The epithelial apical membrane Na+/H+ exchangers [NHE (sodium hydrogen exchanger)2 and NHE3] and Cl−/HCO3− exchangers [DRA (down-regulated in adenoma) and PAT-1 (putative anion transporter 1)] are key luminal membrane transporters involved in electroneutral NaCl absorption in the mammalian intestine. During the last decade, there has been a surge of studies focusing on the short-term regulation of these electrolyte transporters, particularly for NHE3 regulation. However, the long-term regulation of the electrolyte transporters, involving transcriptional mechanisms and transcription factors that govern their basal regulation or dysregulation in diseased states, has only now started to unfold with the cloning and characterization of their gene promoters. The present review provides a detailed analysis of the core promoters of NHE2, NHE3, DRA and PAT-1 and outlines the transcription factors involved in their basal regulation as well as in response to both physiological (butyrate, protein kinases and probiotics) and pathophysiological (cytokines and high levels of serotonin) stimuli. The information available on the transcriptional regulation of the recently identified NHE8 isoform is also highlighted. Therefore the present review bridges a gap in our knowledge of the transcriptional mechanisms underlying the alterations in the gene expression of intestinal epithelial luminal membrane Na+ and Cl− transporters involved in electroneutral NaCl absorption. An understanding of the mechanisms of the modulation of gene expression of these transporters is important for a better assessment of the pathophysiology of diarrhoea associated with inflammatory and infectious diseases and may aid in designing better management protocols.
Electroneutral NaCl absorption is the predominant mechanism underlying Na+, Cl− and fluid absorption in the ileum and colon of the mammalian gastrointestinal tract and involves coupling of Na+/H+ and Cl−/HCO3− exchangers [1–3]. The molecular identity of the proteins involved in this process has only recently become apparent. It is now well established that the luminal-membrane sodium hydrogen exchange is mediated through SLC9A2 [solute carrier family 9 member A2; also called NHE2 (sodium hydrogen exchanger 2)] and SLC9A3 (also called NHE3) of the SLC9 gene family, whereas the luminal membrane Cl−/HCO3− exchange is mediated by SLC26A3 [also called DRA (down-regulated in adenoma)] and SLC26A6 [also called PAT-1 (putative anion transporter 1)] of the SLC26 gene family [3–6]. Recent studies have also identified the presence of NHE8 as another luminal-membrane NHE of IECs (intestinal epithelial cells) suggested to be important during early life . These proteins, specifically NHE2, NHE3, PAT1 and DRA, have been well characterized for their functional roles as well as their short-term regulation by in vitro studies [8–13] and knockout mouse models [14–19]. Since the normal function of these transporters is vital to intestinal fluid absorption, and disturbances in their function and expression have been associated with pathophysiology of diarrhoeal diseases, it is important to understand the molecular basis of the regulation of their gene expression in health and disease. Understanding the long-term regulation of the gene expression of these transporters is also necessary to gain insights into the mechanisms underlying tissue-specific, and the intestinal-region and vertical-axis (crypt–villus), expression of these genes. In this regard, knowledge about the regulation of the gene expression of these important transporters has started to emerge only very recently. To date, however, no comprehensive review detailing the mechanisms of transcriptional regulation of these transporters has been available. Therefore the present review focuses on advances in the promoter characterization as well as the mechanisms underlying transcriptional regulation of these transporters under basal conditions and their modulation by physiological and pathophysiological agents.
Members of the NHE gene family are the key transporters involved in the predominant Na+-absorptive pathway in the ileum and proximal colon by mediating electroneutral transport of an extracellular Na+ for a cytosolic H+. Of the nine members of the NHE gene family identified to date, NHE1, NHE2, NHE3 and NHE8 are expressed in the mammalian intestinal tract [20,21]. NHE1 is localized to the basolateral membrane of IECs, where it serves housekeeping functions such as regulation of pHi (intracellular pH) and cell-volume homoeostasis . NHE2 and NHE3 are localized to the apical membrane and play important roles in intestinal Na+ absorption with varying activities in different segments of the gastrointestinal tract . For example, NHE2 is predominantly expressed in the colon, whereas NHE3 is expressed in the ileum [14,20,23]. The importance of NHE3 in Na+ absorption and water homoeostasis in the intestine became evident from NHE3 knockout mice, which developed mild diarrhoea, low blood pressure and mild metabolic acidosis . Surprisingly, NHE2 knockout mice exhibited no apparent abnormality in intestinal Na+ absorption [16,25]. NHE8, the recently cloned isoform [21,26], has been localized to the brush-border membranes in both the kidney and IECs [21,27]. NHE8 is differentially expressed along the human gastrointestinal tract from the stomach to the colon . However, its functional role in Na+ absorption in the adult intestine is not clear at present.
Studies during the last two decades have implicated dysregulation of Na+ absorption and NHE3 down-regulation in various diarrhoeal disorders, including IBD (inflammatory bowel diseases) [29,30]. The majority of the studies on NHE regulation have focused on short-term regulation, generating a wealth of information on post-translational events of NHE3 regulation involving protein phosphorylation, trafficking, protein turnover, signal transduction pathways and interaction with other regulatory proteins [8,9]. However, the long-term regulation afforded by transcriptional mechanisms provides a basis for adaptation, and is relevant to disease states or disease intervention as well as understanding the basis for differential expression of NHE isoforms along the length of the intestine. The next section highlights our current understanding of the transcriptional mechanisms that affect the expression of NHE2, NHE3 and NHE8, which are expressed on the apical membrane of IECs. The cloning and characterization of NHE2, NHE3 and NHE8 promoters have advanced our knowledge of transcriptional regulation and have provided critical insights into NHE gene regulation.
hNHE2 (human NHE2), encoded by a single-copy gene located on chromosome 2q11.2, is composed of 11 introns and 12 exons and occupies ~90 kb of the genome . NHE2 mRNA is expressed at high levels in skeletal muscle, colon and kidney and at low levels in the testis, prostate, ovary and small intestine, indicating its tissue-specific expression . The protein product encoded by the hNHE2 gene is 812 amino acids long and when expressed in an in vitro cell-free system it shows a band of ~75 kDa , representing the unglycosylated immature form, while the mature form expressed in human intestinal C2BBE1 cells [a subclone of Caco-2 (human colon adenocarcinoma) cells] is ~85 kDa .
To date, the human and rat NHE2 promoters have been cloned [31,34]. The TIS (transcription initiation site) of hNHE2 was localized to the adenine residue 316 nt upstream from the ATG translation start codon. Comparison of the nucleotide sequences of the cloned region with the promoter databases revealed the presence of numerous potential transcription-factor-binding sites, including Sp1 (specificity protein 1), Egr-1 (early growth response protein 1), AP-2 (activator protein 2), GATA, NF-κB (nuclear factor κB), Oct-1 (octameric transcription factor 1), Cdx2 (caudal-related homeobox 2) and a number of half-sites for glucocorticoid and thyroid hormone receptors, suggesting that regulation of this gene involves a complex array of regulatory factors  (Figure 1). The human and rat NHE2 promoter sequences exhibited an overall 59% sequence homology with several conserved binding sites for transcription factors AP-2, Sp1, Egr-1, CACCC, NF-κB and Oct-1 . There were also unique motifs for other transcription-factor-binding sites, suggesting that distinct mechanisms may also be involved in the regulation of these orthologues . The human and rat NHE2 promoters both lack TATA and CAAT boxes. As is typical of all other TATA-less and CAAT-less promoters, the NHE2 promoter is highly GC-rich and contains multiple GC boxes. These motifs are targeted by Sp1 transcription factor family members as well as other transcription factors that show high affinity for GC-rich sequences, such as Egr-1, Krüpple-like factors, ZBP-89 (zinc-binding protein 89) and MAZ (myc-associated zinc finger) [35,36].
Schematic diagrams of the human NHE2 and NHE3 promoters
NHE2 core promoter region
Functional characterization of 5′-deletion constructs of the hNHE2 promoter localized the core promoter region to nucleotides −40 to +150 bp (Figure 1), suggesting that cis elements required for maximal basal transcriptional activity are located in this region. Further 5′-deletions, which caused a 70% reduction in promoter activity, indicated the critical role of GC elements contained in the deleted region (−40 to −20 bp) in promoter regulation. EMSAs (electrophoretic mobility-shift assays) and ChIP (chromatin immunoprecipitation) assays demonstrated that Sp1 and Sp3 transcription factors bound to a GC element centred at the −25 bp region [37,38]. Competition and mutational analyses identified the Sp1-b- and Sp1-c-binding sites to be essential for interaction with Sp1 and Sp3, and also suggested a co-operative role between Sp1-b/Sp1-c and downstream sequences for maximal NHE2 promoter activity  (Figure 1). The Inr (initiator) element, a functional analogue of the TATA box, in conjunction with or without a TATA box can promote transcription initiation . The sequences surrounding the NHE2 transcription initiation site exhibit a weak homology with the Inr consensus sequence  and were found to interact with the transcription factors Sp1 and Sp3 . In addition to Sp1, other proteins such as USF1 (upstream stimulatory factor 1)  and YY1 (Yin Yang 1)  may also interact with the Inr element and enhance transcription. A USF-binding site is present in the 5′-UTR (5′-untranslated region) of the hNHE2 promoter. Deletion of this site diminished basal NHE2 promoter activity .
Regulation of NHE2 by Sp1 and Sp3
Sp1 binds with high affinity to the consensus GC-box element 5′-G/TGGGCGGG/AG/AC/T-3′ [43,44]. Consistent with the involvement of Sp1 and Sp3 in the regulation of basal NHE2 transcription, multiple binding sites for Sp1 have been identified in both the human and rat NHE2 promoters [31,34,38,45,46]. The importance of Sp1 and Sp3 in promoter activity of NHE2 was also shown in co-transfection assays in Drosophila SL2 (Schneider cell line 2) cells, which are devoid of endogenous Sp1-related factors . Both Sp1 and Sp3 were capable of transactivating the NHE2 promoter, and Sp3 stimulated NHE2 to a greater extent than Sp1 .
In contrast with the stimulatory effects of Sp1 or Sp3 on the hNHE2 promoter in IECs, Sp3 inhibited, whereas Sp1 stimulated, rNHE2 (rat NHE2) transcription in renal cells . Although the rNHE2 minimal promoter (−36 to +116 bp) was actively expressed in the mouse renal cell line mIMCD-3, it was insufficient to drive transcription in either RIE cells (rat intestinal epithelial cells) or a human intestinal cell line (Caco-2) . A larger rNHE2 promoter fragment (−69 to +116 bp) was required for basal promoter activity in RIE cells. Similar to the hNHE2 promoter, both Sp1 and Sp3 displayed positive effects on rNHE2 promoter activity in IECs, whereby Sp3 exhibited a higher transactivation potential than Sp1. The lack of promoter activity of the rNHE2 −36 to +116 bp region in IECs may stem from cell-specific expression (mIMCD-3 compared with human intestinal C2BBE1 cells), as species differences did not appear to be involved. Regulation of the human and rat NHE genes by Sp1/Sp3 appears to be mediated through distinct response elements [38,46]. This is evident by the functional diversity of Sp1-binding sites in the human −40 to +150 bp and rat −36 to +116 bp promoter regions. Furthermore, the rat Sp1 elements at −69 to −36 bp, which are responsible for its expression in the IECs, are not conserved in the hNHE2 promoter sequence . Based on the current results, the basal regulation of NHE2 appears to involve various Sp1-binding sites.
The hNHE3 gene, located on chromosome 5p15.3 , is expressed in a tissue-specific manner. Northern blot analysis detected hNHE3 mRNA in decreasing amounts in the following order: kidney>small intestine>testes>ovary>colon≥prostate > thymus > peripheral leucocyte ≥ brain > spleen >placenta. The cDNA encodes a protein of 834 amino acids with a calculated molecular mass of 92906 Da . A single TIS was identified 116 nt upstream from the hNHE3 translation start site . The genomic organization of the hNHE3 gene is not fully characterized. However, in silico analysis of the hNHE3 cDNA sequence revealed that the hNHE3 gene spans >50 kb of chromosome 5 and contains 17 exons and 16 introns (J. Malakooti, unpublished work). The organization of exons and introns of the hNHE3 gene is similar to the reported rNHE3 gene [51,52]. The genomic organization of NHE8 has not been defined.
The hNHE3 promoter was characterized by cloning a ~3.2 kb human genomic DNA spanning the region −3117 to +131 bp . Analysis of the nucleotide sequences of a 1.5 kb promoter fragment upstream from the TIS showed potential consensus sequences for an atypical TATA box and multiple GC elements containing overlapping binding sites for Sp1, Egr-1 and AP-2 in the core promoter region . Further upstream, putative binding sites with relevance to the regulation of intestinal genes or tissue-specific and developmentally controlled genes, such as Cdx2, Sp1, AP-2, USF, MyoD (myogenic differentiation 1), CREBP (cAMP-response-element-binding protein), VDR (vitamin D3 receptor), TRE (thyroid hormone receptor response element), half-sites for GRE (glucocorticoid response element), and CACCC, were identified  (Figure 1).
The rNHE3 promoter was cloned by two different groups and potential transcription factor binding sites were reported [51,52]. A comparison of the nucleotide sequences of the human and rat NHE3 promoters revealed an overall sequence homology of 38%, whereas significant conservation of the nucleotide sequences (79%) and transcription-factor-binding sites were found within the proximal promoter region . Both the human and rat NHE3 promoter regions lack an authentic TATA box and CAAT box in close proximity to the TIS. Furthermore, this region is highly GC-rich in both promoters, which is typical of many developmentally and differentially regulated gene promoters . The consensus sequences for Sp1, AP-2 and CACCC, and the atypical TATA-like sequences, appeared at the same positions in both species in this region.
NHE3 core promoter
Deletion analyses of the hNHE3-promoter–reporter constructs and expression in C2BBE1 cells identified the core promoter region that extends from −88 to +5 bp and confers maximal promoter activity [50,53]. The promoter activity was reduced by 60% with further deletion to −76 bp. The sequence downstream from −76 bp represents the minimal promoter, which confers ~40% of the maximal basal promoter activity [50,53] (Figure 1).
Regulation of NHE3 by Sp1 and Sp3
The basal activity of the hNHE3 promoter is driven by the functional overlapping Egr-1/Sp1 motif contained within the −89 to −69 bp region and overlapping Sp1/AP-2/Sp1 sequences at −72 to −49 bp. Deletion or disruption of these motifs significantly decreased the promoter activity [50,53]. DNA-binding assays demonstrated that the Egr-1/Sp1 element at −89 to −69 bp binds to Sp1 and Sp3 at basal growth conditions. This motif in conjunction with downstream cis elements confers maximal transcriptional activity to the hNHE3 promoter. This site is of central importance to the basal, as well as regulated, expression of the NHE3 promoter [50,53–56].
The Sp1/AP-2/Sp1 element at −72 to −49 bp of the hNHE3 promoter was shown to bind AP-2 . The critical role of Sp1 and Sp3 in the basal expression of hNHE3 was demonstrated by siRNA (small interfering RNA) knockdown of these transcription factors in C2BBE1 cells, which caused a dramatic decrease in the basal hNHE3 promoter activity . Further, transactivation of the hNHE3 promoter by Sp1 and Sp3 was confirmed by exogenous overexpression of Sp1 and Sp3 in SL2 cells . Kiela et al.  investigated the mechanism of rNHE3 gene regulation in transiently transfected Caco-2, IEC-6, QT6 and Drosophila SL2 cells. Deletion and mutational analysis showed that the atypical TATA element in the proximal promoter region did not play a role in the transcriptional activation of the rNHE3 promoter . Three potential Sp1 motifs in the promoter region downstream from −81 bp were shown to be critical for rNHE3 promoter activity. It was also shown that Sp1 and Sp3 interact synergistically with a GATA-5 site in exon 1, leading to enhanced promoter activity, which may be important for a gradient of intestinal NHE3 expression along the crypt–villus axis . The hNHE3 promoter lacks the GATA motif in the 5′-UTR. However, the exon 1 sequence harbours two sets of direct repeat elements  and a 7-amino-acid long mini-cistron . These elements could be involved in down-regulation of NHE3 gene expression, as deletion of this 5′-UTR resulted in a moderate increase in hNHE3 promoter activity .
NHE3 expression in cultured cells has been shown to be increased during cell confluence in a STAT3 (signal transducer and activator of transcription 3)-dependent manner, which appeared to be critical for dome formation in polarized epithelial cells . Furthermore, a co-operative interaction between STAT3 and Sp1/Sp3 has been shown in this effect of STAT3 on NHE3 expression . This further emphasizes the importance of Sp1/Sp3 in NHE3 expression during cell confluence in polarized epithelial cells.
NHE8, the newly identified apical-membrane Na+/H+ exchanger, was initially cloned from a mouse kidney cDNA library . The rNHE8 cDNA encodes a protein of 575 amino acids. The protein product of this gene was detected at 65 kDa in BBM (brush-border membrane) preparations from the rat jejunal mucosa by Western blot analysis . The genomic organization of the NHE8 gene has not been characterized.
NHE8 is present intracellularly in most cells , but also in the apical membrane of epithelial cells of the intestine  and kidney , serving as a Na+/H+ exchanger . Contrary to the expression levels of NHE2 and NHE3 during development, NHE8 mRNA expression was higher in 2–3-week-old rats and lower in adult rats [21,59,60]. This suggests an important role of NHE8 in the early stages of development.
The human NHE8 promoter was recently cloned . The TIS was mapped to a guanine nucleotide 95 bp upstream from the translation initiation codon by 5′-RACE (rapid amplification of cDNA ends) analysis. Characterization of the promoter region localized the minimal promoter to a fragment spanning −32 to +17 bp with respect to the TIS, which interacts with Sp3 .
MODULATION OF NHE GENE EXPRESSION
The following sections highlight the mechanisms underlying the modulation of NHE gene expression by physiological and pathophysiological agents (Table 1).
|Butyrate||NE ||↑ [55,67–69]||ND||↑ ||ND|
|EGF||↑ [33,75]||NE ||↓ ||ND||ND|
|Glucocorticoids||NE ||↑ [51,52,70]||↓ ||ND||ND|
|PMA||↑ ||↑ ||ND||ND||ND|
|TNF||↓ ||↓ ||↓ ||ND||ND|
|IFNγ||↓ ||↓ [54, 81]||ND||↓ ||↓ |
|Butyrate||NE ||↑ [55,67–69]||ND||↑ ||ND|
|EGF||↑ [33,75]||NE ||↓ ||ND||ND|
|Glucocorticoids||NE ||↑ [51,52,70]||↓ ||ND||ND|
|PMA||↑ ||↑ ||ND||ND||ND|
|TNF||↓ ||↓ ||↓ ||ND||ND|
|IFNγ||↓ ||↓ [54, 81]||ND||↓ ||↓ |
Butyrate, a SCFA (short-chain fatty acid), is produced in the colon by microbial fermentation of dietary fibre. Butyrate exerts diverse effects on cellular functions and has been shown to regulate cell proliferation, differentiation and apoptosis . Butyrate is also implicated in suppressing inflammation and carcinogenesis . It enhances colonic NaCl absorption [65,66], and a butyrate-dependent increase in Na+ absorption was demonstrated to be mediated by both NHE2 and NHE3 in rat distal colon . Musch et al.  demonstrated that butyrate enhanced NHE3 activity in C2BBE1 cells through elevated expression of NHE3 mRNA and protein. Further, in vivo studies with rats supplemented with fibre for 2 days displayed increased 22Na+ uptake in colon secondary to enhanced NHE3 mRNA and protein levels . These findings indicated transcriptional activation of NHE3 by butyrate as the likely mechanism for the NHE3 response to butyrate. Amin et al.  demonstrated that butyrate-induced enhancement of hNHE3 mRNA expression and promoter activity in C2BBE1 cells required Sp1 and Sp3 binding to the NHE3 core promoter region (−89 to −69 bp). siRNA silencing and studies using pharmacological inhibitors of specific protein phosphatases further suggested that dephosphorylation of Sp1/Sp3 in response to butyrate may play a role in their enhanced binding affinity to the hNHE3 promoter and stimulation of NHE3 by sodium butyrate.
Sodium butyrate also activates the rNHE3 gene promoter through a serine/threonine-kinase-dependent mechanism  and involved response elements within the −320 to −34 bp region of the rNHE3 promoter. Subsequent studies identified Sp1-binding sites at −58 to −55 bp as the critical cis elements for sodium-butyrate-induced rNHE3 promoter activity. Sp1 and Sp3 were found to interact with this motif under basal growth conditions; however, DAPA (DNA affinity precipitation assay) studies with butyrate-treated nuclear proteins showed decreased Sp1 and increased Sp3 protein co-precipitation with this motif. Furthermore, butyrate treatment led to increased phosphorylation of Sp1 and acetylation of Sp3, with the latter suggested to be involved in mediating the effects of butyrate on the rNHE3 promoter activity . To date, no detailed studies are available on the effects of butyrate on NHE2 or NHE8 gene expression.
Glucocorticoids increase intestinal salt and water absorption, and NHE3 activity and mRNA abundance, in the intestine and kidney [70,71]. Methylprednisolone, a commonly used treatment for IBD, has been shown to reverse the inhibition of electrolyte and nutrient transport in animal models of IBD or patients with CD (Crohn's disease) and UC (ulcerative colitis) [72,73]. Putative cis elements for half-sites for GREs were identified in the 5′-flanking region of the hNHE3 promoter . These GREs were also found in the rNHE3 promoter, and dexamethasone, a synthetic glucocorticoid, was shown to activate the rNHE3 promoter in transiently transfected OK (opossum kidney) and pig LLC-PK1 renal epithelial cells [51,52]. Studies by Yun et al.  showed that long-term treatment of rabbits with methylprednisolone resulted in enhanced NHE3 mRNA levels in the ileum, whereas NHE1 and NHE2 expression did not change . These results suggested that the glucocorticoid effects on NHE3 were mediated by a transcriptional mechanism.
In contrast, recent studies showed that NHE8 expression was decreased in response to glucocorticoids in jejunal and ileal mucosa from rats and in Caco-2 cells . Functional analyses of the hNHE8 promoter in Caco-2 cells localized the GRE to the −89 to −32 bp region. Site-directed mutagenesis and EMSA demonstrated that Pax-5, a member of the paired box family of transcription factors, interacts with the promoter region at −75 to −50 bp and represses the promoter activity .
EGF (epidermal growth factor)
EGF administration to rats has been demonstrated to increase NHE2 mRNA in jejunal mucosa and also enhance NHE2 activity in BBMVs (brush-border membrane vesicles) prepared from rat jejunum. . Similar results were obtained in vitro in RIE cells. Furthermore, actinomycin D, a transcription inhibitor, blocked not only NHE2 mRNA expression, but also EGF-induced NHE2 activity, indicating that a transcriptional mechanism is probably responsible for the effect of EGF on NHE2 activity. Consistently, EGF also enhanced rNHE2-promoter–reporter activity in transiently transfected RIE cells . Similarly, hNHE2 transcription was also stimulated by EGF . In contrast, EGF reduced hNHE8 mRNA expression and promoter activity in Caco-2 cells . EGF-mediated repression of NHE8 promoter activity in Caco-2 cells was shown to involve the MAPKs (mitogen-activated protein kinases) ERK1/2 (extracellular-signal-regulated kinase 1/2) and Sp3 DNA-binding.
PMA, a diacylglycerol analogue, modulates diverse cellular functions through stimulation of signalling pathways such as PKC (protein kinase C) and MAPK. Long-term exposure to a low concentration of PMA was shown to stimulate the expression of both NHE2 and NHE3 mRNA and enhance their promoter activity in C2BBE1 cells [37,53]. Functional analyses and DNA-binding assays showed localization of the PREs (PMA response elements) to the −339 to −324 bp region of NHE2 and −89 to −69 bp of NHE3 promoters (Figure 1). The PREs, 5′-GCGGGGGCGGG-3′, were composed of overlapping binding sites for Egr-1 and Sp1 transcription factors, which were also identified in the promoters of other PMA target genes [77,78]. DNA-binding assays showed that Sp1 and Sp3 in the basal growth conditions and Egr-1 in the PMA-treated nuclear extract interact with PREs of both NHE2 and NHE3 promoters [37,53]. Binding of PMA-induced Egr-1 to the NHE2 and NHE3 PREs eliminated the Sp1/Sp3 interactions with these motifs, indicating that newly synthesized Egr-1 could displace the prebound Sp1/Sp3 and interact with the overlapping Egr-1/Sp1 motif. Egr-1-overexpression studies showed that Egr-1 was sufficient to induce NHE2 and NHE3 promoter activities. siRNA targeted to Egr-1 diminished the stimulatory effects of PMA on NHE3 mRNA expression [37,53].
The production of various pro-inflammatory cytokines, such as IFNγ (interferon γ), TNFα (tumour necrosis factor α) and IL-1β (interleukin 1β), in the intestine is one of the major contributing factors to the pathogenesis of inflammation-associated diarrhoea in IBD. The expression and activity of NHE3 has been shown to be suppressed by colonic inflammation  and the pro-inflammatory mediators TNFα and IFNγ . Rocha et al.  demonstrated that both in rats and C2BBE1 cells, the expression and activity of NHE2 and NHE3 were down-regulated by IFNγ. It has been shown that simultaneous addition of both TNFα and IFNγ resulted in significant reduction of NHE3 promoter activity that involved the −95 to +5 bp region of the NHE3 promoter . TNFα/IFNγ treatment led to diminished binding of Sp1/Sp3 to the Egr-1/Sp1 motif at −89 to −69 bp, whereas the level of Sp1/Sp3 protein expression was not altered. Sp1/Sp3 binding to the NHE3 proximal promoter region was also confirmed by ChIP assays . The inhibitory effect of TNFα/IFNγ on the NHE3 promoter was completely blocked by inhibitors of PKA (protein kinase A), implicating cytokine-dependent activation of PKA in the repression of NHE3 . Sp1 is known to be phosphorylated by different protein kinases, including PKA , and dephosphorylated by protein phosphatases . In this study, the binding affinity of Sp1 and Sp3 proteins for the NHE3 promoter region was found to be significantly decreased after in vitro phosphorylation of nuclear proteins by the α-catalytic subunit of PKA . These results suggested that Sp1/Sp3 were essential for NHE3 promoter activity and their phosphorylation by PKA may be involved in repression of NHE3 transcription by cytokines (Figure 2). Along the same lines, butyrate was found to restore the cytokine-induced inhibition of the NHE3 transcriptional activity and mRNA expression . These findings are important as restoration of NHE3 function in conditions associated with decreased NHE3 activity could have therapeutic relevance if defective Na+-absorptive processes could be corrected. A recent study by Yeruva et al.  found no significant difference in NHE3 mRNA and protein abundance, but NHE3 function was reduced in UC patients. However, this is in contrast with earlier studies showing decreased NHE3 mRNA and protein levels in UC . These results suggest that transcriptional regulation, in part, may be responsible for impaired Na+ absorption in IBD.
Potential mechanisms regulating hNHE2 and hNHE3 expression by pro-inflammatory cytokines in C2BBE1 cells
NHE2 mRNA and protein levels as well as its activity are repressed by long-term exposure to IFNγ  in the rat intestine and C2BBE1 cells, and to TNFα  in C2BBE1 cells. Studies using actinomycin D plus TNFα further confirmed transcriptional regulation of NHE2 . Consistent with these results, NHE2 promoter activity was also diminished by TNFα treatment. Functional characterization of the 5′-regulatory region of the NHE2 promoter localized the TNFα-responsive region and identified an NF-κB motif as the TNFα response element (−578 to −568 bp)  (Figure 1). TNFα-mediated repression of NHE2 mRNA was accompanied by reduced NHE2 protein abundance and activity (Figure 2).
NHE8 mRNA and protein levels were also reduced in jejunal mucosa from TNBS (2,4,6-trinitrobenzenesulfonic acid)- or LPS (lipopolysaccharide)-treated rats , suggesting that this isoform is also down-regulated by inflammation. In vitro studies in Caco-2 cells demonstrated TNFα-induced down-regulation of hNHE8 promoter activity through reduction of the DNA-binding activity of Sp3 . Reduced expression of NHEs in inflammation suggests that this may contribute to the diarrhoea associated with inflammatory conditions.
Serotonin [also called 5-HT (5-hydroxytryptamine)]
Serotonin, an important neurotransmitter predominantly present in the gut and brain, plays a critical role in motility, secretion and absorption in the gut . Increased levels of serotonin in the gut have been implicated in the pathogenesis of diarrhoea associated with irritable bowel syndrome and UC . Under acute conditions, serotonin decreases NHE2 and NHE3 activities in Caco-2 cells . Long-term treatment of C2BBE1 cells with serotonin resulted in a significant reduction in NHE3 mRNA and protein expression and promoter activity . This reduction in promoter activity appears to be due to reduced DNA-binding activity of Sp1/Sp3 transcription factors to the −89 to −69 bp region of the NHE3 proximal promoter. The negative impact of serotonin on NHE3 could be blocked by the PKCα antagonist Gö6976, implicating the PKCα pathway in regulation of the repressive effects of serotonin on NHE3 . To date, no data are available on the long-term effects of serotonin on NHE2 and NHE8.
Studies over the last decade have shown that DRA and PAT-1 are the candidate genes mediating intestinal-luminal Cl−/HCO3− exchange [3,6]. DRA, identified as a gene down-regulated in colonic adenomas , was shown to be the major Cl−/HCO3− exchanger involved in electroneutral NaCl absorption in the intestine [12,19]. Mutations in hDRA have been shown to result in CLD (congenital chloride diarrhoea), an autosomal recessive disorder characterized by a watery stool with high chloride concentration and metabolic alkalosis [89–91]. Similarly, the phenotype of DRA knockout mice was similar to CLD in humans . PAT-1 knockout mice showed a decrease in apical Cl−/HCO3− exchange activity, but did not exhibit a diarrhoeal phenotype . It appears that, although PAT-1 is involved in mediating Cl− absorption, it is not directly coupled to water movement as seen with DRA.
Studies have shown that the expression of both DRA and PAT-1 varies along the length of the intestine [19,93] and crypt–villus axis [93–96]. DRA protein is predominantly expressed in the colon compared with much lower levels in the small intestine [3,19,93] and is localized to the apical membranes of IECs [19,90,96]. However, the expression pattern of PAT-1 is the opposite to that of DRA expression in the intestine. PAT-1 is abundantly expressed in the small intestine compared with the colon , suggesting that PAT-1 is important for the apical Cl−/HCO3− exchange in the small intestine, whereas DRA is important in the colon . The protein product encoded by the DRA gene is a glycoprotein of 764 amino acids . The PAT-1 gene encodes an integral membrane protein of 738 amino acids . With respect to the crypt–villus–surface axis distribution, in situ hybridization studies showed expression of DRA in the surface epithelium and upper crypt of the rat and human colon . These observations correlate well with the recent studies showing that DRA Cl−/HCO3− exchange activity was higher in the upper region (surface) of the colonic crypts compared with the lower region (middle and base) . However, PAT-1 was expressed at very low levels in the crypts . Ontogenic studies in mice showed that DRA mRNA expression in the colon is low at birth, but increases during postnatal development . However, the small-intestine DRA mRNA expression remained lower throughout postnatal development . Similarly, well-differentiated post-confluent Caco-2 cells showed higher expression of DRA, with no expression in pre-confluent cells . The above studies suggest that regional, crypt–villus axis or ontogenic expression of DRA and/or PAT-1 in the intestine must be under transcriptional regulation. However, very limited studies are available regarding (long-term) transcriptional regulation of DRA and PAT-1 through changes in gene transcription. For example, a few studies have shown an inhibition of DRA expression in intestinal inflammation and in response to Citrobacter rodentium infection [93,96,100,100a]. PAT-1 expression was also decreased in response to the pro-inflammatory cytokine IFNγ . In contrast, DRA expression was shown to be increased in response to anti-inflammatory/pro-absorptive agents, such as probiotics  and butyrate . New insights into the transcriptional mechanisms of DRA and PAT-1 regulation are beginning to emerge with the cloning and characterization of the promoter regions of human DRA and PAT-1. The following sections will discuss in detail: (i) the basal characterization and regulation of DRA and PAT-1 promoters; and (ii) the transcriptional regulation of DRA and PAT-1 under both physiological and pathophysiological conditions.
A 3765 bp portion of the 5′-regulatory region of the DRA gene (promoter) was cloned by hybridization screening of a human BAC (bacterial artificial chromosome) genomic library . The DRA promoter [p-3765–Luc (luciferase)] was found to be significantly active in human LS174T colonic cancer cells  and intestinal Caco-2 cells . However, in the non-intestinal cells, HepG2 (human hepatocellular liver carcinoma) and NIH 3T3 mouse embryonic fibroblasts, the p-3765–Luc DRA promoter construct showed no activity, indicating that DRA promoter activity is intestinal-cell-line dependent. Sequence analysis of the DRA promoter and comparison with transcription-factor databases revealed the presence of potential binding sites for transcription factors such as AP-1, c-Jun, HNF4 (hepatocyte nuclear factor 4), GATA, YY1 and STAT1 (Figure 3).
Schematic diagrams of the human DRA and PAT1 promoters
Core promoter region
Luciferase reporter assays of the progressive 5′-deletions of the hDRA promoter suggested that cis elements required for basal transcriptional activity are located in the DNA region between −398 and +114 bp . Sequence analysis of the core promoter region indicated the presence of potential binding sites for transcription factors such as AP-1, c-Jun and HNF4 (Figure 3). DNase I footprint analysis and EMSA showed HNF4 may be important in driving the basal activity of the DRA promoter in LS174T human colonic cells . HNF4 binds to the consensus sequence 5′-AGGTCANAGGTCA-3′ in order to activate transcription. The role of AP-1 and c-Jun in the basal regulation of DRA promoter activity has not been studied, and future studies are needed to elucidate in detail their involvement in the basal regulation of the DRA promoter. The physiological significance of the regulation of DRA promoter activity was further validated in an in vivo mouse model . Expression of hGH (human growth hormone) driven by the DRA promoter in transgenic mice was seen specifically in the villus epithelial cells of the small intestine and in surface epithelial cells of the colon , but not in other non-intestinal tissues, suggesting that expression of DRA is specific to the intestine and differentiation status of the epithelium.
The PAT-1 gene is composed of 21 exons and 21 introns, and spans 9.7 kb of chromosome 3p21.3 . The SLC26A6 gene encodes an integral membrane protein of 738 amino acids with a predicted topology of 11 transmembrane helices and an intracellular –NH2 and –COOH terminus [98,105]. The human SLC26A6 gene was also found to have three alternatively spliced variants, named SLC26A6a, c and d. With regard to their tissue distribution, RT (reverse transcription)–PCR studies indicated that SLC26A6a, but not c or d, is the spliced variant expressed in the human small intestine and colon . The human PAT1 isoforms a, c and d are composed of 12, 8 and 12 membrane-spanning domains respectively.
The 5′-flanking region of the PAT-1 gene was cloned by PCR, using human genomic DNA and gene-specific primers . The TIS was localized to a guanine residue 99 nt upstream from the ATG translation start codon. Studies showed that PAT-1 promoter activity (1120 bp; p-964/+156 bp) was high in the Caco-2 cell line (which, on differentiation, exhibits a small-intestinal phenotype) compared with NCM460 (normal human colon mucosal epithelial) and HEK (human embryonic kidney)-293 cells, consistent with the fact that PAT-1 is expressed predominantly in the small intestine . Sequence analysis of the PAT-1 promoter showed consensus sites for various transcription factors, such as HNF1α, YY1, Sp1, CdxA, AP-2, Mzf1 (myeloid zinc finger 1), MyoD and Egr-1 (Figure 3).
Core promoter region
Progressive deletions from the 5′-flanking region demonstrated that deletions from −714 to −214 bp did not exhibit any change in promoter activity compared with the full-length PAT-1 promoter construct (−964 to +156). Further deletion to −44 bp significantly decreased promoter activity by ~80%, indicating that the region between −214 and +156 bp harbours cis elements important for the basal activity of the PAT-1 promoter . Sequence analysis of this region revealed the presence of potential binding sites for HNF1α, YY1 and Sp1 (Figure 3). More detailed studies are needed to further define the regulation of PAT-1 by these transcription factors. Besides human promoters, there are no reports on the cloning and characterization of DRA or PAT-1 promoters in other species.
MODULATION OF DRA AND PAT1 GENE EXPRESSION
Long-term regulation of gene expression generally mimics the prolonged physiological or pathophysiological changes that occur in the intestine, such as in response to the luminal SCFA butyrate or intestinal diseases (e.g. IBD). The following section will focus on the transcriptional regulation of DRA and PAT-1 gene expression in response to both physiological and pathophysiological stimuli (Table 1).
A previous study has shown that butyrate stimulates DRA expression and promoter activity in LS174T colonic cells . Progressive 5′-deletions of the DRA promoter showed that the butyrate-responsive region was located between −688 and −398 bp. DNase I footprint analysis and EMSA identified a region from −468 to −487 bp that harboured two overlapping potential binding sites for YY1 and GATA, indicating their involvement in butyrate-induced stimulation of DRA promoter activity . Competition studies showed that the YY1 consensus oligonucleotide completely abolished YY1 binding and also decreased GATA binding. On the other hand, the GATA consensus oligonucleotide did abolish GATA binding but increased YY1 binding, indicating that YY1 and GATA may influence the binding of each other. Mutations in the potential YY1- and GATA-binding sites attenuated the stimulatory effects of butyrate on promoter activity, further suggesting that the binding of YY1 and GATA transcription factors is crucial for butyrate-induced stimulation of the DRA promoter . Interestingly, YY1 binding to the DRA promoter was competed by a 20 bp fragment from the promoter region of the fabp (fatty-acid-binding protein) gene that was previously known to silence the expression of FABP in the crypt epithelium of the small intestine . It seems that YY1 may be critical for silencing the expression of DRA in proliferating undifferentiated IECs and may play an important role in determining their expression pattern along the crypt–surface axis in the colon. GATA-5 was shown to modulate the promoter activity of rat NHE3 and was suggested to play a role in its differential expression along the crypt–villus axis . The GATA isoform involved in butyrate-induced effects on DRA promoter activity is not known . Future studies are needed to elucidate the interplay of GATA and YY1 and to identify the isoform of GATA involved in butyrate-induced effects on DRA promoter activity. Also, the mechanism of regulation of PAT-1 gene expression by butyrate is not known.
LA (Lactobacillus acidophilus)
Lactobacilli are one of the predominant commensal bacteria in the gut microflora and have previously been used for the prevention and treatment of diarrhoeal disorders . Recent studies using Caco-2 monolayers have shown that LA stimulated DRA mRNA and protein expression . However, PAT-1 mRNA and protein expression remained unaltered upon LA treatment. In vivo mouse studies also demonstrated a significant increase in DRA mRNA and protein levels  in response to LA. CS (culture supernatants) of live LA bacteria showed similar effects to live bacteria and stimulated both DRA expression and function in Caco-2 monolayers . The effects of LA CS were also seen at the transcriptional level. LA CS significantly increased DRA promoter activity in Caco-2 cells . These studies suggested that stimulation of DRA promoter activity and expression by LA-secreted soluble effector molecules may contribute to the up-regulation of intestinal Cl− absorption and provide novel insights into the mechanisms underlying the potential antidiarrhoeal effects of LA. However, the molecular mechanisms involved in the transcriptional regulation of DRA by LA-secreted soluble factors are not known. Further studies are needed to identify the cis elements and transcription factors involved in the regulation of DRA promoter activity by LA-secreted soluble factors and to elucidate the identity of soluble factors involved in the stimulation of DRA promoter activity.
A recent study has demonstrated that chloride absorption is reduced in patients with UC . The reduction in Cl− absorption in the surface of the colonic crypts was consistent with a decrease in DRA mRNA expression in UC patients compared with normal . A decrease in DRA mRNA expression has also been reported in the caecum and colonic regions of two different animal models of colitis: IL-10 knockout mice and HLA-B27/β2M transgenic rats . In vitro studies of the response to pro-inflammatory cytokines using Caco-2 cells have also shown a decrease in DRA expression by IL-1β  and a decrease in both DRA and PAT-1 expression by IFNγ [100,101]. The transcriptional mechanisms underlying repression of DRA and PAT1 by IFNγ have been investigated in detail as outlined below.
IFNγ elicits its effects through the induction of a signal transduction pathway involving JAK1 (Janus kinase 1), JAK2 and STAT1. The binding of IFNγ to its surface receptor activates the receptor-associated tyrosine kinases JAK1 and JAK2. JAKs tyrosine-phosphorylate and activate the latent cytosolic STAT1, which then dimerizes and translocates to the nucleus , where it binds to IFNγ response elements [109,110]. IFNγ decreased DRA promoter activity in Caco-2 cells via a JAK/STAT1 pathway . Progressive deletions from the 5′-flanking region of the DRA promoter showed that the IFNγ-responsive region was located in the −1183 to −790 bp region . Sequence analysis of this region identified one potential GAS (γ-activated site) cis element flanking the region −933 to −925 bp. The GAS element is an 8–10 bp inverted-repeat DNA element with a consensus sequence of 5′-TT(N4-6) AA-3′ . Additionally, an EMSA showed increased protein binding to the oligonucleotide spanning the potential GAS element (−933 to −925 bp) in response to IFNγ, which was blocked by an anti-STAT1 antibody, suggesting that the identified protein is STAT1 . Also, mutational studies and in vivo ChIP assays showed that the GAS element is required for the inhibitory effects of IFNγ on DRA promoter activity. These findings provided evidence for the role of STAT1 in the inhibition of DRA promoter activity by IFNγ  (Figure 4). Increased expression and activation of STAT1 has been observed in the colonic mucosa of patients with UC and, to a lesser degree, in CD . In fact, the anti-inflammatory effects of glucocorticoids have been suggested to occur through inhibition of STAT1 in IBD patients . Therefore the development of novel therapeutic modalities against STAT1 may be of importance in the treatment of colonic inflammation where DRA expression and function are down-regulated.
Proposed model for inhibition of DRA and PAT-1 promoter activity by IFNγ
In contrast with the role of STAT1 in IFNγ-mediated effects on DRA promoter activity, PAT-1 promoter activity was also decreased by IFNγ, albeit via a distinct cis element/transcription factor . Progressive deletions from the 5′-flanking region of the PAT-1 promoter showed the presence of a potential IFNγ response element(s) region between −414 and −214 bp . Sequence analysis of the IFNγ-responsive region identified one potential ISRE (interferon-stimulated response element) binding site flanking the region −318 to −300 bp. ISREs, usually defined by palindromic TTTC sequences separated by two or three nucleotides, are located in the promoter region of many IFNγ-inducible genes . EMSAs also showed increased protein binding to the oligonucleotide spanning the potential ISRE site (−318 to −300 bp) in the presence of IFNγ. Additionally, supershift assays with antibody against IRF-1 (interferon regulatory factor 1) transcription factor showed that the DNA-protein binding was blocked, indicating that IRF-1 binds to the ISRE site . This was further confirmed by ChIP assays showing that the association of IRF-1 with endogenous PAT-1 promoter (containing the ISRE site) was significantly enhanced by IFNγ. Mutations in the potential ISRE cis element completely attenuated the inhibitory effects of IFNγ on promoter activity, further suggesting the involvement of IRF-1 in the regulation of PAT-1 promoter activity by IFNγ  (Figure 4). IRF-1 expression is increased in the lamina propria of patients with active CD . Since, CD commonly affects the ileum, it is plausible to speculate that IRF-1 plays a key role in the inflammation of ileum. Immunosuppressive treatment (azathioprine or 6-mercaptopurine for 24 h) of patients with CD has been shown to reduce IRF-1 . Although, the role of PAT-1 in intestinal inflammation has not been established, targeting of IRF-1 as a therapeutic alternative may be important in the treatment of ileal inflammation where PAT-1 expression and function (Cl− absorption) are inhibited.
The advent of cloning and the use of transgenic mouse models have significantly enhanced our understanding of the molecular mechanisms underlying NaCl absorption and its regulation in health and disease. It is now accepted that NHE2, NHE3, DRA and PAT1 are the predominant luminal membrane Na+- and Cl−-absorbing proteins involved in vectorial NaCl absorption in the adult mammalian intestine. Recent studies have also provided strong evidence that in addition to short-term regulation of these transporters, long-term regulation has strong implications in tissue-, region- and differentiation-specific expression of these genes under physiological conditions. Studies from disease models and patients have further shown that repression of NHE3 and DRA expression are associated with infectious diarrhoea or IBD. Until recently, detailed mechanistic understanding of the long-term regulation of these transporters was very limited. However, with the cloning of the promoters of these transporters, new data on their transcriptional regulation have now started to emerge. These data have identified and characterized the core promoter, the predicted transcription-factor-binding sites and the transcription factors involved in basal transcription and regulation of these genes by various physiological and pathophysiological agents. These studies demonstrate that basal transcriptional regulation of both hNHE2 and hNHE3 involves the transcription factors Sp1 and Sp3. Although AP-2 was also found to interact with the NHE3 promoter, the functional significance of this binding remains to be investigated. The studies further demonstrated that diverse effector molecules, e.g. butyrate, phorbol esters, IFNγ, TNFα and serotonin, converge at a site containing the overlapping Egr-1/Sp1 motif in the NHE3 promoter to trigger distinct outcomes. However, the detailed molecular mechanisms as to how these agents exert distinct effects on the NHE3 promoter remains to be investigated. In contrast with NHE3, NHE2 repression by inflammatory cytokines was found to involve activation of the NF-κB pathway. However, it is unclear whether NF-κB represses NHE2 promoter activity via direct binding or through a putative downstream transcription factor. With respect to transcriptional regulation of DRA and PAT-1, the available data are even more sketchy. This has been mainly due to the very recent identification of their role in intestinal chloride absorption. In addition, the cloning of their promoters was accomplished only during the last 5 years. It is now known that HNF4 is involved in the basal transcription of DRA, although its stimulation by butyrate involved GATA and YY1 factors. However, the exact GATA isoform involved in the regulation of DRA promoter and the interplay between GATA and YY1 needs more extensive investigation. The basal transcriptional regulation of PAT-1 has not been characterized; however, sequence analysis of the promoter region predicted that HNF1α, YY1 and Sp1 might play important roles. Studies have further shown that the cytokine IFNγ down-regulated the expression of both the DRA and PAT-1 genes, albeit via distinct mechanisms. Whereas the activation of STAT1 via the JAK–STAT pathway and its direct binding to the GAS element mediated the effect of IFNγ on the DRA promoter, the activation of IRF-1 by STAT1 and subsequent binding of IRF-1 to the ISRE was involved in the suppression of the PAT1 promoter by IFNγ. Repression of both PAT1 or DRA and NHE3 promoter activities in response to IFNγ may lead to decreased NaCl absorption in the ileum and colon. The overall decrease in NaCl absorption may play a crucial role in the pathophysiology of diarrhoea associated with IBD. Taken together, these results indicate that down-regulation of NHE2, NHE3, DRA and PAT1 expression and the decrease in the promoter activities in response to inflammatory mediators may contribute to pathophysiology of diarrhoea associated with inflammatory disorders of the gut. On the other hand, mechanisms involved in the induction of gene expression of NHE3 or DRA by agents such as butyrate, soluble factors secreted by probiotics or glucocorticoids need to be investigated in detail, as these could be exploited to improve the treatment modalities of IBD. Furthermore, a detailed characterization of the newly identified NHE8 may also add more to our understanding of NaCl absorption and its regulation in health and disease as well as in developing and adult intestine. Finally, the role of epigenetic mechanisms in regulating the expression of intestinal Na+ and Cl− transporters is not fully understood and warrants future investigations.
We thank Alip Borthakur, Ph.D. and Waddah A. Alrefai, M.D. for critical editing of the review.
activator protein 2
- Caco-2 cell
human colon adenocarcinoma cell
caudal-related homeobox 2
congenital chloride diarrhoea
down-regulated in adenoma
epidermal growth factor
early growth response protein 1
electrophoretic mobility-shift assay
glucocorticoid response element
hepatocyte nuclear factor
inflammatory bowel diseases
intestinal epithelial cell
interferon regulatory factor 1
interferon-stimulated response element
mitogen-activated protein kinase
myc-associated zinc finger
myogenic differentiation 1
myeloid zinc finger 1
nuclear factor κB
sodium hydrogen exchanger
octameric transcription factor 1
putative anion transporter 1
protein kinase A
protein kinase C
PMA response element
- RIE cell
rat intestinal epithelial cell
short-chain fatty acid
small interfering RNA
specificity protein 1
signal transducer and activator of transcription
transcription initiation site
tumour necrosis factor α
thyroid hormone receptor response element
upstream stimulatory factor
Yin Yang 1
Studies in the authors' laboratories were supported in part by the U.S. Department of Veterans Affairs and the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) [grant numbers DK 33349 (to J.M.), DK 54016 and DK 81858 (to P.D.), P01 DK 067887 (to P.D. and J.M.) and DK 74458 (to R.G.)] and the Crohn's and Colitis Foundation of America (CCFA) [grant number 1942 (to S.S.)].
These authors contributed equally to this work as senior authors.