A Direct Link Implicating Loss of SLC26A6 to Gut Microbial Dysbiosis, Compromised Barrier Integrity, and Inflammation.

BACKGROUND & AIMS: Putative anion transporter-1 (PAT1, SLC26A6) plays a key role in intestinal oxalate and bicarbonate secretion. PAT1 knockout (PKO) mice exhibit hyperoxaluria and nephrolithiasis. Notably, diseases such as inflammatory bowel disease are also associated with higher risk of hyperoxaluria and nephrolithiasis. However, the potential role of PAT1 deficiency in gut-barrier integrity and susceptibility to colitis is currently elusive. METHODS: Age-matched PKO and wild-type littermates were administered 3.5% dextran sulfate sodium in drinking water for 6 days. Ileum and colon of control and treated mice were harvested. Messenger RNA and protein expression of tight junction proteins were determined by reverse transcription polymerase chain reaction and western blotting. Severity of inflammation was assessed by measuring diarrheal phenotype, cytokine expression, and hematoxylin and eosin staining. Gut microbiome and associated metabolome were analyzed by 16S ribosomal RNA sequencing and mass spectrometry, respectively. RESULTS: PKO mice exhibited significantly higher loss of body weight, gut permeability, colonic inflammation, and diarrhea in response to dextran sulfate sodium treatment. In addition, PKO mice showed microbial dysbiosis and significantly reduced levels of butyrate and butyrate-producing microbes compared with controls. Co-housing wild-type and PKO mice for 4 weeks resulted in PKO-like signatures on the expression of tight junction proteins in the colons of wild-type mice. CONCLUSIONS: Our data demonstrate that loss of PAT1 disrupts gut microbiome and related metabolites, decreases gut-barrier integrity, and increases host susceptibility to intestinal inflammation. These findings, thus, highlight a novel role of the oxalate transporter PAT1 in promoting gut-barrier integrity, and its deficiency appears to contribute to the pathogenesis of inflammatory bowel diseases.

Serotonin Transporter Deficiency Induces Metabolic Alterations in the Ileal Mucosa.

Serotonin transporter (SERT) deficiency has been implicated in metabolic syndrome, intestinal inflammation, and microbial dysbiosis. Interestingly, changes in microbiome metabolic capacity and several alterations in host gene expression, including lipid metabolism, were previously observed in SERT-/- mice ileal mucosa. However, the precise host or microbial metabolites altered by SERT deficiency that may contribute to the pleiotropic phenotype of SERT KO mice are not yet understood. This study investigated the hypothesis that SERT deficiency impacts lipid and microbial metabolite abundances in the ileal mucosa, where SERT is highly expressed. Ileal mucosal metabolomics was performed by Metabolon on wild-type (WT) and homozygous SERT knockout (KO) mice. Fluorescent-activated cell sorting (FACS) was utilized to measure immune cell populations in ileal lamina propria to assess immunomodulatory effects caused by SERT deficiency. SERT KO mice exhibited a unique ileal mucosal metabolomic signature, with the most differentially altered metabolites being lipids. Such changes included increased diacylglycerols and decreased monoacylglycerols in the ileal mucosa of SERT KO mice compared to WT mice. Further, the ileal mucosa of SERT KO mice exhibited several changes in microbial-related metabolites known to play roles in intestinal inflammation and insulin resistance. SERT KO mice also had a significant reduction in the abundance of ileal group 3 innate lymphoid cells (ILC3). In conclusion, SERT deficiency induces complex alterations in the ileal mucosal environment, indicating potential links between serotonergic signaling, gut microbiota, mucosal immunity, intestinal inflammation, and metabolic syndrome.

Downregulation of NHE-3 (SLC9A3) expression by MicroRNAs in intestinal epithelial cells.

Na+/H+ exchanger-3 (NHE-3) is the major apical membrane transporter involved in vectorial Na+ absorption in the intestine. Dysregulation of NHE-3 expression and/or function has been implicated in pathophysiology of diarrhea associated with gut inflammation and infections. Therefore, it is critical to understand the mechanisms involved in the regulation of NHE-3 expression. MicroRNAs (miRNAs) are highly conserved small RNAs that can regulate gene expression at the posttranscriptional level. To date, however, very little is known about the regulation of NHE-3 expression by microRNAs. Therefore, current studies were undertaken to examine the potential miRNA candidates that can regulate the expression of NHE-3 in intestinal epithelial cells. In silico analysis, using different algorithms, predicted several miRNAs that target NHE-3. MicroRNAs with highest context and target score, miR-326, miR-744-5p, and miR-330-5p, were selected for the current study. Human NHE-3 gene 3' untranslated region [3'UTR; 160 base pair (bp)] was cloned into pmirGLO vector upstream of luciferase reporter and transiently transfected with mimics of miR-326, miR-744-5p, and miR-330-5p into Caco-2, HT-29, and SK-CO15 cells. Cotransfection of NHE-3 3' UTR with miR-326 and -miR-330-5p mimics resulted in a significant decrease in relative luciferase activity. Transfection of miR-326 and -330-5p mimics into SK-CO15 cells significantly decreased the NHE-3 protein expression, with no change in NHE-3 messenger ribonucleic acid (mRNA) levels. Our findings demonstrate a novel mechanism for posttranscriptional regulation of NHE-3 by miR-326 and -330-5p by translational repression. We speculate that miR-326 and -330-5p dependent pathways may be involved in modulating NHE-3 expression under physiological and pathophysiological conditions.

A Novel Role of SLC26A3 in the Maintenance of Intestinal Epithelial Barrier Integrity.

BACKGROUND & AIMS: The down-regulated in adenoma (DRA) protein, encoded by SLC26A3, a key intestinal chloride anion exchanger, has recently been identified as a novel susceptibility gene for inflammatory bowel disease (IBD). However, the mechanisms underlying the increased susceptibility to inflammation induced by the loss of DRA remain elusive. Compromised barrier is a key event in IBD pathogenesis. The current studies were undertaken to elucidate the impact of DRA deficiency on epithelial barrier integrity and to define underlying mechanisms. METHODS: Wild-type and DRA-knockout (KO) mice and crypt-derived colonoids were used as models for intestinal epithelial response. Paracellular permeability was measured by using fluorescein isothiocyanate-dextran flux. Immunoblotting, immunofluorescence, immunohistochemistry, and ribonucleoprotein immunoprecipitation assays were performed. Gut microbiome analysis was conducted to investigate the impact of DRA deficiency on gut microbial communities. RESULTS: DRA-KO mice exhibited an increased colonic paracellular permeability with significantly decreased levels of tight junction/adherens junction proteins, including ZO-1, occludin, and E-cadherin. A similar expression pattern of occludin and E-cadherin was observed in colonoids derived from DRA-KO mice and short hairpin RNA-mediated DRA knockdown in Caco-2 cells. Microbial analysis showed gut dysbiosis in DRA-KO mice. However, cohousing studies showed that dysbiosis played only a partial role in maintaining tight junction protein expression. Furthermore, our results showed increased binding of RNA-binding protein CUGBP1 with occludin and E-cadherin genes in DRA-KO mouse colon, suggesting that posttranscriptional mechanisms play a key role in gut barrier dysfunction. CONCLUSIONS: To our knowledge, our studies demonstrate a novel role of DRA in maintaining the intestinal epithelial barrier function and potential implications of its dysregulation in IBD pathogenesis.

Cryptosporidium parvum infection induces autophagy in intestinal epithelial cells.

Autophagy, a process of degradation and recycling of macromolecules and organelles to maintain cellular homeostasis, has also been shown to help eliminate invading pathogens. Conversely, various pathogens including parasites have been shown to modulate/exploit host autophagy facilitating their intracellular infectious cycle. In this regard, Cryptosporidium parvum (CP), a protozoan parasite of small intestine is emerging as a major global health challenge. However, the pathophysiology of cryptosporidiosis is mostly unknown. We have recently demonstrated CP-induced epithelial barrier disruption via decreasing the expression of specific tight junction (TJ) and adherens junction (AJ) proteins such as occludin, claudin-4 and E-cadherin. Therefore, we utilised confluent Caco-2 cell monolayers as in vitro model of intestinal epithelial cells (IECs) to investigate the potential role of autophagy in the pathophysiology of cryptosporidiosis. Autophagy was assessed by increase in the ratio of LC3II (microtubule associated protein 1 light chain 3) to LC3I protein and decrease in p62/SQSTM1 protein levels. CP treatment of Caco-2 cells for 24 hr induced autophagy with a maximum effect observed with 0.5 × 106 oocyst/well. CP decreased mTOR (mammalian target of rapamycin, a suppressor of autophagy) phosphorylation, suggesting autophagy induction via mTOR inactivation. Measurement of autophagic flux utilizing the lysosomal inhibitor chloroquine (CQ) showed more pronounced increase in LC3II level in cells co-treated with CP + CQ as compared to CP or CQ alone, suggesting that CP-induced increase in LC3II was due to enhanced autophagosome formation rather than impaired lysosomal clearance. CP infection did not alter ATG7, a key autophagy protein. However, the decrease in occludin, claudin-4 and E-cadherin by CP was partially blocked following siRNA silencing of ATG7, suggesting the role of autophagy in CP-induced decrease in these TJ/AJ proteins. Our results provide novel evidence of autophagy induction by CP in host IECs that could alter important host cell processes contributing to the pathophysiology of cryptosporidiosis.

NPC1L1-dependent transport of 27-alkyne cholesterol in intestinal epithelial cells.

Niemann-Pick C1 Like-1 (NPC1L1) mediates the uptake of micellar cholesterol by intestinal epithelial cells and is the molecular target of the cholesterol-lowering drug ezetimibe (EZE). The detailed mechanisms responsible for intracellular shuttling of micellar cholesterol are not fully understood due to the lack of a suitable NPC1L1 substrate that can be traced by fluorescence imaging and biochemical methods. 27-Alkyne cholesterol has been previously shown to serve as a substrate for different cellular processes similar to native cholesterol. However, it is not known whether alkyne cholesterol is absorbed via an NPC1L1-dependent pathway. We aimed to determine whether alkyne cholesterol is a substrate for NPC1L1 in intestinal cells. Human intestinal epithelial Caco2 cells were incubated with micelles containing alkyne cholesterol in the presence or absence of EZE. Small intestinal closed loops in C57BL/6J mice were injected with micelles containing alkyne cholesterol with or without EZE. Alkyne cholesterol esterification in Caco2 cells was significantly inhibited by EZE and by inhibitor of clathrin-mediated endocytosis Pitstop 2. The esterification was similarly reduced by inhibitors of the acyl-CoA cholesterol acyltransferase (ACAT). Alkyne cholesterol efficiently labeled the apical membrane of Caco2 cells and the amount retained on the membrane was significantly increased by EZE as judged by accessibility to exogenous cholesterol oxidase. In mouse small intestine, the presence of EZE reduced total alkyne cholesterol uptake by ∼75%. These data show that alkyne cholesterol acts as a substrate for NPC1L1 and may serve as a nonradioactive tracer to measure cholesterol absorption in both in vitro and in vivo models.

S-acylation modulates the function of the apical sodium-dependent bile acid transporter in human cells.

The ileal apical sodium-dependent bile acid transporter (ASBT) is crucial for the enterohepatic circulation of bile acids. ASBT function is rapidly regulated by several posttranslational modifications. One reversible posttranslational modification is S-acylation, involving the covalent attachment of fatty acids to cysteine residues in proteins. However, whether S-acylation affects ASBT function and membrane expression has not been determined. Using the acyl resin-assisted capture method, we found that the majority of ASBT (∼80%) was S-acylated in ileal brush border membrane vesicles from human organ donors, as well as in HEK293 cells stably transfected with ASBT (2BT cells). Metabolic labeling with alkyne-palmitic acid (100 µm for 15 h) also showed that ASBT is S-acylated in 2BT cells. Incubation with the acyltransferase inhibitor 2-bromopalmitate (25 µm for 15 h) significantly reduced ASBT S-acylation, function, and levels on the plasma membrane. Treatment of 2BT cells with saturated palmitic acid (100 µm for 15 h) increased ASBT function, whereas treatment with unsaturated oleic acid significantly reduced ASBT function. Metabolic labeling with alkyne-oleic acid (100 µm for 15 h) revealed that oleic acid attaches to ASBT, suggesting that unsaturated fatty acids may decrease ASBT's function via a direct covalent interaction with ASBT. We also identified Cys-314 as a potential S-acylation site. In conclusion, these results provide evidence that S-acylation is involved in the modulation of ASBT function. These findings underscore the potential for unsaturated fatty acids to reduce ASBT function, which may be useful in disorders in which bile acid toxicity is implicated.

miR-29a, b, and c regulate SLC5A8 expression in intestinal epithelial cells.

Short-chain fatty acids (SCFAs) produced by bacterial fermentation of dietary fiber exert myriad of beneficial effects including the amelioration of inflammation. SCFAs exist as anions at luminal pH; their entry into the cells depends on the expression and function of monocarboxylate transporters. In this regard, sodium-coupled monocarboxylate transporter-1 (SMCT-1) is one of the major proteins involved in the absorption of SCFA in the mammalian colon. However, very little is known about the mechanisms of regulation of SMCT-1 expression in health and disease. MicroRNAs (miRs) are known to play a key role in modulating gene expression. In silico analysis showed miR-29a, b, and c with highest context score and its binding region was conserved among mammals. The 3'-untranslated region (UTR) of human SMCT-1 gene was cloned into pmirGLO vector upstream of luciferase reporter and transiently transfected with miR-29a, b, and c mimics into Caco-2 and/or T-84 cells. The presence of UTR of this gene significantly decreased luciferase activity compared with empty vector. Cotransfection with miR-29a, b, or c resulted in further decrease in 3'-UTR activity of SMCT-1 luciferase constructs. Mimic transfection significantly decreased SMCT-1 protein expression without altering mRNA expression. Furthermore, the expression of miR-29a and c were significantly lower in mouse colon compared with small intestine, consistent with higher levels of SMCT-1 protein in the colon. Our studies demonstrated a novel finding in which miR-29a, b, and c downregulate SMCT-1 expression in colonic epithelial cells and may partly explain the differential expression of these transporters along the length of the gastrointestinal (GI) tract.NEW & NOTEWORTHY Our study for the first time reports the posttranscriptional regulation of SMCT-1 by miR-29a, b, and c in colonic epithelial cells. We also demonstrate that the expression of these microRNAs is lower in the mouse proximal and distal colon which partially explains the higher expression level of SMCT-1 in the colon compared with small intestine.

Hepatocyte nuclear factor-4α regulates expression of the serotonin transporter in intestinal epithelial cells.

The serotonin transporter (SERT) functions to regulate the availability of serotonin (5-HT) in the brain and intestine. An intestine-specific mRNA variant arising from a unique transcription start site and alternative promoter in the SERT gene has been identified (iSERT; spanning exon 1C). A decrease in SERT is implicated in several gut disorders, including inflammatory bowel diseases (IBD). However, little is known about mechanisms regulating the iSERT variant, and a clearer understanding is warranted for targeting SERT for the treatment of gut disorders. The current studies examined the expression of iSERT across different human intestinal regions and investigated its regulation by HNF4α (hepatic nuclear factor-4α), a transcription factor important for diverse cellular functions. iSERT mRNA abundance was highest in the human ileum and Caco-2 cell line. iSERT mRNA expression was downregulated by loss of HNF4α (but not HNF1α, HNF1ß, or FOXA1) in Caco-2 cells. Overexpression of HNF4α increased iSERT mRNA concomitant with an increase in SERT protein. Progressive promoter deletion and site-directed mutagenesis revealed that the HNF4α response element spans nucleotides -1,163 to -1150 relative to the translation start site. SERT mRNA levels in the intestine were drastically reduced in the intestine-specific HNF4α-knockout mice relative to HNF4αFL/FL mice. Both HNF4α and SERT mRNA levels were also downregulated in mouse model of ileitis (SAMP) compared with AKR control mice. These results establish the transcriptional regulation of iSERT at the gut-specific internal promoter (hSERTp2) and have identified HNF4α as a critical modulator of basal SERT expression in the intestine.

Serotonin Modulates AhR Activation by Interfering with CYP1A1-Mediated Clearance of AhR Ligands.

BACKGROUND/AIMS: Serotonin (5-hydroxytryptamine, 5-HT) is a neurotransmitter and hormone with important physiological functions in many organs, including the intestine. We have previously shown that 5-HT activates the aryl hydrocarbon receptor (AhR) in intestinal epithelial cells (IECs) via a serotonin transporter (SERT)-dependent mechanism. AhR is a nuclear receptor that binds a variety of molecules including tryptophan (TRP) metabolites to regulate physiological processes in the intestine including xenobiotic detoxification and immune modulation. We hypothesized that 5-HT activates AhR indirectly by interfering with metabolic clearance of AhR ligands by cytochrome P450 1A1 (CYP1A1). METHODS: Inhibition of CYP1A1 activity by 5-HT was assessed in the human intestinal epithelial cell line Caco-2 and recombinant CYP1A1 microsomes using both luciferase and LC-MS/MS. Degradation of 5-HT by recombinant CYP1A1 was measured by LC-MS/MS. For in vitro studies, CYP1A1 and CYP1B1 mRNA expression levels were measured by RT-PCR and CYP1A1 activity was measured by ethoxyresorufin-O-deethylase (EROD) assays. For in vivo studies, AhR ligands were administered to SERT KO mice and WT littermates and intestinal mucosa CYP1A1 mRNA was measured. RESULTS: We show that 5-HT inhibits metabolism of both the pro-luciferin CYP1A1 substrate Luc-CEE as well as the high affinity AhR ligand 6-formylindolo[3,2-b] carbazole (FICZ). Recombinant CYP1A1 assays revealed that 5-HT is metabolized by CYP1A1 in an NADPH dependent manner. Treatment with 5-HT in TRP-free medium, which is devoid of trace AhR ligands, showed that 5-HT requires the presence of AhR ligands to activate AhR. Cotreatment with 5-HT and FICZ confirmed that 5-HT potentiates induction of AhR target genes by AhR ligands. However, this was only true for ligands which are CYP1A1 substrates such as FICZ. Administration of ß-napthoflavone by gavage or indole-3-carbinol via diet to SERT KO mice revealed that lack of SERT impairs intestinal AhR activation. CONCLUSION: Our studies provide novel evidence of crosstalk between serotonergic and AhR signaling where 5-HT can influence the ability of AhR ligands to activate the receptor in the intestine.

Mechanisms of Niemann-Pick type C1 Like 1 protein degradation in intestinal epithelial cells.

Intestinal Niemann-Pick C1 Like 1 (NPC1L1) protein plays a key role in cholesterol absorption. A decrease in NPC1L1 expression has been implicated in lowering plasma cholesterol and mitigating the risk for coronary heart disease. Little is known about the mechanisms responsible for NPC1L1 protein degradation that upon activation may lead to a reduction in NPC1L1 protein levels in intestinal epithelial cells (IECs). In current studies, the human intestinal Caco-2 and HuTu-80 cell lines expressing NPC1L1-hemagglutinin fusion protein were used to investigate the mechanisms of NPC1L1 protein degradation. Incubation with the proteasome inhibitors MG-132 and lactacystin (10 µM, 24 h) significantly increased NPC1L1 protein levels in IECs. Also, the inhibition of the lysosomal pathway with bafilomycin A1 (80 nM, 24 h) resulted in a significant increase in NPC1L1 protein levels. Immunoprecipitation studies showed that NPC1L1 protein is both a poly- and monoubiquinated polypeptide and that the inhibition of the proteasomal pathway remarkably increased the level of the polyubiquinated NPC1L1. The surface expression of NPC1L1 was increased by the inhibition of both proteasomal and lysosomal pathways. Furthermore, the pharmacological inhibition of mitogen-activated protein kinase pathway (PD-98059, 15 µM, 24 h) and siRNA silencing of ERK1/2 resulted in a significant decrease in NPC1L1 protein levels in IECs. In conclusion, our results showed that basal level of intestinal cholesterol transporter NPC1L1 protein is modulated by both ubiquitin proteasome- and lysosome-dependent degradation as well as by ERK1/2-dependent pathway. The modulation of these pathways may provide novel clues for therapeutic intervention to inhibit cholesterol absorption and lower plasma cholesterol.

miR-125a-5p: a novel regulator of SLC26A6 expression in intestinal epithelial cells.

Putative anion transporter 1 (PAT1, SLC26A6), an intestinal epithelial Cl-/ HCO3- exchanger, also plays a key role in oxalate homeostasis via mediating intestinal oxalate secretion. Indeed, Slc26a6-null mice showed defect in intestinal oxalate secretion and high incidence of kidney stones. Recent emergence of PAT-1 as a novel therapeutic target for nephrolithiasis warrants detailed understanding of the mechanisms of PAT-1 regulation in health and disease. Therefore, we investigated the regulation of PAT-1 expression by microRNAs (miRNA), as they have been shown to play key role in modulating expression of other ion transporters. In silico analysis of PAT-1 3'-untranslated region (UTR) revealed potential binding sites for several miRNAs, suggesting the role of miRNAs in modulating PAT1 expression. miRNAs showing highest context scores (125a-5p, 339-5p, 423-5p, 485-5p, and 501-3p) were selected as candidates for their effects on the activity of a 263-bp PAT-1 3'-untranslated region (UTR) fragment cloned into pmirGLO vector upstream of luciferase. The 3'-UTR activity was measured by dual luciferase reporter assay in Caco-2, T-84, HT-29, and SK-CO15 cells. Transient transfection of PAT-1 3'-UTR significantly decreased the relative luciferase activity compared with the empty vector suggesting binding of potential miRNA(s) to the PAT-1 3'-UTR. Among all the selected candidates, cotransfection with miRNA mimics 125a-5p and 423-5p further decreased PAT-1 3'-UTR activity. Furthermore, increasing miR-125a-5p abundance via mimic transfection in Caco-2 cells decreased both mRNA and protein levels of PAT-1. Our results demonstrate a novel regulatory mechanism of intestinal PAT-1 expression via miR-125a-5p that could be of therapeutic importance in disorders associated with decreased PAT-1 expression and function.

Decreased SLC26A3 expression and function in intestinal epithelial cells in response to Cryptosporidium parvum infection.

The protozoan parasite Cryptosporidium parvum (CP) causes cryptosporidiosis, a diarrheal disease worldwide. Infection in immunocompetent hosts typically results in acute, self-limiting, or recurrent diarrhea. However, in immunocompromised individuals infection can cause fulminant diarrhea, extraintestinal manifestations, and death. To date, the mechanisms underlying CP-induced diarrheal pathogenesis are poorly understood. Diarrheal diseases most commonly involve increased secretion and/or decreased absorption of fluid and electrolytes. We and others have previously shown impaired chloride absorption in infectious diarrhea due to dysregulation of SLC26A3 [downregulated in adenoma (DRA)], the human intestinal apical membrane Cl-/HCO3- exchanger protein. However, there are no studies on the effects of CP infection on DRA activity. Therefore, we examined the expression and function of DRA in intestinal epithelial cells in response to CP infection in vitro and in vivo. CP infection (0.5 × 106 oocysts/well in 24-well plates, 24 h) of Caco-2 cell monolayers significantly decreased Cl-/HCO3- exchange activity (measured as DIDS-sensitive 125I uptake) as well as DRA mRNA and protein levels. Substantial downregulation of DRA mRNA and protein was also observed following CP infection ex vivo in mouse enteroid-derived monolayers and in vivo in the ileal and jejunal mucosa of C57BL/6 mice for 24 h. However, at 48 h after infection in vivo, the effects on DRA mRNA and protein were attenuated and at 5 days after infection DRA returned to normal levels. Our results suggest that impaired chloride absorption due to downregulation of DRA could be one of the contributing factors to CP-induced acute, self-limiting diarrhea in immunocompetent hosts.

Cryptosporidium parvum disrupts intestinal epithelial barrier function via altering expression of key tight junction and adherens junction proteins.

Infection with the protozoan parasite Cryptosporidium parvum (CP) causes cryptosporidiosis, a widespread diarrhoeal disease. Impaired intestinal epithelial barrier function and increased permeability are most commonly associated with diarrhoeal diseases caused by enteric infections. However, studies on barrier disruption and underlying mechanisms in cryptosporidiosis are extremely limited. Epithelial tight junctions (TJs) and adherens junctions (AJs) are important in maintaining barrier integrity. Therefore, we examined the effects of CP infection on paracellular permeability and on the expression of the major TJ and AJ proteins utilising in vitro, ex vivo, and in vivo models. CP infection (0.5 × 106  oocysts/well in Transwell inserts, 24 hr) increased paracellular permeability (FITC-dextran flux) in Caco-2 cell monolayers and substantially decreased the protein levels of occludin, claudin 4, and E-cadherin. Claudin 3, zonula occludens-1 (ZO1) and α-catenin were also significantly decreased, whereas claudins 1 and 2 and ß-catenin were not altered. Substantial downregulation of occludin, claudin 4, and E-cadherin was also observed in response to CP infection ex vivo in mouse enteroid-derived monolayers and in vivo in the ileal and jejunal mocosa of C57BL/6 mice. The mRNA levels of these proteins were also significantly decreased in CP-infected mouse ileum and jejunum but were unaltered in Caco-2 cells. Further, bafilomycin-A, an inhibitor of lysosomal proton pump, partially abrogated CP effects on occludin expression in Caco-2 cells, suggesting a potential role of posttranslational mechanisms, such as induction of protein degradation pathways, in mediating the effects of the parasite. Our studies suggest that disruption of barrier function via downregulation of specific key components of TJ and AJ could be a major mechanism underlying CP infection-induced diarrhoea.

A novel bioluminescence-based method to investigate uptake of bile acids in living cells.

Bile acid transporters, including the ileal apical sodium-dependent bile acid transporter (ASBT) and the hepatic sodium-taurocholate cotransporting polypeptide (NTCP), are crucial for the enterohepatic circulation of bile acids. Our objective was to develop a method for measuring bile acid transporter activity in real time to precisely evaluate rapid changes in their function. We designed a reporter system relying on a novel probe: cholic acid attached to luciferin via a disulfide-containing, self-immolating linker (CA-SS-Luc). Incubation of human embryonic kidney-293 cells coexpressing luciferase and ASBT with different concentrations of CA-SS-Luc (0.01-1 µM) resulted in bioluminescence with an intensity that was concentration- and time-dependent. The bioluminescence measured during incubation with 1 µM CA-SS-Luc was dependent on the levels of ASBT or NTCP expressed in the cells. Coincubation of CA-SS-Luc with natural bile acids enhanced the bioluminescence in a concentration-dependent manner with kinetic parameters for ASBT similar to those previously reported using conventional methods. These findings suggest that this method faithfully assesses ASBT function. Further, incubation with tyrosine phosphatase inhibitor III (PTPIII) led to significantly increased bioluminescence in cells expressing ASBT, consistent with previous studies showing an increase in ASBT function by PTPIII. We then investigated CA-SS-Luc in isolated mouse intestinal epithelial cells. Ileal enterocytes displayed significantly higher luminescence compared with jejunal enterocytes, indicating a transport process mediated by ileal ASBT. In conclusion, we have developed a novel method to monitor the activity of bile acid transporters in real time that has potential applications both for in vitro and in vivo studies. NEW & NOTEWORTHY This article reports the development of a real-time method for measuring the uptake of bile acids using a bioluminescent bile acid-based probe. This method has been validated for measuring uptake via the apical sodium-dependent bile acid transporter and the sodium-taurocholate cotransporting polypeptide in cell culture and ex vivo intestinal models.

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.

Clostridium difficile toxins A and B decrease intestinal SLC26A3 protein expression.

Clostridium difficile infection (CDI) is the primary cause of nosocomial diarrhea in the United States. Although C. difficile toxins A and B are the primary mediators of CDI, the overall pathophysiology underlying C. difficile-associated diarrhea remains poorly understood. Studies have shown that a decrease in both NHE3 (Na+/H+ exchanger) and DRA (downregulated in adenoma, Cl-/[Formula: see text] exchanger), resulting in decreased electrolyte absorption, is implicated in infectious and inflammatory diarrhea. Furthermore, studies have shown that NHE3 is depleted at the apical surface of intestinal epithelial cells and downregulated in patients with CDI, but the role of DRA in CDI remains unknown. In the current studies, we examined the effects of C. difficile toxins TcdA and TcdB on DRA protein and mRNA levels in intestinal epithelial cells (IECs). Our data demonstrated that DRA protein levels were significantly reduced in response to TcdA and TcdB in IECs in culture. This effect was also specific to DRA, as NHE3 and PAT-1 (putative anion transporter 1) protein levels were unaffected by TcdA and TcdB. Additionally, purified TcdA and TcdA + TcdB, but not TcdB, resulted in a decrease in colonic DRA protein levels in a toxigenic mouse model of CDI. Finally, patients with recurrent CDI also exhibited significantly reduced expression of colonic DRA protein. Together, these findings indicate that C. difficile toxins markedly downregulate intestinal expression of DRA which may contribute to the diarrheal phenotype of CDI. NEW & NOTEWORTHY Our studies demonstrate, for the first time, that C. difficile toxins reduce DRA protein, but not mRNA, levels in intestinal epithelial cells. These findings suggest that a downregulation of DRA may be a critical factor in C. difficile infection-associated diarrhea.

Activation of Nuclear Factor-κB by Tumor Necrosis Factor in Intestinal Epithelial Cells and Mouse Intestinal Epithelia Reduces Expression of the Chloride Transporter SLC26A3.

BACKGROUND & AIMS: Diarrhea associated with inflammatory bowel diseases has been associated with increased levels of inflammatory cytokines, including tumor necrosis factor (TNF). The intestinal mucosa of patients with inflammatory bowel diseases has reduced expression of solute carrier family 26 member 3 (SLC26A3, also called DRA). We investigated whether TNF directly affects expression of DRA in human intestinal epithelial cells (IECs) and in the intestines of mice, and studied the mechanisms of these effects. METHODS: We performed quantitative reverse transcription polymerase chain reaction, immunofluorescence, and immunoblot analyses in Caco-2, HT-29, and T-84 cells human IECs cultured in 2 or 3 dimensions with or without TNF (50 ng/mL for 6-24 hours). We purified nuclear extracts and quantified nuclear factor-κB (NF-κB) activation and DNA binding. We isolated intestinal crypts from C57BL/6 mice, cultured enteroids, incubated these with TNF (50 ng/mL, 24 hours), and quantified messenger RNAs. DRA-mediated exchange of Cl- for HCO3- was measured by uptake of 125I. Expression of the NF-κB inhibitor α (IkBa) was knocked down in Caco-2 cells with small interfering RNAs. Activation of NF-κB in response to TNF was measured by luciferase reporter assays; binding of the NF-κB subunit p65 in cells was analyzed in chromatin immunoprecipitation assays. DRA promoter activity was measured in a luciferase reporter assay. C57BL/6 mice were injected with TNF (5 µg/mouse for 3-6 hours) or vehicle (control); intestines were collected and analyzed by immunofluorescence, or RNA and protein were collected from the mucosa. RESULTS: Incubation of IECs with TNF reduced expression of DRA. Knockdown of NF-κB inhibitor α in IECs led to nuclear translocation of the NF-κB subunit p65 and reduced levels of DRA messenger RNA and protein. Expression of a transgene encoding p65 or p50 in IECs led to significant reductions in the promoter activity of DRA and its expression. In chromatin immunoprecipitation assays, p65 bound directly to the promoter of DRA, at the regions of -935 to -629 and -375 to -84. Injection of mice with TNF or incubation of crypt-derived enteroids with TNF reduced their expression of DRA messenger RNA and protein. CONCLUSIONS: In human IECs and intestinal tissues from mice, we found TNF to activate NF-κB, which reduced expression of the Cl- / HCO3- exchanger DRA (SLC26A3), via direct binding to the promoter of DRA. This pathway is an important therapeutic target for inflammatory bowel disease-associated diarrhea.

Role of SHP2 protein tyrosine phosphatase in SERT inhibition by enteropathogenic E. coli (EPEC).

Enteropathogenic Escherichia coli (EPEC), one of the diarrheagenic E. coli pathotypes, is among the most important food-borne pathogens infecting children worldwide. Inhibition of serotonin transporter (SERT), which regulates extracellular availability of serotonin (5-HT), has been implicated previously in EPEC-associated diarrhea. EPEC was shown to inhibit SERT via activation of protein tyrosine phosphatase (PTPase), albeit the specific PTPase involved is not known. Current studies aimed to identify EPEC-activated PTPase and its role in SERT inhibition. Infection of Caco-2 monolayers with EPEC strain E2348/69 for 30 min increased the activity of Src-homology-2 domain containing PTPase (SHP2) but not SHP1 or PTPase 1B. Similarly, Western blot studies showed increased tyrosine phosphorylation of (p-tyrosine) SHP2, indicative of its activation. Concomitantly, EPEC infection decreased SERT p-tyrosine levels. This was associated with increased interaction of SHP2 with SERT, as evidenced by coimmunoprecipitation studies. To examine whether SHP2 directly influences SERT phosphorylation status or function, SHP2 cDNA plasmid constructs (wild type, constitutively active, or dominant negative) were overexpressed in Caco-2 cells by Amaxa electroporation. In the cells overexpressing constitutively active SHP2, SERT polypeptide showed complete loss of p-tyrosine. In addition, there was a decrease in SERT function, as measured by Na+Cl--sensitive [3H]5-HT uptake, and an increase in association of SERT with SHP2 in Caco-2 cells expressing constitutively active SHP2 compared with dominant-negative SHP2. Our data demonstrate that intestinal SERT is a target of SHP2 and reveal a novel mechanism by which a common food-borne pathogen uses cellular SHP2 to inhibit SERT.NEW & NOTEWORTHY The data presented in the current study reveal that intestinal serotonin transporter (SERT) is a target of the tyrosine phosphatase SHP2 and show a novel mechanism by which a common diarrheagenic pathogen, EPEC, activates cellular SHP2 to inhibit SERT function. These studies highlight host-pathogen interactions, which may be of therapeutic relevance in the management of diarrhea associated with enteric infections.

Expression and localization of VPAC1, the major receptor of vasoactive intestinal peptide along the length of the intestine.

Vasoactive intestinal peptide (VIP) is an endogenous neuropeptide with a broad array of physiological functions in many organs including the intestine. Its actions are mediated via G protein-coupled receptors, and vasoactive intestinal peptide receptor 1 (VPAC1) is the key receptor responsible for majority of VIP's biological activity. The distribution of VPAC1 along the length of the gastrointestinal tract and its subcellular localization in intestinal epithelial cells have not been fully characterized. The current studies were undertaken to determine VPAC1 distribution and localization so that VIP-based therapies can be targeted to specific regions of the intestine. The results indicated that the mRNA levels of VPAC1 showed an abundance pattern of colon > ileum > jejunum in the mouse intestine. In parallel, the VPAC1 protein levels were higher in the mouse colon, followed by the ileum and jejunum. Immunofluorescence studies in mouse colon demonstrated that the receptor was specifically localized to the luminal surface, as was evident by colocalization with the apical marker villin but not with the basolateral marker Na+/K+-ATPase. In the human intestine, VPAC1 mRNA expression exhibited a distribution similar to that in mouse intestine and was highest in the sigmoid colon. Furthermore, in the human colon, VPAC1 also showed predominantly apical localization. The physiological relevance of the expression and apical localization of VPAC1 remains elusive. We speculate that apical VPAC1 in intestinal epithelial cells may have relevance in recognizing secreted peptides in the intestinal lumen and therefore supports the feasibility of potential therapeutic and targeting use of VIP formulations via oral route to treat gastrointestinal diseases.NEW & NOTEWORTHY These studies for the first time present comprehensive data on the relative characterization of vasoactive intestinal peptide (VIP) receptors in the intestinal mucosa. Vasoactive intestinal peptide receptor 1 (VPAC1) was identified as the predominant receptor with higher levels in the colon compared with the small intestine and was mainly localized to the apical membrane. In addition, the findings in the human tissues were consistent with VPAC1 expression in the mouse intestine and open possibilities to target colonic tissues with VIP for treating diseases such as inflammatory bowel disease.