NHE3 Promoter Research Articles

G protein‐coupled receptor 37L1 participates in epigenetic gene regulation in human renal proximal tubule cells

G protein‐coupled receptors (GPCRs) in the kidney regulate the reabsorption of essential nutrients, ions, and water from the glomerular filtrate. Abnormality in the renal luminal sodium transport plays an important role in the pathogenesis of essential hypertension. There are a growing number of GPCRs and their interacting protein components that are localized on the nuclear membrane or within the nucleus, where they retain their signaling functions. In most cases, nuclear GPCRs are constitutively expressed on the nuclear membrane. The functions of these nuclear GPCRs are not well understood. Recently we showed that G protein‐coupled receptor 37L1 (GPR37L1), expressed on the apical membrane of renal proximal tubules (RPT), regulates luminal sodium transport and blood pressure via modulating NHE3 function. However, the GPR37L1 signaling pathway in the RPT has not been studied. Co‐immunoprecipitation (Co‐IP), combined with mass spectrometry analyses, revealed the association of GPR37L1 with nuclear import proteins: IPO7, IPO8, KPNA2, KPNB1; nuclear export proteins: XPO1 and XPO7; nuclear pore complex proteins: NUP210; kinases involved in signaling: mTOR, PRKDC, IRS4; and a DNA (Cytosine‐5‐)‐Methyltransferase 1 (DNMT1) in the GPR37L1 protein complex. Immunoblot analyses of nuclear preparations from the human RPT cells (hRPTCs) confirmed the presence of GPR37L1 in the nucleus. Confocal fluorescence imaging of hRPTCs expressing GPR37L1 tagged with green fluorescence protein (GPR37L1‐GFP) showed distinct nuclear membrane expression of GPR37L1. Association of GPR37L1 with mTOR, PRKDC, and DNMT1 proteins was confirmed by Co‐IP followed by immunoblot analyses. Overexpression of GPR37L1 in hRPTCs increased the phosphorylation of PI3, and AKT and decreased the expression of DNMT1, and NHE3 promoter methylation. Our data show that GPR37L1 signals via the PI3K/AKT/mTOR pathway to decrease the expression of DNMT1. Decreased DNMT1 function reduces CpG methylation on the NHE3 promoter that in turn increases NHE3 transcription. This is the first report showing the direct role of a nuclear GPCR in the regulation of renal sodium transport through epigenetic gene regulation.Support or Funding InformationR56DK116828: Prasad Konkalmatt

ChREBP deficiency leads to diarrhea-predominant irritable bowel syndrome

ObjectiveFructose malabsorption is a common digestive disorder in which absorption of fructose in the small intestine is impaired. An abnormality of the main intestinal fructose transporter proteins has been proposed as a cause for fructose malabsorption. However the underlying molecular mechanism for this remains unclear. In this study, we investigated whether carbohydrate response element-binding protein (ChREBP) plays a role in intestinal fructose absorption through the regulation of genes involved in fructose transport and metabolism and ion transport. MethodsWild type (WT) and Chrebp knockout (KO) mice (6 or 8 weeks old) were fed a control diet (55% starch, 15% maltodextrin 10) or high-fructose diet (HFrD, 60% fructose, 10% starch) for 3–12 days. Body weight and food intake were measured, signs of fructose malabsorption were monitored, and the expression of genes involved in fructose transport/metabolism and ion transport was evaluated. Furthermore, transient transfection and chromatin immunoprecipitation were performed to show the direct interaction between ChREBP and carbohydrate response elements in the promoter of Slc2A5, which encodes the fructose transporter GLUT5. ResultsChrebp KO mice fed the control diet maintained a constant body weight, whereas those fed a HFrD showed significant weight loss within 3–5 days. In addition, Chrebp KO mice fed the HFrD exhibited a markedly distended cecum and proximal colon containing both fluid and gas, suggesting incomplete fructose absorption. Fructose-induced increases of genes involved in fructose transport (GLUT5), fructose metabolism (fructokinase, aldolase B, triokinase, and lactate dehydrogenase), and gluconeogenesis (glucose-6-phosphatase and fructose-1,6-bisphosphatase) were observed in the intestine of WT but not of Chrebp KO mice. Moreover the Na+/H+ exchanger NHE3, which is involved in Na+ and water absorption in the intestine, was significantly decreased in HFrD-fed Chrebp KO mice. Consistent with this finding, the high-fructose diet-fed Chrebp KO mice developed severe diarrhea. Results of chromatin immunoprecipitation assays showed a direct interaction of ChREBP with the Glut5 promoter, but not the Nhe3 promoter, in the small intestine. Ectopic co-expression of ChREBP and its heterodimer partner Max-like protein X activated the Glut5 promoter in Caco-2BBE cells. ConclusionsChREBP plays a key role in the dietary fructose transport as well as conversion into lactate and glucose through direct transcriptional control of genes involved in fructose transport, fructolysis, and gluconeogenesis. Moreover, ablation of Chrebp results in a severe diarrhea in mice fed a high-fructose diet, which is associated with the insufficient induction of GLUT5 in the intestine.

Epigenetic modulation of intestinal Na+/H+ exchanger-3 expression.

Na+/H+ exchanger-3 (NHE3) is crucial for intestinal Na+ absorption, and its reduction has been implicated in infectious and inflammatory bowel diseases (IBD)-associated diarrhea. Epigenetic mechanisms such as DNA methylation are involved in the pathophysiology of IBD. Whether changes in DNA methylation are involved in modulating intestinal NHE3 gene expression is not known. Caco-2 and HuTu 80 cells were used as models of human intestinal epithelial cells. Normal C57/BL6, wild-type, or growth arrest and DNA damage-inducible 45b (GADD45b) knockout (KO) mice were used as in vivo models. NHE3 gene DNA methylation levels were assessed by MBDCap (MethyMiner) assays. Results demonstrated that in vitro methylation of NHE3 promoter construct (p-1509/+127) cloned into a cytosine guanine dinucleotide-free lucia vector decreased the promoter activity in Caco-2 cells. DNA methyltransferase inhibitor 5-azacytidine (10 μM, 24 h) caused a significant decrease in DNA methylation of the NHE3 gene and concomitantly increased NHE3 expression in Caco-2 cells. Similarly, 5-azacytidine treatment increased NHE3 mRNA levels in HuTu 80 cells. 5-Azacytidine treatment for 3 wk (10 mg/kg body wt ip, 3 times/wk) also resulted in an increase in NHE3 expression in the mouse ileum and colon. Small-interfering RNA knockdown of GADD45b (protein involved in DNA demethylation) in Caco-2 cells decreased NHE3 mRNA expression. Furthermore, there was a significant decrease in NHE3 mRNA and protein expression in the ileum and colon of GADD45b KO mice. Our findings demonstrate that NHE3 gene expression is regulated by changes in its DNA methylation. NEW & NOTEWORTHY Our studies for the first time demonstrate that Na+/H+ exchanger-3 gene expression is regulated by an epigenetic mechanism involving DNA methylation.

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.

Epigenetic Modulation of Intestinal NHE3 Expression by DNA Methylation

NHE3 plays an important role in intestinal Na+ absorption and its down-regulation has been implicated in inflammatory diarrhea. Recent evidence also indicates a novel role of epigenetic mechanisms, such as DNA methylation in the pathophysiology of IBD. Whether changes in DNA methylation are involved in modulating intestinal NHE3 gene expression is not known. To address this, methyl-CpG binding domain-based capture assay was performed in Caco2 cells treated with the pro-inflammatory cytokines. IL6 (100 ng/ml) or IL1β (50 ng/ml) for 48h increased DNA methylation at the NHE3 gene promoter, indicating that DNA methylation may contribute to the decrease in NHE3 expression during intestinal inflammation. In parallel, in vitro methylation of NHE3 promoter (p-1507/+131) cloned into a CpG free lucia vector decreased the promoter activity. Conversely, DNA methyltransferase (DNMT) inhibitor, 5-azacytidine (10 μM, 24h) caused a significant decrease in DNA methylation of NHE3 gene. Also, 5-aza increased NHE3 expression in Caco2 & duodenal HUTU-80 cells by ~2-3 fold (p<0.05). Treatment of mice with 5-aza for 3 wks resulted in an increase in NHE3 expression in the ileum and colon (~2 fold, p<0.05). Further, the role of the demethylase GADD45β was examined. siRNA knockdown of GADD45β in Caco2 cells decreased NHE3 mRNA expression by ~50% (p<0.05). Immunostaining showed a decrease in NHE3 expression in the ileum & colon of GADD45β -/- mice. These findings suggest that changes in DNA methylation may be involved in the inhibition of NHE3 gene expression in intestinal inflammation contributing to the pathophysiology of IBD-associated diarrhea. (Supported by VA/NIH)

Peroxisome Proliferator-Activated Receptor α Protects Renal Tubular Cells from Gentamicin-Induced Apoptosis via Upregulating Na+/H+ Exchanger NHE1.

Peroxisome proliferator-activated receptor (PPAR)-α is a transcription factor that has been reported to inhibit gentamicin-induced apoptosis in renal tubular cells. However, the antiapoptotic mechanism of PPARα is still unknown. In this study, we found that PPARα overexpression induced Na+/H+ exchanger-1 (NHE1) expression in the rat renal tubular cells NRK-52E. Beraprost, a PPARα ligand, also increased NHE1 expression in the renal tubules in normal mice, but not in PPARα knockout mice. Chromatin immunoprecipitation assays revealed that two PPARα binding elements were located in the rat NHE1 promoter region. Na+/H+ exchanger activity also increased in the PPARα-overexpressed cells. Flow cytometry showed that the PPARα-overexpressed cells were resistant to apoptosis-induced shrinkage. Cariporide, a selective NHE1 inhibitor, inhibited the antiapoptotic effect of PPARα in the gentamicin-treated cells. The interaction between NHE1 and ezrin/radixin/moesin (ERM) and between ERM and phosphatidylinositol 4,5-bisphosphate in the PPARα-overexpressed cells was more than in the control cells. ERM short interfering RNA (siRNA) transfection inhibited the PPARα-induced antiapoptotic effect. PPARα overexpression also increased the phosphoinositide 3-kinase (PI3K) expression, which is dependent on NHE1 activity. Increased PI3K further increased the phosphorylation of the prosurvival kinase Akt in the PPARα-overexpressed cells. Wortmannin, a PI3K inhibitor, inhibited PPARα-induced Akt activity and the antiapoptotic effect. We conclude that PPARα induces NHE1 expression and then recruits ERM to promote PI3K/Akt-mediated cell survival in renal tubular cells. The application of PPARα activation reduces the nephrotoxicity of gentamicin and may expand the clinical use of gentamicin.

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.

5 Acid-Induced Stimulation of the NHE2 Promoter Activity is Mediated by Transcription Factor EGR-1 in the Human Intestinal Epithelial Cell Line C2BBe1

NHE8 is a member of the SLC9 family. This Na+/H+ exchanger is expressed on the apical membrane of intestinal epithelial cells. Although NHE8 has been shown to be important for intestinal sodium absorption early in development, its physiological role in the gastrointestinal tract remains unknown. By using NHE8 knockout (NHE8KO) mouse model, we discovered that NHE8 plays an important role in protecting the gastric mucosa against gastric acid. Stomach tissue was isolated from NHE8KO mice and their wild-type littermates. Localization of NHE8 expression in the stomach was detected using Immunohistochemistry. Mucosal surface pH was measured using a surface pH meter. Histology of the stomach of NHE8KOmice was also analyzed. RNA was isolated and used for PCR to detect the expression of PAT1 and DRA. Total tissue lysate was prepared for Western detection of NHE8, PAT1 and DRA. Immunohistochemistry indicated that NHE8 is expressed on the apical membrane of gastric epithelial cells. Histological evaluation showed no abnormality of the stomach in NHE8KO mice. Interestingly, higher incidence of gastric bleeding was observed in NHE8KO mice. The appearance of ulcers was seen in the glandular stomach. Mucosal surface pH measurement indicated that NHE8KO mice had decreased mucosal surface pH compared to wild-type mice (3.72 ± 0.13 in NHE8KO vs. 4.26 ± 0.09 in wild-type). Lower surface pH of NHE8KO gastric mucosa suggests that HCO3secretion may be altered. Therefore, we studied the expression of PAT1 and DRA in NHE8KO mice. Indeed, PAT1 and DRA mRNA expression in the stomach of NHE8KO mice were significantly reduced by ~30% and ~90%, respectively, in NHE8KO mice compared to their wild-type littermates. At the protein expression level, PAT1 protein abundance was reduced by ~70% and DRA protein abundance was nearly abolished in NHE8KO mice. Thus NHE8 may play an important role in mucosal protection by participating bicarbonate secretion.

Differential responses of proximal tubule Na+/H+ exchanger NHE3 to low pH: comparison between metabolic and respiratory acidosis

The Na+/H+ exchanger isoform 3 (NHE3) is essential for HCO3 − reabsorption in renal proximal tubules. The goal of this work was to evaluate whether there are differences between metabolic and respiratory acidosis with regard to NHE3 modulation and to identify which are the relevant parameters that may trigger these distinct adaptative responses. Metabolic acidosis was achieved by lowering [HCO3 −] in the cell culture medium and respiratory acidosis by increasing pCO2 in the incubator chamber. We found that cell‐surface NHE3 expression increased in response to metabolic and respiratory acidosis. Mild (pH 7.21±0.02) and severe (6.95±0.07) metabolic acidosis increased Nhe3 mRNA levels, while mild (7.11±0.03) and severe (6.86±0.01) respiratory acidosis did not up‐regulate Nhe3 expression. Analyses of the Nhe3 promoter suggested that the regulatory elements sensitive to metabolic acidosis are located in the region spanning −466 to −153 pb. Two SP1 consensus binding sites were localized in this region and metabolic acidosis caused an increment in the SP1 protein levels. We conclude that low pH might be related to NHE3 displacement to the apical membrane, however extracellular pH does not seem to be the parameter that up‐regulates NHE3 expression. Moreover, SP1 was identified as a potential transcription factor that mediates the response to metabolic acidosis in proximal tubule cells. Financial support: CNPq and FAPESP.

Distinct mechanisms underlie adaptation of proximal tubule Na+/H+ exchanger isoform 3 in response to chronic metabolic and respiratory acidosis

The Na(+/)H(+) exchanger isoform 3 (NHE3) is essential for HCO(3)(-) reabsorption in renal proximal tubules. The expression and function of NHE3 must adapt to acid-base conditions. The goal of this study was to elucidate the mechanisms responsible for higher proton secretion in proximal tubules during acidosis and to evaluate whether there are differences between metabolic and respiratory acidosis with regard to NHE3 modulation and, if so, to identify the relevant parameters that may trigger these distinct adaptive responses. We achieved metabolic acidosis by lowering HCO(3)(-) concentration in the cell culture medium and respiratory acidosis by increasing CO(2) tension in the incubator chamber. We found that cell-surface NHE3 expression was increased in response to both forms of acidosis. Mild (pH 7.21 ± 0.02) and severe (6.95 ± 0.07) metabolic acidosis increased mRNA levels, at least in part due to up-regulation of transcription, whilst mild (7.11 ± 0.03) and severe (6.86 ± 0.01) respiratory acidosis did not up-regulate NHE3 expression. Analyses of the Nhe3 promoter region suggested that the regulatory elements sensitive to metabolic acidosis are located between -466 and -153 bp, where two consensus binding sites for SP1, a transcription factor up-regulated in metabolic acidosis, were localised. We conclude that metabolic acidosis induces Nhe3 promoter activation, which results in higher mRNA and total protein level. At the plasma membrane surface, NHE3 expression was increased in metabolic and respiratory acidosis alike, suggesting that low pH is responsible for NHE3 displacement to the cell surface.

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.

Essential regulatory elements for NHE3 gene transcription in renal proximal tubule cells

The objectives of the present study were to identify the cis-elements of the promoter absolutely required for the efficient rat NHE3 gene transcription and to locate positive and negative regulatory elements in the 5’-flanking sequence (5’FS), which might modulate the gene expression in proximal tubules, and to compare this result to those reported for intestinal cell lines. We analyzed the promoter activity of different 5’FS segments of the rat NHE3 gene, in the OKP renal proximal tubule cell line by measuring the activity of the reporter gene luciferase. Because the segment spanning the first 157 bp of 5’FS was the most active it was studied in more detail by sequential deletions, point mutations, and gel shift assays. The essential elements for gene transcription are in the region -85 to -33, where we can identify consensual binding sites for Sp1 and EGR-1, which are relevant to NHE3 gene basal transcription. Although a low level of transcription is still possible when the first 25 bp of the 5’FS are used as promoter, efficient transcription only occurs with 44 bp of 5’FS. There are negative regulatory elements in the segments spanning -1196 to -889 and -467 to -152, and positive enhancers between -889 and -479 bp of 5’FS. Transcription factors in the OKP cell nuclear extract efficiently bound to DNA elements of rat NHE3 promoter as demonstrated by gel shift assays, suggesting a high level of similarity between transcription factors of both species, including Sp1 and EGR-1.

Transcriptional regulation of the Na+/H+ exchanger NHE3 by chronic exposure to angiotensin II in renal epithelial cells

Angiotensin II (Ang II) exerts an acute bimodal effect on proximal tubule NHE3: while low doses stimulate the exchanger, high doses inhibit it. In the present study, we have investigated the chronic effects of Ang II on NHE3 expression and transcriptional regulation. Treatment of a tubular epithelial cell line, OKP, with Ang II 10−11M significantly increased NHE protein expression and mRNA levels, without evidence of bimodal effect. No change in mRNA half-life was detected, but transient transfection studies showed a significant increase in NHE3 promoter activity. Binding sites for Sp1/Egr-1 and AP2 transcription factors of the NHE3 proximal promoter were mutated and we observed that the Sp1/Egr-1 binding site integrity is necessary for Ang II stimulatory effects. Inhibition of cytochrome P450, PI3K, PKA and MAPK pathways prevented the Ang II stimulatory effect on the NHE3 promoter activity. Taking all the results together, our data reveal that chronic Ang II treatment exerts a stimulatory effect on NHE3 expression and promoter activity. The Ang II up-regulation of the NHE3 promoter activity appears to involve the Sp1/Egr-1 binding site and the interplay of several intracellular signaling pathways.

Essential regulatory elements for NHE3 gene transcription in renal proximal tubule cells

The objectives of the present study were to identify the cis-elements of the promoter absolutely required for the efficient rat NHE3 gene transcription and to locate positive and negative regulatory elements in the 5'-flanking sequence (5'FS), which might modulate the gene expression in proximal tubules, and to compare this result to those reported for intestinal cell lines. We analyzed the promoter activity of different 5'FS segments of the rat NHE3 gene, in the OKP renal proximal tubule cell line by measuring the activity of the reporter gene luciferase. Because the segment spanning the first 157 bp of 5'FS was the most active it was studied in more detail by sequential deletions, point mutations, and gel shift assays. The essential elements for gene transcription are in the region -85 to -33, where we can identify consensual binding sites for Sp1 and EGR-1, which are relevant to NHE3 gene basal transcription. Although a low level of transcription is still possible when the first 25 bp of the 5'FS are used as promoter, efficient transcription only occurs with 44 bp of 5'FS. There are negative regulatory elements in the segments spanning -1196 to -889 and -467 to -152, and positive enhancers between -889 and -479 bp of 5'FS. Transcription factors in the OKP cell nuclear extract efficiently bound to DNA elements of rat NHE3 promoter as demonstrated by gel shift assays, suggesting a high level of similarity between transcription factors of both species, including Sp1 and EGR-1.

Chronic effects of angiotensin II on NHE3 promoter involve several intracellular signaling pathways and Sp1/Egr‐1 binding site

Previous studies have shown that 24h exposure to 10−11 M angiotensin II (AII) stimulated NHE3 activity and increased NHE3 protein content and mRNA levels in OKP cells. The purpose of this study was to verify the effects of AII on the NHE3 gene promoter activity and its regulatory mechanisms. OKP cells were used in transient transfection assays. Four fragments of NHE3 promoter cloned into a vector containing the reporter gene Firefly luciferase were studied (−2095/+55, −152/+55, −85/+31 and −65/+31). The promoter activity of the fragment −65/+31 was significantly increased after 24h‐treatment with 10−11M AII. This increase was abolished by pretreatment with losartan (AT1R inhibitor). Two mutations were performed in this fragment. The mutation at the Sp1/Egr‐1 binding site completely abolishes the AII stimulatory effect, whereas the mutation at the AP2 binding site partially revert this effect. In order to verify the mechanisms involved in this stimulatory response, we used intracellular signaling pathways inhibitors. Our results showed that cytochrome P450, PI3K, PKA and MAPK pathways are involved in this process, while PLC and JAK/STAT pathways do not participate. These data reveal that AII chronic stimulatory effects on NHE3 proximal promoter occur through AT1R and are dependent on the Sp1/Egr‐1 binding site integrity. This response involves activation of different intracellular signaling pathways.Financial Support: FAPESP and CNPq

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

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. 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. 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. 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.

Abstract 1266: TNF-alpha mediated down-regulation of Na+/H+ exchanger 1 (NHE1) in breast cancer MCF7 cells and its relevance to apoptosis

Abstract Intracellular alkalinization is a hallmark of cancer cells shown to confer resistance against caspase mediated apoptosis. Na+/H+ exchanger 1 (NHE1), an ion exchanger important in the maintenance of intracellular pH and cell volume homeostasis had been implicated in this intracellular alkalinization. Increased NHE1 protein level and activity has been found in many cancer cells types where increase in intracellular pH by constitutively active NHE1 confers resistance to apoptosis in these cells. Silencing of NHE1 protein in cancer cells results in reduced cell growth and increased in sensitivity to death triggers. TNF-α can induce H2O2 production in cells to mediate apoptosis with characteristic cell shrinkage. H2O2 had recently been shown to downregulate NHE1 expression and increase cells’ sensitivity to apoptosis. This study was undertaken to study the role of caspases and H2O2 in the regulation of NHE1 protein and gene expression by TNF-α. Results showed that TNF-α had both cytostatic and cytotoxic effects on MCF-7 cells with concurrent decrease in intracellular pH and NHE1 activity. NHE1 was found to be downregulated both at the protein and mRNA level following TNF-α treatment. Gelshift assay showed that transcription factors binding to the promoter of NHE1 could be blocked by pre-incubation with H2O2, suggesting that transcription of NHE1 could be redox-regulated. Indeed, we also show by using a luciferase reporter assay that NHE1 promoter is down-regulated in presence of H2O2. Various caspases were activated in TNF-α treated cells and the inhibition of caspases prevented the downregulation of NHE1. ROS was elevated in MCF-7 within 2 hours of treatment. Incubation of cells with catalase increased their survival in response to TNF-α, thus implicating H2O2 as a mediator of TNF-α induced cell death. Overexpression of catalase in MCF-7 reduced the downregulation of NHE1 by TNF-α. Our results suggest by TNF-α mediate cell death through decreasing NHE1 activity and down-regulating NHE1 expression in a ROS mediated manner. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1266.

Impact of Sodium/Proton Exchanger 3 Gene Variants on Sudden Infant Death Syndrome

To determine the contribution of variations in the sodium/proton exchanger 3 (NHE3) gene in sudden infant death syndrome (SIDS). Variations in the exons and promoter of the NHE3 gene were analyzed with direct sequencing analysis and mini sequencing (SNaPshot analysis) in 251 cases of SIDS, plus 50 infant control subjects who had died of other causes, and 170 healthy adults. The C2405T variant (exon 16) and 2 polymorphisms in the promoter (G1131A and C1197T) were encountered significantly more frequently in cases of SIDS than in control subjects. At least 1 of these 3 variants was detected in 49% of SIDS cases, but only in 30% of control subjects. Our findings suggest the involvement of polymorphisms in the NHE3 gene and promoter in cases of SIDS, which may result in an overexpression of NHE3 in the medulla oblongata and which possibly leads to a disturbance in breathing control. Furthermore, our results underline the heterogeneous character of SIDS.

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 μM) resulted in a significant decrease in NHE3 mRNA and protein expression. In transient transfection studies, 5-HT repressed the NHE3 promoter activity by ∼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 PKCα and modulation of DNA-binding affinities of Sp1 and Sp3.