation or repression of ~7% of the analyzed sequences (28). It was interesting that in the latter study, TSA induced more limited changes with different time characteristics than those observed after NaB treatment (28). Although this might be explained by the differences in the kinetics of HDAC inhibition by butyrate and TSA (28), it may also indicate that histone hyperacetylation is not the only mechanism of action of NaB on gene transcription.
It has been speculated that in addition to chromatin structure alterations, transcriptional effects of NaB may also be due to posttranslational modifications of transcription factors (11). Only a few investigations, however, attempted to characterize the signal-transduction pathway involved in NaB-mediated regulation of gene expression and to focus on the role of cellular phosphatases and kinases. In one study, the induction of histone H1° and c-myc gene expression by NaB in hepatoma cells could be prevented by a cotreatment with calyculin A and okadaic acid, inhibitors of Ser/Thr phosphatases (11). In our experiments, however, we demonstrated that neither Tyr phosphatases (inhibited with sodium orthovanadate) nor Ser/Thr phosphatases (inhibited with the same concentration of okadaic acid as in the above-cited study) play a significant role in the NaB-mediated increase in NHE3 promoter activity. This discrepancy may be explained by cell- and gene-specific mechanisms of NaB action.
Modulation of tyrosine kinase activity has also been postulated to be an important signal-transduction event in the mediation of the cellular effects of NaB (15). Tyrosine kinases have been implicated in NaB stimulation of alkaline phosphatase in Caco-2 cells (2). In this investigation, a high concentration of genistein (75 µg/ml; 277 µM) reversed NaB stimulation of alkaline phosphatase activity. Results derived from the use of high concentrations of genistein have to be, however, interpreted with great caution, because high concentrations of this inhibitor may inhibit other kinases as well as kinase-independent systems (the latter includes lactate transport, respiratory chain activity, and aldehyde dehydrogenase) (49). In our experiments, concentrations of genistein up to 100 µM (typical concentrations found in the literature) did not affect the fold induction of promoter activity, and only 277 µM genistein reversed the effects of NaB. Considering other possible effects (not related to Tyr kinase inhibition) of genistein in this experimental setup, we tested possible involvement of protein Ser/Thr kinases using a broad-spectrum inhibitor of this family of kinases, H-7. In these experiments, cotreatment of cells with H-7 completely reversed the effect of NaB on the NHE3 promoter. We therefore demonstrated that the stimulatory action of NaB on NHE3 promoter activity requires Ser/Thr kinase activity. H-7 failed, however, to inhibit an induction of NHE3 promoter activity by a specific HDAC inhibitor, TSA, thus implicating different mechanisms of action of these two compounds. Nevertheless, the effects of TSA and NaB were not synergistic, which could indicate that although initial regulatory events for the two compounds may be different (with NaB effects being dependent on Ser/Thr kinases), TSA and NaB may share a key regulatory downstream mechanism.
Further experiments aimed at deciphering which of the Ser/Thr kinases may be involved in the stimulation of the NHE3 promoter by NaB indicated a role of cAMP-dependent PKA, because two specific inhibitors of PKA, Rp-cAMPS and H-89, as well as cotransfection with a dominant-negative form of the regulatory subunit of PKA, inhibited the induction of the promoter by NaB. Surprisingly, we detected only a very minor increase in nuclear PKA activity in NaB-treated Caco-2 cells (~10%). Experiments with CREB-dependent reporter vectors, however, indicated that even these small changes in PKA activity were sufficient to influence gene transcription. It may, therefore be speculated, that PKA activity is one of the initial elements in the signaling mechanism and that the effects of NaB are further amplified by downstream events.
Additionally, stimulation of PKA with 8-Br-cAMP or forskolin, which stimulated CREB-driven reporter gene expression (a control for effective PKA stimulation), did not stimulate NHE3 promoter activity and further did not show any synergistic effects with NaB. These experiments indicate that PKA plays a permissive role in the mechanism of NaB action, and its activation is not alone sufficient to reproduce the effects of NaB. Although this is a novel observation and it needs to be confirmed with other genes and cell systems, we speculate that PKA-dependent phosphorylation may not be the only critical signal-transduction event in the hierarchy of occurrences that leads to NaB stimulation of gene transcription.
Preliminary mapping of cis elements in the NHE3 promoter
that are involved in induction of transcription initiation by NaB indicated the sequence located between 34 and
320 bp relative to
the transcription initiation site, with elements between
34 and
118
and
119 to
320 bp being equally important. Prediction analyses of
this region of the NHE3 promoter indicated the presence of putative
binding sites for activator proteins 2 and 4 (AP2 and AP4,
respectively), hepatic nuclear factor 3b, NF
B, and Sp1. A sequence
with a weak homology (~60%) to the butyrate-response element found
in human cyclin D1 (25) and mouse calbindin-D28k (16) gene promoters was also found, partially overlapping
one of the putative AP4 binding sites. Recently, Sp1 sites of the p21/WAF1/Cip1 gene promoter have been identified as mediating the
stimulatory effect of NaB on the transcription of this gene (31), indicating that regulation of the activity of Sp
transcription factors may be part of the mechanism of NaB-mediated
regulation of transcriptional initiation. PKA has been shown to promote
phosphorylation of Sp1, increase its DNA binding activity, and promote
Sp1-dependent reporter gene expression in Sf9 cells (40).
Further experiments, however, will be needed to identify the
transcription factor(s) and the link between its activity and
PKA-dependent signaling.
In summary, we demonstrate that butyrate is a potent stimulator of the transcriptional initiation of the rat NHE3 gene promoter. We also provide evidence that this stimulation depends on the activity of protein kinase A. We further show that the mechanism of NaB action on the NHE3 promoter is distinct from that of another inhibitor of HDACs, TSA, which does not involve PKA. Activation of PKA alone is not sufficient to induce transcription of NHE3 gene promoter. This indicates that the mechanism of NaB action on the NHE3 gene transcription involves a pathway more complex than direct activation of a stimulatory transcription factor via PKA-dependent phosphorylation. Full understanding of this mechanism, and of regulation of the NHE3 promoter activity in particular, requires further investigation. The accumulating literature indicates an increasing interest in the physiological and therapeutic role of butyrate. Its role in regulating electrolyte fluxes in the mammalian intestine as well as the ability to regulate colonic butyrate production by dietary interventions and through the use of probiotics warrant more extensive studies on the influence of SCFA on colonic biology and gene expression.
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ACKNOWLEDGEMENTS |
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We thank Drs. L. Whitesell and J. Martinez for helpful discussions during preparation of this manuscript.
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FOOTNOTES |
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This investigation was funded by National Institutes of Health Grant #2R01-DK41274 and by the W.M. Keck Foundation grant.
Address for reprint requests and other correspondence: F. K. Ghishan, Dept. of Pediatrics, Director, Steele Memorial Children's Research Center, Univ. of Arizona Health Sciences Center, 1501 N. Campbell Ave., Tucson, AZ 85724 (E-mail: fghishan{at}peds.arizona.edu).
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.
Received 27 March 2001; accepted in final form 27 June 2001.
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