Hepatocyte nuclear factor 4α regulates the expression of intestinal epithelial Na+/H+ exchanger isoform 3.

Na+/H+ exchanger isoform 3 (NHE3) plays a key role in coupled electroneutral NaCl absorption in the mammalian intestine. Reduced NHE3 expression or function has been implicated in the pathogenesis of diarrhea associated with inflammatory bowel disease (IBD) or enteric infections. Our previous studies revealed transcriptional regulation of NHE3 by various agents such as TNF-α, IFN-γ, and butyrate involving transcription factors Sp1 and Sp3. In silico analysis revealed that the NHE3 core promoter also contains a hepatocyte nuclear factor 4α (HNF-4α) binding site that is evolutionarily conserved in several species suggesting that HNF-4α has a role in NHE3 regulation. Nhe3 mRNA levels were reduced in intestine-specific Hnf4α-null mice. However, detailed mechanisms of NHE3 regulation by HNF-4α are not known. We investigated the regulation of NHE3 gene expression by HNF-4α in vitro in the human intestinal epithelial cell line C2BBe1 and in vivo in intestine-specific Hnf4α-null ( Hnf4αΔIEpC) and control ( Hnf4αfl/fl) mice. HNF-4α knockdown by short interfering RNA in C2BBe1 cells significantly decreased NHE3 mRNA and NHE3 protein levels. Gel mobility shift and chromatin immunoprecipitation assays revealed that HNF-4α directly interacts with the HNF-4α motif in the NHE3 core promoter. Site-specific mutagenesis on the HNF-4α motif decreased, whereas ectopic overexpression of HNF-4α increased, NHE3 promoter activity. Furthermore, loss of HNF-4α in Hnf4αΔIEpC mice decreased colonic Nhe3 mRNA and NHE3 protein levels. Our results demonstrate a novel role for HNF-4α in basal regulation of NHE3 expression. These studies represent an important and novel target for therapeutic intervention in IBD-associated diarrhea. NEW & NOTEWORTHY Our studies for the first time show that hepatocyte nuclear factor 4α directly regulates NHE3 promoter activity and its basal expression in the intestine.

Translational repression of SLC26A3 by miR-494 in intestinal epithelial cells.

SLC26A3 [downregulated in adenoma (DRA)] is a Cl(-)/HCO3(-) exchanger involved in electroneutral NaCl absorption in the mammalian intestine. Altered DRA expression levels are associated with infectious and inflammatory diarrheal diseases. Therefore, it is critical to understand the regulation of DRA expression. MicroRNAs (miRNAs) are endogenous, small RNAs that regulate protein expression via blocking the translation and/or promoting mRNA degradation. To investigate potential modulation of DRA expression by miRNA, five different in silico algorithms were used to predict the miRNAs that target DRA. Of these miRNAs, miR-494 was shown to have a highly conserved putative binding site in the DRA 3'-untranslated region (3'-UTR) compared with other DRA-targeting miRNAs in vertebrates. Transfection with pmirGLO dual luciferase vector containing DRA 3'-UTR (pmirGLO-3'-UTR DRA) resulted in a significant decrease in relative luciferase activity compared with empty vector. Cotransfection of the DRA 3'-UTR luciferase vector with a miR-494 mimic further decreased luciferase activity compared with cells transfected with negative control. The transfection of a miR-494 mimic into Caco-2 and T-84 cells significantly increased the expression of miR-494 and concomitantly decreased the DRA protein expression. Mutation of the seed sequences for miR-494 in 3'-UTR of DRA abrogated the effect of miR-494 on 3'-UTR. These data demonstrate a novel regulatory mechanism of DRA expression via miR-494 and indicate that targeting this microRNA may serve to be a potential therapeutic strategy for diarrheal diseases.

Extracellular acidosis stimulates NHE2 expression through activation of transcription factor Egr-1 in the intestinal epithelial cells.

Na(+)/H(+) exchangers (NHEs) play important roles in regulating internal pH (pHi), cell volume and neutral Na(+) absorption in the human intestine. Earlier studies have shown that low extracellular pH (pHe) and metabolic acidosis increases the expression and function of NHE1-3 genes. However, transcriptional mechanisms involved remained unknown. Therefore, we investigated the molecular mechanisms underlying acid-induced NHE2 expression in C2BBe1 and SK-CO15 intestinal epithelial cells. Assessing total RNA and protein by RT-PCR and Western blot analysis, respectively, displayed significant increases in the NHE2 mRNA and protein levels in cells exposed to acidic media (pH 6.5 and 6.7) compared to normal medium. Acid treatment was also associated with a significant enhancement in NHE2 transport activity. Quantification of the heterogeneous nuclear RNA indicated that the rate of NHE2 transcription was increased in response to acid. Furthermore, acid caused a significant increase in NHE2 promoter activity confirming transcriptional upregulation. Through functional and mutational studies the acid-response element was mapped to a 15-nucleotide GC-rich sequence at bp -337 to -323 upstream from the transcription start site. We previously identified this element as an overlapping Egr-1/Sp1/Egr-1 motif that was essential for the NHE2 upregulation by mitogen-induced transcription factor Egr-1. Cells exposed to acid exhibited a temporal increase in Egr-1 mRNA and protein expression. These events were followed by Egr-1 nuclear accumulation, as detected by immunofluorescence microscopy, and potentiated its in vitro and in vivo interaction with the NHE2 promoter. Disruption of ESE motif and knockdown of Egr-1 expression by targeted small interfering RNA abrogated the acid-induced NHE2 transcriptional activity. These data indicate that the acid-dependent NHE2 stimulation is implemented by transcriptional upregulation of NHE2 via acid-induced Egr-1 in the intestinal epithelial cells.

Lactobacillus acidophilus upregulates intestinal NHE3 expression and function.

A major mechanism of electroneutral NaCl absorption in the human ileum and colon involves coupling of Na(+)/H(+) and Cl(-)/HCO(3)(-) exchangers. Disturbances in these mechanisms have been implicated in diarrheal conditions. Probiotics such as Lactobacillus have been indicated to be beneficial in the management of gastrointestinal disorders, including diarrhea. However, the molecular mechanisms underlying antidiarrheal effects of probiotics have not been fully understood. We have previously demonstrated Lactobacillus acidophilus (LA) to stimulate Cl(-)/HCO3- exchange activity via an increase in the surface levels and expression of the Cl(-)/HCO3- exchanger DRA in vitro and in vivo. However, the effects of LA on NHE3, the Na(+)/H(+) exchanger involved in the coupled electroneutral NaCl absorption, are not known. Current studies were, therefore, undertaken to investigate the effects of LA on the function and expression of NHE3 and to determine the mechanisms involved. Treatment of Caco2 cells with LA or its conditioned culture supernatant (CS) for 8-24 h resulted in a significant increase in Na(+)/H(+) exchange activity, mRNA, and protein levels of NHE3. LA-CS upregulation of NHE3 function and expression was also observed in SK-CO15 cells, a human colonic adenocarcinoma cell line. Additionally, LA treatment increased NHE3 promoter activity, suggesting involvement of transcriptional mechanisms. In vivo, mice gavaged with live LA showed significant increase in NHE3 mRNA and protein expression in the ileum and colonic regions. In conclusion, LA-induced increase in NHE3 expression may contribute to the upregulation of intestinal electrolyte absorption and might underlie the potential antidiarrheal effects of probiotics.

PKCδ-dependent activation of ERK1/2 leads to upregulation of the human NHE2 transcriptional activity in intestinal epithelial cell line C2BBe1.

The apical Na+/H+ exchanger (NHE) isoform NHE2 is involved in transepithelial Na+ absorption in the intestine. Our earlier studies have shown that mitogenic agent phorbol 12-myristate 13-acetate (PMA) induces the expression of NHE2 through activation of transcription factor early growth response-1 (Egr-1) and its interactions with the NHE2 promoter. However, the signaling pathways involved in transcriptional stimulation of NHE2 in response to PMA in the intestinal epithelial cells are not known. Chemical inhibitors and genetic approaches were used to investigate the signaling pathways responsible for the stimulation of NHE2 expression by PMA via Egr-1 induction. We show that, in response to PMA, PKCδ, a member of novel PKC isozymes, and MEK-ERK1/2 pathway of mitogen-activated protein kinases stimulate the NHE2 expression in C2BBe1 intestinal epithelial cells. PMA rapidly and transiently induced activation of PKCδ. Small inhibitory RNA-mediated knockdown of PKCδ blocked the stimulatory effect of PMA on the NHE2 promoter activity. In addition, blockade of PKCδ by rottlerin, a PKCδ-specific inhibitor, and ERK1/2 by U0126, a MEK-ERK inhibitor, abrogated PMA-induced Egr-1 expression. Immunofluorescence studies revealed that inhibition of ERK1/2 activation prevents translocation of PMA-induced Egr-1 into the nucleus. Consistent with these data, PMA-induced Egr-1 interaction with the NHE2 promoter region was prevented in nuclear extracts from U0126-pretreated cells. In conclusion, our data provide the first evidence that the stimulatory effect of PMA on NHE2 expression is mediated through the initial activation of PKCδ, subsequent PKCδ-dependent activation of MEK-ERK1/2 signaling pathway, and stimulation of Egr-1 expression. Furthermore, we show that transcription factor Egr-1 acts as an intermediate effector molecule that links the upstream signaling cues to the long-term stimulation of NHE2 expression by PMA in C2BBe1 cells.

Transcriptional regulation of the intestinal luminal Na⁺ and Cl⁻ transporters.

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.

Epidermal growth factor upregulates serotonin transporter in human intestinal epithelial cells via transcriptional mechanisms.

Serotonin transporter (SERT) regulates extracellular availability of serotonin and is a potential pharmacological target for gastrointestinal disorders. A decrease in SERT has been implicated in intestinal inflammatory and diarrheal disorders. However, little is known regarding regulation of SERT in the intestine. Epidermal growth factor (EGF) is known to influence intestinal electrolyte and nutrient transport processes and has protective effects on intestinal mucosa. Whether EGF regulates SERT in the human intestine is not known. The present studies examined the regulation of SERT by EGF, utilizing Caco-2 cells grown on Transwell inserts as an in vitro model. Treatment with EGF from the basolateral side (10 ng/ml, 24 h) significantly stimulated SERT activity (∼2-fold, P < 0.01) and mRNA levels compared with control. EGF increased the activities of the two alternate promoter constructs for human SERT gene: SERT promoter 1 (hSERTp1, upstream of exon 1a) and SERT promoter 2 (hSERTp2, upstream of exon 2). Inhibition of EGF receptor (EGFR) tyrosine kinase activity by PD168393 (1 nM) blocked the stimulatory effects of EGF on SERT promoters. Progressive deletions of the SERT promoter indicated that the putative EGF-responsive elements are present in the -672/-472 region of the hSERTp1 and regions spanning -1195/-738 and -152/+123 of hSERTp2. EGF markedly increased the binding of Caco-2 nuclear proteins to the potential AP-1 cis-elements present in EGF-responsive regions of hSERTp1 and p2. Overexpression of c-jun but not c-fos specifically transactivated hSERTp2, with no effects on hSERTp1. Our findings define novel mechanisms of transcriptional regulation of SERT by EGF via EGFR at the promoter level that may contribute to the beneficial effects of EGF in gut disorders.

Tumor necrosis factor-α represses the expression of NHE2 through NF-κB activation in intestinal epithelial cell model, C2BBe1.

BACKGROUND: High levels of proinflammatory cytokines are linked to pathogenesis of diarrhea in inflammatory bowel disease (IBD). Na(+) absorption is compromised in IBD. The studies were designed to determine the effect of tumor necrosis factor-α (TNF-α) on the expression and activity of NHE2, a Na(+) /H(+) exchanger (NHE) that is involved in transepithelial Na(+) absorption in intestinal epithelial cells. METHODS: NHE2 regulation was examined in TNF-α-treated C2BBe1 cells by reverse-transcription polymerase chain reaction (RT-PCR), reporter gene assays, and Western blot analysis. NHE isoform activities were measured as ethyl-isopropyl-amiloride- and HOE694-sensitive (22) Na-uptake. In vitro and in vivo protein-DNA interactions were assessed by gel mobility shift assays and chromatin immunoprecipitation studies. RESULTS: TNF-α treatment of C2BBe1 cells led to repression of NHE2 promoter activity, mRNA, and protein levels; and inhibited both NHE2 and NHE3 mediated (22) Na-uptake. 5'-deletion analysis of the NHE2 promoter-reporter constructs identified basepair -621 to -471 as the TNF-α-responsive region (TNF-RE). TNF-α activated NF-κB subunits, p50 and p65, and their DNA-binding to a putative NF-κB motif within TNF-RE. Mutations in the NF-κB motif abolished NF-κB-DNA interactions and abrogated TNF-α-induced repression. Ectopic overexpression of NF-κB resulted in repression of NHE2 expression. Two functionally distinct inhibitors of NF-κB blocked the inhibitory effect of TNF-α. CONCLUSIONS: The human NHE2 isoform is a direct target of transcription factor NF-κB. TNF-α-mediated activation of NF-κB decreases the expression and activity of NHE2 in the intestinal epithelial cell line, C2BBe1. These findings implicate NF-κB in the modulation of Na(+) absorption during intestinal inflammatory conditions such as IBD where a high level of TNF-α is detected.

Transcriptional regulation of the human Na+/H+ exchanger NHE3 by serotonin in intestinal epithelial cells.

Serotonin (5-HT) decreases NHE2 and NHE3 activities under acute conditions in human intestinal epithelial cells. Here, we have investigated the effects of 5-HT on expression of the human NHE3 gene and the mechanisms underlying its transcriptional regulation in differentiated C2BBe1 cells. Treatment of the human intestinal epithelial cell line, C2BBe1, with 5-HT (20 microM) resulted in a significant decrease in NHE3 mRNA and protein expression. In transient transfection studies, 5-HT repressed the NHE3 promoter activity by approximately 55%. The repression of the NHE3 promoter activity in response to 5-HT was accompanied by reduced DNA-binding activity of transcription factors Sp1 and Sp3 to the NHE3 promoter without alteration in their nuclear levels. Pharmacological inhibitors of protein kinase C reversed the inhibitory effect of 5-HT on the promoter activity. Our data indicate that 5-HT suppresses the transcriptional activity of the NHE3 promoter and this effect may be mediated by PKCalpha and modulation of DNA-binding affinities of Sp1 and Sp3.

PKC-dependent stimulation of the human MCT1 promoter involves transcription factor AP2.

Monocarboxylate transporter (MCT1) plays an important role in the absorption of short-chain fatty acids (SCFA) such as butyrate in the human colon. Previous studies from our laboratory have demonstrated that phorbol ester, PMA (1 microM, 24 h), upregulates butyrate transport and MCT1 protein expression in human intestinal Caco-2 cells. However, the molecular mechanisms involved in the transcriptional regulation of MCT1 gene expression by PMA in the intestine are not known. In the present study, we showed that PMA (0.1 microM, 24 h) increased the MCT1 promoter activity (-871/+91) by approximately fourfold. A corresponding increase in MCT1 mRNA abundance in response to PMA was also observed. PMA-induced stimulation of MCT1 promoter activity was observed as early as 1 h and persisted until 24 h, suggesting that the effects of PMA are attributable to initial PKC activation. Kinase inhibitor and phosphorylation studies indicated that these effects may be mediated through activation of the atypical PKC-zeta isoform. 5'-deletion studies demonstrated that the MCT1 core promoter region (-229/+91) is the PMA-responsive region. Site-directed mutagenesis studies showed the predominant involvement of potential activator protein 2 (AP2) binding site in the activation of MCT1 promoter activity by PMA. In addition, overexpression of AP2 in Caco-2 cells significantly increased MCT1 promoter activity in a dose-dependent manner. These findings showing the regulation of MCT1 promoter by PKC and AP2 are of significant importance for an understanding of the molecular regulation of SCFA absorption in the human intestine.

Involvement of Sp1 and Sp3 in differential regulation of human NHE3 promoter activity by sodium butyrate and IFN-gamma/TNF-alpha.

Previously, we reported that IFN-gamma and TNF-alpha downregulate the expression of the human Na(+)/H(+) exchanger (NHE)3 gene by modulating Sp1/Sp3 transcription factors in C2BBe1 cells. It is reported that butyrate inhibits IFN-gamma and TNF-alpha signaling pathways. In this study, we have investigated the effect of sodium butyrate (NaB) and IFN-gamma/TNF-alpha on human NHE3 promoter activity. In transient transfection studies, NaB (5 mM) led to 10-fold stimulation of NHE3 promoter activity after incubation for 24 h. With 5'-deletion analysis, the NaB-responsive region was mapped to the NHE3 core promoter, bp -95 to + 5, which we had shown previously to confer responsiveness to IFN-gamma/TNF-alpha. The stimulatory effect of NaB on the NHE3 promoter was reduced by 60% in the presence of IFN-gamma/TNF-alpha. Mutually, the repressive effect of these cytokines was attenuated by NaB. Knockdown of Sp1 and Sp3 expression with small interfering RNA (siRNA) resulted in a significant resistance to NaB effects. NaB treatment showed no effect on Sp1 and Sp3 protein expression as assessed by Western blot analyses. Gel mobility shift assays with nuclear proteins from NaB-treated cells showed enhanced binding of Sp1 and Sp3 to the NHE3 promoter. The phosphatase inhibitor okadaic acid (200 nM) blocked the stimulatory effect of NaB on the NHE3 promoter. NaB effects on the NHE3 promoter were significantly attenuated by protein phosphatase (PP)1alpha- and PP2Aalpha-specific siRNA transfection. Our data suggest that the differential regulation of NHE3 gene expression by NaB and IFN-gamma/TNF-alpha is mediated through alternative pathways that converge on Sp1/Sp3.

Sp1 and Sp3 control constitutive expression of the human NHE2 promoter by interactions with the proximal promoter and the transcription initiation site.

We have previously cloned the human Na+/H+ exchanger NHE2 gene and its promoter region. In the present study, the regulatory elements responsible for the constitutive expression of NHE2 were studied. Transient transfection assays revealed that the -40/+150 promoter region contains the core promoter responsible for the optimal promoter activity. A smaller fragment, -10/+40, containing the TIS (transcription initiation site) showed minimal activity. We identified a palindrome that overlaps the TIS and binds to the transcription factors Sp1 and Sp3. Mutations in the 5' flank of the palindrome abolished the Sp1/Sp3 interaction and reduced promoter activity by approx. 45%. In addition, a conserved GC-box centered at -25 was found to play a critical role in basal promoter activity and also interacted with Sp1 and Sp3. An internal deletion in the GC-box severely reduced the promoter activity. Sp1/Sp3 binding to these elements was established using gel-mobility shift assays, confirmed by chromatin immunoprecipitation and co-transfections in Drosophila SL2 cells. Furthermore, we identified two positive regulatory elements in the DNA region corresponding to the 5'-UTR (5'-untranslated region). The results in the present study indicate that Sp1 and Sp3 are required for constitutive NHE2 expression and that the positive regulatory elements of the 5'-UTR may co-operate with the 5'-flanking region to achieve the optimal promoter activity.

IFN-gamma and TNF-alpha regulate human NHE3 gene expression by modulating the Sp family transcription factors in human intestinal epithelial cell line C2BBe1.

Diarrhea associated with inflammatory bowel disease has been attributed to stimulated secretion of proinflammatory cytokines like IFN-gamma and TNF-alpha, which have been shown to downregulate the expression of the sodium-hydrogen exchanger-3 (NHE3) gene. In this study, we have investigated the mechanism of NHE3 gene regulation by IFN-gamma and TNF-alpha in C2BBe1 cells. In response to both IFN-gamma (30 ng/ml) and TNF-alpha (20 ng/ml), the construct containing the bp -95 to +5 region of the human NHE3 promoter, which harbors a number of cis-elements including four potential Sp1 binding sites, showed a maximum repression of 60%. Knockdown of Sp1 and Sp3 expression using small interfering RNA resulted in a significant inhibition of the NHE3 promoter activity and resistance to cytokines effects. These cytokines showed no effects on the expression of Sp1 and Sp3 mRNA and protein levels as assessed by RT-PCR and Western blot analyses, respectively. After treatment with cytokines, the binding of Sp1 and Sp3 proteins to NHE3 promoter decreased significantly, as seen by gel mobility shift assays and chromatin immunoprecipitation assays. The inhibitory effects of both cytokines on the NHE3 promoter were completely blocked by the broad-range kinase inhibitor staurosporine and the selective protein kinase A (PKA) inhibitor 8-bromoadenosine-3',5'-cyclic monophosphorothioate, Rp-isomer. The binding affinity of Sp1 and Sp3 proteins for NHE3 Sp1 probe was significantly decreased after in vitro phosphorylation of nuclear proteins by the alpha-catalytic subunit of PKA. Our data indicate that IFN-gamma and TNF-alpha may repress the NHE3 promoter activity in C2BBe1 cells by PKA-mediated phosphorylation of Sp1 and Sp3 transcription factors.

Transcriptional stimulation of the human NHE3 promoter activity by PMA: PKC independence and involvement of the transcription factor EGR-1.

NHE3 (Na+/H+ exchanger 3) is essential for Na+ absorption in the ileum and is expressed in a cell-specific manner in the apical membrane of the intestinal epithelial cells. In the present study, we report the stimulatory effect of PMA on the hNHE3 (human NHE3) transcription. Pretreatment with actinomycin D or cycloheximide blocked the up-regulation of the NHE3 mRNA by PMA, indicating that the increased level of NHE3 mRNA expression is regulated by transcriptional activation and is dependent on de novo protein synthesis. 5'-Deletion of the promoter region and transfection analysis in C2BBe1 cells revealed that the PMA effect is mediated through a GC-rich DNA region between nt -88 and -69. Gel mobility-shift assays demonstrated that in nuclear extracts from C2BBe1 cells grown under the basal growth conditions, Sp1 (stimulating protein-1) and Sp3 interact with this GC-rich DNA region, while, in PMA-treated nuclear extracts, PMA-induced EGR-1 (early growth response gene product 1) transcription factor binds to the same site. Binding of EGR-1 diminished the Sp1 and Sp3 interactions with this promoter region significantly. Co-transfection of Sp1 or Sp3 into SL2 cells activated the NHE3-reporter constructs, suggesting that Sp1 and Sp3 act as positive regulators of the NHE3 expression. In addition, overexpression of EGR-1 was sufficient to transactivate the NHE3-reporter gene activity, and knockdown of EGR-1 with gene-specific small interfering RNA resulted in inhibition of the PMA-induced up-regulation of the endogenous NHE3 mRNA expression. Furthermore, the PKC (protein kinase C) inhibitor chelerythrine chloride did not affect PMA-induced NHE3 promoter activity, suggesting that PMA stimulation of the hNHE3 gene expression may be PKC-independent.

Zinc finger transcription factor Egr-1 is involved in stimulation of NHE2 gene expression by phorbol 12-myristate 13-acetate.

The apical membrane Na(+)/H(+) exchanger isoforms NHE2 and NHE3 are involved in transepithelial Na(+) absorption in the intestine. However, they exhibit differences in their pattern of tissue expression and regulation of their activity by various molecular signals. To study the mechanisms involved in the transcriptional regulation of these genes, we characterized cis-acting elements within the human NHE2 promoter that regulate NHE2 promoter expression in C2BBe1 cells. A small DNA region (-85/+249) was involved in the regulation of basal transcriptional activity of the NHE2 promoter as determined by transient transfection assays. RT-PCR analysis showed that NHE2 mRNA was upregulated in response to phorbol 12-myristate 13-acetate (PMA). Results from actinomycin D-treated cells indicated that the regulation of the NHE2 gene by PMA occurs in part at the transcriptional level. Furthermore, PMA treatment led to a 100% increase in promoter activity through elements located on the -415/+249 DNA fragment. A PMA-induced nuclear factor that bound to the NHE2 promoter was identified as the transcription factor Egr-1. We identified two PMA response elements in the -415/+1 promoter region that bind to Sp1 and Sp3 in untreated nuclear extracts and to Egr-1 in PMA-treated nuclear extracts. In cotransfection experiments, Egr-1 was able to transactivate the NHE2 promoter. Our data indicate that Egr-1 may play a key role in regulated expression of the human NHE2 gene.

Serotonin inhibits Na+/H+ exchange activity via 5-HT4 receptors and activation of PKC alpha in human intestinal epithelial cells.

BACKGROUND & AIMS: Increased serotonin levels have been implicated in the pathophysiology of diarrhea associated with celiac and inflammatory diseases. However, the effects of serotonin on Na+ /H+ exchange (NHE) activity in the human intestine have not been investigated fully. The present studies examined the acute effects of 5-hydroxytryptamine (5-HT) on NHE activity using Caco-2 cells as an in vitro model. METHODS: Caco-2 cells were treated with 5-HT (.1 micromol/L, 1 h) and NHE activity was measured as ethyl-isopropyl-amiloride (EIPA)-sensitive 22Na uptake. The effect of 5-HT receptor-specific agonists and antagonists was examined. The role of signaling intermediates in 5-HT-mediated effects on NHE activity was elucidated using pharmacologic inhibitors and immunoblotting. RESULTS: NHE activity was inhibited significantly (approximately 50%-75%, P < .05) by .1 micromol/L 5-HT via inhibition of maximal velocity (Vmax) without any changes in apparent affinity (Km) for the substrate Na+ . NHE inhibition involved a decrease of both NHE2 and NHE3 activities. Studies using specific inhibitors and agonists showed that the effects of 5-HT were mediated by 5-HT4 receptors. 5-HT-mediated inhibition of NHE activity was dependent on phosphorylation of phospholipase C gamma 1 (PLC gamma 1) via activation of src-kinases. Signaling pathways downstream of PLC gamma 1 involved increase of intracellular Ca 2+ levels and subsequent activation of protein kinase C alpha (PKC alpha). The effects of 5-HT on NHE activity were not cell-line specific because T84 cells also showed NHE inhibition. CONCLUSIONS: A better understanding of the regulation of Na+ absorption by 5-HT offers the potential for providing insights into molecular and cellular mechanisms involved in various diarrheal and inflammatory disorders.

Role of USF1 and USF2 as potential repressor proteins for human intestinal monocarboxylate transporter 1 promoter.

Butyrate, a short-chain fatty acid, is the major energy fuel for the colonocytes. We have previously reported that monocarboxylate transporter isoform 1 (MCT1) mediates uptake of butyrate by human colonic Caco-2 cells. To better understand the mechanisms of MCT1 expression and regulation in the human intestine, we examined the activity and regulation of MCT1 promoter in Caco-2 cells. The transcription initiation site in the MCT1 promoter was identified as a guanine nucleotide 281 bp upstream from the translation initiation site and is surrounded by a guanine-cytosine-rich area. The promoter was found to be highly active when transfected into Caco-2 cells, and its activity decreased with deletions at its 5'-end. Gel mobility shift experiments showed binding of the transcription factors upstream stimulatory factor (USF)1 and 2 to the site -114 to -119 of the MCT1 promoter. With the use of site-directed mutagenesis and promoter activity in Caco-2 cells, the USF proteins appeared to have a repressor role on the MCT1 promoter, which was further confirmed by cotransfecting expression vectors encoding USF1 and 2 in Caco-2 cells and determining endogenous MCT1 expression in USF2 overexpressed cells. The two potential SP1 binding sites found in the same region of the promoter were found not to be involved in its regulation.

Differential regulation of Na+/H+ exchange isoform activities by enteropathogenic E. coli in human intestinal epithelial cells.

Enteropathogenic Escherichia coli (EPEC) is an important human intestinal foodborne pathogen associated with diarrhea, especially in infants and young children. Although EPEC produces characteristic attaching and effacing lesions and loss of microvilli, the pathophysiology of EPEC-associated diarrhea, particularly during early infection, remains elusive. The present studies were designed to examine the direct effects of EPEC infection on intestinal absorption via Na(+)/H(+) exchanger (NHE) isoforms. Caco-2 cells were infected with EPEC strain E2348/69 or nonpathogenic E. coli HB101 for a period of 60 to 120 min. Total NHE activity was significantly increased at 60 min, reaching approximately threefold increase after 90 min of EPEC infection. Similar findings were seen in HT-29 cells and T84 cells indicating that the response was not cell-line specific. Most surprising was the differential regulation of NHE2 and NHE3 by EPEC. Marked activation of NHE2 (300%) occurred, whereas significant inhibition ( approximately 50%) of NHE3 activity was induced. The activity of basolateral isoform NHE1 was also significantly increased in response to EPEC infection. Mutations that disrupted the type III secretion system (TTSS) ablated the effect of EPEC on the activity of both NHE2 and NHE3. These results suggest that EPEC, through a TTSS-dependent mechanism, exerts differential effects on NHE isoform activity in intestinal epithelial cells. Additionally, NHEs do not appear to play any role in EPEC-mediated inflammation, because the NHE inhibitors amiloride and 5-(N-ethyl-N-isopropyl)amiloride did not prevent EPEC-mediated IkappaBalpha degradation.

Regulation of NHE3 by nitric oxide in Caco-2 cells.

The effect of nitric oxide (NO) on Na+/H+ exchange (NHE) activity was investigated utilizing Caco-2 cells as an experimental model. Incubation of Caco-2 cells with 10(-3) M S-nitroso-N-acetylpenicillamine (SNAP), a conventional donor of NO, for 20 min resulted in a approximately 45% dose-dependent decrease in NHE activity, as determined by assay of ethylisopropylamiloride-sensitive 22Na uptake. A similar decrease in NHE activity was observed utilizing another NO-specific donor, sodium nitroprusside. SNAP-mediated inhibition of NHE activity was not secondary to a loss of cell viability. NHE3 activity was significantly reduced by SNAP (P < 0.05), whereas NHE2 activity was essentially unaltered. The effects of SNAP were mediated by the cGMP-dependent signal transduction pathway as follows: 1) LY-83583 and 1H-(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one (ODQ), specific inhibitors of soluble guanylate cyclase, blocked the inhibitory effect of SNAP on NHE; 2) 8-bromo-cGMP mimicked the effects of SNAP on NHE activity; 3) the SNAP-induced decrease in NHE activity was counteracted by a specific protein kinase G inhibitor, KT-5823 (1 microM); 4) chelerythrine chloride (2 microM) or calphostin C (200 nM), specific protein kinase C inhibitors, did not affect inhibition of NHE activity by SNAP; 5) there was no cross activation by the protein kinase A-dependent pathway, as the inhibitory effects of SNAP were not blocked by Rp-cAMPS (25 microM), a specific protein kinase A inhibitor. These data provide novel evidence that NO inhibits NHE3 activity via activation of soluble guanylate cyclase, resulting in an increase in intracellular cGMP levels and activation of protein kinase G.

Molecular cloning and functional analysis of the human Na(+)/H(+) exchanger NHE3 promoter.

Na(+)/H(+) exchanger (NHE) isoforms NHE2 and NHE3, colocalized to the brush border membrane of the epithelial cells, exhibit differences in their pattern of tissue expression and regulation by various molecular signals. To investigate the mechanisms involved in regulation of NHE3 gene expression, the human NHE3 promoter region was cloned and characterized. Primer extension experiments located the transcription start site to a position 116 nucleotides upstream from the translation start codon. The 5'-flanking region lacked a CCAAT box but contained a TATA-like sequence. Nucleotide sequencing of the 5'-flanking region revealed the presence of a number of cis elements including Sp1, AP-2, MZF-1, CdxA, Cdx-2, steroid and nonsteroid hormone receptor half sites, and a phorbol 12-myristate 13-acetate-response element. Transient transfection experiments using C2/bbe cell line defined a maximal promoter activity in -95/+5 region. The regulatory response elements clustered within this region include a potential transcription factor IID (TF IID), a CACCC, two Sp1, and two AP-2 motifs. Deletion of a fragment containing the AP-2 and Sp1 motifs resulted in a drastic decrease in promoter activity. In gel mobility shift assays, an oligonucleotide spanning from -78 to -56 bp bound a recombinant AP-2, and the corresponding binding activity in nuclear extracts was supershifted with anti-AP2alpha antibody. Our studies suggest that the NHE3 expression is regulated by a combination of cis elements and their cognate transcription factors that include the AP-2 and Sp1 family members.