BBe Cells Research Articles

SLC26A3 (DRA) is stimulated in a synergistic, intracellular Ca2+-dependent manner by cAMP and ATP in intestinal epithelial cells.

In polarized intestinal epithelial cells, downregulated in adenoma (DRA) is an apical Cl-/[Formula: see text] exchanger that is part of neutral NaCl absorption under baseline conditions, but in cyclic adenosine monophosphate (cAMP)-driven diarrheas, it is stimulated and contributes to increased anion secretion. To further understand the regulation of DRA in conditions mimicking some diarrheal diseases, Caco-2/BBE cells were exposed to forskolin (FSK) and adenosine 5'-triphosphate (ATP). FSK and ATP stimulated DRA in a concentration-dependent manner, with ATP acting via P2Y1 receptors. FSK at 1 µM and ATP at 0.25 µM had minimal to no effect on DRA given individually; however, together, they stimulated DRA to levels seen with maximum concentrations of FSK and ATP alone. In Caco-2/BBE cells expressing the Ca2+ indicator GCaMP6s, ATP increased intracellular Ca2+ (Ca2+i) in a concentration-dependent manner, whereas FSK (1 µM), which by itself did not significantly alter Ca2+i, followed by 0.25 µM ATP produced a large increase in Ca2+ that was approximately equal to the elevation caused by 1 µM ATP. 1,2-Bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis(acetoxymethyl ester) (BAPTA-AM) pretreatment prevented the ATP and FSK/ATP synergistically increased the DRA activity and the increase in Ca2+i caused by FSK/ATP. FSK/ATP synergistic stimulation of DRA was similarly observed in human colonoids. In Caco-2/BBE cells, subthreshold concentrations of FSK (cAMP) and ATP (Ca2+) synergistically increased Ca2+i and stimulated DRA activity with both being blocked by BAPTA-AM pretreatment. Diarrheal diseases, such as bile acid diarrhea, in which both cAMP and Ca2+ are elevated, are likely to be associated with stimulated DRA activity contributing to increased anion secretion, whereas separation of DRA from Na+/H+ exchanger isoform-3 (NHE3) contributes to reduced NaCl absorption.NEW & NOTEWORTHY The BB Cl-/[Formula: see text] exchanger DRA takes part in both neutral NaCl absorption and stimulated anion secretion. Using intestinal cell line, Caco-2/BBE high concentrations of cAMP and Ca2+ individually stimulated DRA activity, whereas low concentrations, which had no/minimal effect, synergistically stimulated DRA activity that required a synergistic increase in intracellular Ca2+. This study increases understanding of diarrheal diseases, such as bile salt diarrhea, in which both cAMP and elevated Ca2+ are involved.

The Air-Liquid Interface Reorganizes Membrane Lipids and Enhances the Recruitment of Slc26a3 to Lipid-Rich Domains in Human Colonoid Monolayers.

Cholesterol-rich membrane domains, also called lipid rafts (LRs), are specialized membrane domains that provide a platform for intracellular signal transduction. Membrane proteins often cluster in LRs that further aggregate into larger platform-like structures that are enriched in ceramides and are called ceramide-rich platforms (CRPs). The role of CRPs in the regulation of intestinal epithelial functions remains unknown. Down-regulated in adenoma (DRA) is an intestinal Cl-/HCO3- antiporter that is enriched in LRs. However, little is known regarding the mechanisms involved in the regulation of DRA activity. The air-liquid interface (ALI) was created by removing apical media for a specified number of days; from 12-14 days post-confluency, Caco-2/BBe cells or a colonoid monolayer were grown as submerged cultures. Confocal imaging was used to examine the dimensions of membrane microdomains that contained DRA. DRA expression and activity were enhanced in Caco-2/BBe cells and human colonoids using an ALI culture method. ALI causes an increase in acid sphingomyelinase (ASMase) activity, an enzyme responsible for enhancing ceramide content in the plasma membrane. ALI cultures expressed a larger number of DRA-containing platforms with dimensions >2 µm compared to cells grown as submerged cultures. ASMase inhibitor, desipramine, disrupted CRPs and reduced the ALI-induced increase in DRA expression in the apical membrane. Exposing normal human colonoid monolayers to ALI increased the ASMase activity and enhanced the differentiation of colonoids along with basal and forskolin-stimulated DRA activities. ALI increases DRA activity and expression by increasing ASMase activity and platform formation in Caco-2/BBe cells and by enhancing the differentiation of colonoids.

Abstract P366: The Impact Of Estrogen Signaling On Gut Epithelial Cells

Cardiovascular disease (CVD) is the leading cause of death in women worldwide. Menopause is associated with increased susceptibility to CVD, but the mechanism is currently unknown. Studies have suggested that loss of estrogen signaling in the intestine during menopause may lead to worse myocardial infarction (MI) outcomes in female mice. Our lab has shown that suppressing inflammation in the gut using pre- and pro-biotics protects the heart from ischemic heart disease caused by MI through unknown means. We propose that alterations in gut microbiota are due to destruction of intestinal pattern recognition receptors (PRRs) such as toll-like receptor 4 (TLR4) and TLR5. Studies show that estrogen signaling modulates PRRs in other tissues, but this has yet to be investigated in gut epithelial cells. To elucidate the mechanisms of estrogen receptor function in the gut as a direct mediator of vascular health, we are utilizing an immortalized human cell line of colorectal adenocarcinoma brush border expressing (Caco2 BBE) cells. We hypothesized that estrogen treatment in gut epithelial cells will lead to increased expression of PRRs and decreased inflammation in Caco2 BBE cell lines. To investigate expression and localization of estrogen receptors (ERs), Caco2 BBE cells at different confluency were treated with estradiol (E2) or DMSO control for 24hrs, then either RNA or protein were harvested for use in qPCR and western blot, or cells were formalin fixed for use in immunohistochemistry. In each case, ERα was not detected, but ERβ was present at varying concentrations. Data also showed increased expression of TLR5 and TLR7 in E2 treated cells, while TNF-α and IL-1 were decreased. In conclusion, data show potential confluency dependent ERβ expression and suggest that ERα may not be present in our Caco2 BBE cell line. Our hypothesis is also supported by data for PPRs and inflammatory markers. Though these results are revealing, future studies are needed to further characterize Caco2 BBE cells in the context of ER signaling.

TRANSFER RNA QUEUOSINE MODIFICATION ENZYME MANIPULATES TIGHT JUNCTION PROTEINS IN INFLAMMATORY BOWEL DISEASE

Abstract Background Transfer RNA (tRNA) queuosine (Q)-modification occur at the wobble anticodon position of four special cellular tRNAs. In eukaryotes, tRNA-Q modification relies on the intestinal microbial product queuine and eukaryotic tRNA-guanine transglycosylase complex contains of Q tRNA ribosyltransferase catalytic subunit 1 (QTRT1). Q-tRNA modification is critical for the fidelity and accuracy to translate RNA to protein. It is known that dysfunction of Q-tRNA associates with cancer proliferation and malignancy. However, mechanisms of how Q and Q-tRNA modifications influence intestinal epithelial biology and chronic inflammation are unknown. Methods We explored the expression of QTRT1 in patients with IBD by investigating human biopsy tissues and re-analyzing gene expression datasets (with both UC and CD patients). To further investigate the QTRT1 role in IBD, the dextran sodium sulfate (DSS) induced colitis mouse model and IL10-deficient mouse model were used to evaluate the molecular mechanism. We further investigated the impact of QTRT1 reduction on intestinal cells by generating QTRT1 knockdown HCT116 and Caco-2 BBE cells using a Double Nickase Plasmid. Results We found that the QTRT1 expression in IHC staining was significantly downregulated in the colon of both Ulcerative Colitis (UC) and Crohn’s Disease (CD) patients compared with normal controls. After extracting and analyzing the datasets, we found the gene expression of QTRT1 was significantly suppressed in colonic mucosa of UC and intestinal mucosa of CD, compared with controls. Moreover, the four tRNA-Q-related tRNA synthetase (asparaginyl-tRNA synthetase, aspartyl-tRNA synthetase, histidyl-tRNA synthetase and tyrosyl-tRNA synthetase) were also decreased in IBD patients. The decreased QTRT1-expression was further confirmed in both DSS-induced colitis mice and IL10-defecient mice. Meanwhile, the significantly downregulated beta-catenin and Claudin-5, and upregulated Caludin-2 were identified in these two colitis models. All these molecular alterations, which are important in cell adhesion and tight junction formation, were further verified in protein expression and mRNA level using QTRT1 reduction HCT116 and Caco-2 BBE cells in vitro. Conclusion Our results demonstrate that the QTRT1 plays a novel role in human IBD by altering intestinal cell adhesion and junctions. Microbiome-dependent Q supply can be altered during disease development or when a limited variety of food-types are ingested. Investigations on the gut microbiome-derived tRNA modification in IBD will uncover novel molecular mechanisms for potential prevention and therapy.

AvrA Exerts Inhibition of NF-κB Pathway in Its Naïve Salmonella Serotype through Suppression of p-JNK and Beclin-1 Molecules.

Avian salmonellosis caused by Salmonella enterica serovar Enteritidis (S. Enteritidis) and Pullorum (S. Pullorum) remains a big threat to the poultry industry and public hygiene. AvrA is an effector involved in inhibiting inflammation. Compared to AvrA from S. Enteritidis (SE-AvrA), the AvrA from S. Pullorum (SP-AvrA) lacks ten amino acids at the C-terminal. In this study, we compared the anti-inflammatory response induced by SP-AvrA to that of SE-AvrA. Transient expression of SP-AvrA in epithelial cells resulted in significantly weaker inhibition of NF-κB pathway activation when treated with TNF-α compared to the inhibition by SE-AvrA. SP-AvrA expression in the S. Enteritidis resulted in weaker suppression of NF-κB pathway in infected HeLa cells compared to SE-AvrA expression in the cells, while SP-AvrA expressed in S. Pullorum C79-13 suppressed NF-κB activation in infected HeLa and Caco 2 BBE cells to a greater extent than did SE-AvrA because of the higher expression of SP-AvrA than SE-AvrA in S. Pullorum. Further analysis demonstrated that the inhibition of NF-κB pathway in Salmonella-infected cells corresponded to the downregulation of the p-JNK and Beclin-1 protein molecules. Our study reveals that AvrA modifies the anti-inflammatory response in a manner dependent on the Salmonella serotype through inhibition of NF-κB pathway.

Histone H3K9 methylation regulates chronic stress and IL-6-induced colon epithelial permeability and visceral pain.

Chronic stress is associated with activation of the HPA axis, elevation in pro-inflammatory cytokines, decrease in intestinal epithelial cell tight junction (TJ) proteins, and enhanced visceral pain. It is unknown whether epigenetic regulatory pathways play a role in chronic stress-induced intestinal barrier dysfunction and visceral hyperalgesia. Young adult male rats were subjected to water avoidance stress±H3K9 methylation inhibitors or siRNAs. Visceral pain response was assessed. Differentiated Caco-2/BBE cells and human colonoids were treated with cortisol or IL-6±antagonists. Expression of TJ, IL-6, and H3K9 methylation status at gene promoters was measured. Transepithelial electrical resistance and FITC-dextran permeability were evaluated. Chronic stress induced IL-6 up-regulation prior to a decrease in TJ proteins in the rat colon. The IL-6 level inversely correlated with occludin expression. Treatment with IL-6 decreased occludin and induced visceral hyperalgesia. Chronic stress and IL-6 increased H3K9 methylation and decreased transcriptional GR binding to the occludin gene promoter, leading to down-regulation of protein expression and increase in paracellular permeability. Intrarectal administration of a H3K9 methylation antagonist prevented chronic stress-induced visceral hyperalgesia in the rat. In a human colonoid model, cortisol decreased occludin expression, which was prevented by the GR antagonist RU486, and IL-6 increased H3K9 methylation and decreased TJ protein levels, which were prevented by inhibitors of H3K9 methylation. Our findings support a novel role for methylation of the repressive histone H3K9 to regulate chronic stress, pro-inflammatory cytokine-mediated reduction in colon TJ protein levels, and increase in paracellular permeability and visceral hyperalgesia.

SspH2 as anti-inflammatory candidate effector and its contribution in Salmonella Enteritidis virulence

Salmonella enterica serovar Enteritidis (S. Enteritidis) is a facultative intracellular pathogen deploying the type III secretion system (T3SS) encoded by Salmonella Pathogenicity Island 2 (SPI2) to transfer effector proteins into host cells to modify its functions and accomplish intracellular replication. To study the effect of SspH2 on immune response induced by S. Enteritidis, we generated a deletion mutant of the effector gene sspH2 and a plasmid mediated complementary strain in S. Enteritidis C50336. The results of LD50 showed that SspH2 has no obvious effect on the virulence of S. Enteritidis. However, deletion of sspH2 decreased the invasion and intercellular colonization of the bacteria in Caco2 BBE cells. Using bacteriological counts from tissue homogenates the result of colonization in internal organs showed that in spleen and liver tissues, at 3rd and 4th day p.i. there is a significance decreased number of C50336-ΔsspH2 compared to the C50336-WT and C50336-ΔsspH2-psspH2, respectively. The qRT-PCR analysis results of both in vivo and in vitro experiments clearly showed that the mutant strain C50336ΔsspH2 significantly promoted expression of IL-1β, INF-γ, IL-12, and iNOS cytokines compared to the groups infected with the wild type or complementary strains, while the IL-8 synthesis was decreased in the mutant strain infected group. All of these findings revealed that SspH2 promotes the colonization of S. Enteritidis in host cells, and it is an important anti-inflammatory biased effector in Salmonella.

DOP51 Investigation on the role of Par4-associated cell polarity and associated barrier defects in inflammatory bowel diseases

Abstract Background In inflammatory bowel diseases (IBD), epithelial barrier defects occur as a consequence of chronic inflammation. Recent research suggested that cell polarity alterations may be upstream of barrier defects and additionally play a role in IBD-associated carcinogenesis (colitis-associated and small intestinal carcinoma). Par4 is a gene encoding a protein crucial in the development of cell polarity. LKB1, its human homologue, is mutated in Peutz–Jeghers syndrome (PJS), a genetic condition characterised by a higher risk of epithelial cancers. While the pivotal role of Par4/LKB1 in the development of epithelial cell polarity is established, its involvement in IBD-associated barrier defects and carcinogenesis is yet to be defined. Methods Endoscopic bowel mucosa samples from patients with Crohn’s disease, ulcerative colitis, PJS and controls were analysed to assess expression and localisation of Par4/LKB1. Cryosections were immunostained for Par4/LKB-1 along with a diverse set of markers of cell polarity and intercellular adhesion. The analysis was performed by confocal laser scanning microscopy in order to compare the expression of mucosal as well as the subcellular localisation of Par4/LKB1 in the gut mucosa. A quantitative analysis of protein expression with western blotting was performed as well. The function of Par4/LKB1 in intestinal epithelial cells (IEC) was evaluated by means of a Par4-deficient IEC model as well as in an IEC Par4-overexpression model. Moreover, a preliminary assessment of the possible role of Par4/LKB1-related polarity processes in IBD-associated carcinomas was performed through scanning genetic data from colitis-associated carcinomas for potential Par4-related mutations and altered Par4/LKB1 expression. Results The immunofluorescent staining allowed visualisation of intracellular expression of Par4 in epithelia from PJS, IBD patients and controls. In PJS-polyps, despite the alteration of regular tissue architecture typical of these lesions, the polarity of epithelial cells was maintained - contrary to control tissue, a punctate pattern of the Par4 staining was shown. In IBD tissue, no relevant differences in Par4 expression at confocal microscopy, as well as a quantitative assessment, were observed as compared with controls. No differences were observed in PJS or IBD samples as regards the expression of cell polarity markers such as Par3, CD71, crb3 or adhesion molecules such as ZO-1, e-cadherin, occludin, JAM-A. As regards the IEC model, we observed that Caco-2/BBe cells overexpressing Par4/LKB1 showed enhanced Par4/LKB1 cytosolic staining at immunofluorescence. In this model, a defective epithelial polarity and organisation on permeable supports could be observed in Caco2/BBe cysts in parallel with reduced expression of native LKB1 as seen in western blots. Preliminary analysis of data from our colitis-associated carcinomas database suggested various pathways potentially involving Par4/LKB1, yet no direct analysis of tissue samples for Par4-related mutations was performed yet. Conclusions Altered expression of Par4 in epithelial cells may influence the development of a functional epithelial barrier as suggested by IEC models. Par4/LKB1 as a known oncosuppressor gene is involved in a number of pathways potentially relevant for carcinogenesis in IBD; however, a direct connection between Par4-related alteration of the epithelial barrier and carcinogenesis is yet to be established.

NHERF3 is necessary for Escherichia coli heat-stable enterotoxin-induced inhibition of NHE3: differences in signaling in mouse small intestine and Caco-2 cells.

Enterotoxigenic Escherichia coli (ETEC) is a leading cause of childhood death from diarrhea and the leading cause of Traveler's diarrhea. E. coli heat-stable enterotoxin (ST) is a major virulence factor of ETEC and inhibits the brush border Na/H exchanger NHE3 in producing diarrhea. NHE3 regulation involves multiprotein signaling complexes that form on its COOH terminus. In this study, the hypothesis was tested that ST signals via members of the Na/H exchanger regulatory factor (NHERF) family of scaffolding proteins, NHERF2, which had been previously shown to have a role, and now with concentration on a role for NHERF3. Two models were used: mouse small intestine and Caco-2/BBe cells. In both models, ST rapidly increased intracellular cGMP, inhibited NHE3 activity, and caused a quantitatively similar decrease in apical expression of NHE3. The transport effects were NHERF3 and NHERF2 dependent. Also, mutation of the COOH-terminal amino acids of NHERF3 supported that NHERF3-NHERF2 heterodimerization was likely to account for this dual dependence. The ST increase in cGMP in both models was partially dependent on NHERF3. The intracellular signaling pathways by which ST-cGMP inhibits NHE3 were different in mouse jejunum (activation of cGMP kinase II, cGKII) and Caco-2 cells, which do not express cGKII (elevation of intracellular Ca2+ concentration [Ca2+]i). The ST elevation of [Ca2+]i was from intracellular stores and was dependent on NHERF3-NHERF2. This study shows that intracellular signaling in the same diarrheal model in multiple cell types may be different; this has implications for therapeutic strategies, which often assume that models have similar signaling mechanisms.

Chronic stress and intestinal permeability: Lubiprostone regulates glucocorticoid receptor-mediated changes in colon epithelial tight junction proteins, barrier function, and visceral pain in the rodent and human.

Chronic psychological stress is associated with increased intestinal epithelial permeability and visceral hyperalgesia. Lubiprostone, an agonist for chloride channel-2, promotes secretion and accelerates restoration of injury-induced epithelial barrier dysfunction. The mechanisms underlying how lubiprostone regulates colon epithelial barrier function and visceral hyperalgesia in chronic stress remain unknown. Male rats were subjected to water avoidance stress for 10 consecutive days. Lubiprostone was administered daily during the stress phase. Visceromotor response to colorectal distension was measured. Human colon crypts and cell lines were treated with cortisol and lubiprostone. The transepithelial electrical resistance and FITC-dextran permeability were assayed. Chromatin immunoprecipitation was conducted to assess glucocorticoid receptor binding at tight junction gene promoters. Lubiprostone significantly decreased chronic stress-induced visceral hyperalgesia in the rat (P<0.05; n=6). WA stress decreased occludin and claudin-1 and increased claudin-2 in rat colon crypts, which was prevented by lubiprostone. Cortisol treatment induced similar alterations of tight junction protein expression in Caco-2/BBE cells (P<0.05) and significantly changed paracellular permeability in monolayers (P<0.01). These changes were blocked by lubiprostone. Glucocorticoid receptor and its binding at occludin promoter region were decreased in cortisol-treated cells and human colon crypts, which was largely reversed by lubiprostone. In rat colonic cells, glucocorticoid receptor and its co-chaperone proteins were down-regulated after corticosterone treatment and lubiprostone reversed these changes. Lubiprostone preferentially prevents chronic stress-induced alterations of intestinal epithelial tight junctions, barrier function, and visceral hyperalgesia that was associated with modulation of glucocorticoid receptor expression and function.

Cobalt chloride compromises transepithelial barrier properties of CaCo-2 BBe human gastrointestinal epithelial cell layers

BackgroundElevation of the transcription factor HIF-1 is a prominent mediator of not only processes that accompany hypoxia, but also the tumor microenvironment and tissue regeneration. This study uses mediators of “chemical hypoxia” to ask the question whether HIF-1α elevation in a healthy epithelial cell layer leads to leakiness in its tight junctional seals.MethodsTransepithelial electrical resistance and transepithelial diffusion of 14C–D-mannitol and other radiolabeled probes are used as indicators of transepithelial barrier function of CaCo-2 BBe human gastrointestinal epithelial cell layers cultured on permeable supports. Western immunoblot analyses of integral tight junctional proteins (occludin and claudins) are used as further indicators of barrier function change.ResultsCobalt, an inhibitor of the prolyl hydroxylase enzymes governing HIF-1α breakdown in the cell, induces transepithelial leakiness in CaCo-2 BBe cell layers in a time and concentration-dependent manner. This increased leakiness is accompanied by significant changes in certain specific integral tight junctional (TJ) proteins such as a decreased level of occludin and increased level of claudin-5. Similar results regarding barrier function compromise also occur with other chemical inhibitors of HIF-1α breakdown, namely ciclopiroxolamine (CPX) and dimethyloxalylglycine (DMOG). The increased leak is manifested by both decreased transepithelial electrical resistance (Rt) and increased paracellular diffusion of D-mannitol (Jm). The induced transepithelial leak shows significant size selectivity, consistent with induced effects on TJ permeability. Less-differentiated cell layers were significantly more affected than well-differentiated cell layers regarding induced transepithelial leak. A genetically modified CaCo-2 variant with reduced levels of HIF-1β, showed reduced transepithelial leak in response to cobalt exposure, further indicating that elevation of HIF-1α levels induced by agents of “chemical hypoxia” is responsible for the compromised barrier function of the CaCo-2 BBe cell layers.ConclusionsExposure to inducers of chemical hypoxia elevated HIF-1α levels and increased transepithelial leak. The degree of epithelial differentiation has significant effects on this action, possibly explaining the varying effects of HIF-1 modulation in epithelial and endothelial barrier function in different physiological and pathophysiological conditions.

Krüppel-like factor 5 is essential for maintenance of barrier function in mouse colon.

Krüppel-like factor 5 (KLF5) is a member of the zinc finger family of transcription factors that regulates homeostasis of the intestinal epithelium. Previous studies suggested an indispensable role of KLF5 in maintaining intestinal barrier function. In the current study, we investigated the mechanisms by which KLF5 regulates colonic barrier function in vivo and in vitro. We used an inducible and a constitutive intestine-specific Klf5 knockout mouse models (Villin-CreERT2;Klf5fl/fl designated as Klf5ΔIND and Villin-Cre;Klf5fl/fl as Klf5ΔIS ) and studied an inducible KLF5 knockdown in Caco-2 BBe cells using a lentiviral Tet-on system (Caco-2 BBe KLF5ΔIND). Specific knockout of Klf5 in colonic tissues, either inducible or constitutive, resulted in increased intestinal permeability. The phenotype was accompanied by a significant reduction in Dsg2, which encodes desmoglein-2, a desmosomal cadherin, at both mRNA and protein levels. Transmission electron microscopy showed alterations of desmosomal morphology in both KLF5 knockdown Caco-2 BBe cells and Klf5 knockout mouse colonic tissues. Inducible knockdown of KLF5 in Caco-2BBe cells grown on Transwell plates led to impaired barrier function as evidenced by decreased transepithelial electrical resistance and increased paracellular permeability to fluorescein isothiocyanate-4 kDa dextran. Furthermore, DSG2 was significantly decreased in KLF5 knockdown cells, and DSG2 overexpression partially rescued the impaired barrier function caused by KLF5 knockdown. Electron microscopy studies demonstrated altered desmosomal morphology after KLF5 knockdown. In combination with chromatin immunoprecipitation analysis and promoter study, our data show that KLF5 regulates intestinal barrier function by mediating the transcription of DSG2, a gene encoding a major component of desmosome structures.NEW & NOTEWORTHY The study is original research on the direct function of a Krüppel-like factor on intestinal barrier function, which is commonly exerted by cell junctions, including tight junctions, adherens junctions, and desmosomes. Numerous previous studies were focused on tight junctions and adherens junctions. However, this study provided a new perspective on how the intestinal barrier function is regulated by KLF5 through DSG2, a component of desmosome complexes.

Chronic stress and intestinal barrier dysfunction: Glucocorticoid receptor and transcription repressor HES1 regulate tight junction protein Claudin-1 promoter

Chronic stress and elevated glucocorticoid hormone are associated with decreases in the intestinal epithelial tight junction protein claudin-1 (CLDN1). Human/rat CLDN1 promoters contain glucocorticoid response elements (GREs) and adjacent transcription repressor HES1 binding N-boxes. Notch signaling target HES1 expression was high and glucocorticoid receptor (NR3C1) low at the crypt base and the pattern reversed at the crypt apex. Chronic stress reduced overall rat colon HES1 and NR3C1 that was associated with CLDN1 downregulation. Chromatin-immunoprecipitation experiments showed that HES1 and NR3C1 bind to the CLDN1 promoter in rat colon crypts. The binding of NR3C1 but not HES1 to CLDN1 promoter significantly decreased in chronically stressed animals, which was prevented by the NR3C1 antagonist RU486. We employed the 21-day Caco-2/BBe cell model to replicate cell differentiation along the crypt axis. HES1 siRNA treatment early in differentiation increased CLDN1. In contrast, stress levels of cortisol decreased CLDN1 in late differentiation stage but not in the early stage. HES1 was high, whereas NR3C1 and CLDN1 were low in the early stage which reversed in the late stage, e.g. HES1/NR3C1 binding to CLDN1 promoter demonstrates a dynamic and reciprocal pattern. These results suggest that chronic stress impairs colon epithelium homeostasis and barrier function via different mechanisms along the crypt axis.

Phosphorylation of NHE3-S719 regulates NHE3 activity through the formation of multiple signaling complexes.

Casein kinase 2 (CK2) binds to the NHE3 C-terminus and constitutively phosphorylates a downstream site (S719) that accounts for 40% of basal NHE3 activity. The role of CK2 in regulation of NHE3 activity in polarized Caco-2/bbe cells was further examined by mutation of NHE3-S719 to A (not phosphorylated) or D (phosphomimetic). NHE3-S719A but not -S719D had multiple changes in NHE3 activity: 1) reduced basal NHE3 activity-specifically, inhibition of the PI3K/AKT-dependent component; 2) reduced acute stimulation of NHE3 activity by LPA/LPA5R stimulation; and 3) reduced acute inhibition of NHE3 activity-specifically, elevated Ca2+ related (carbachol/Ca2+ ionophore), but there was normal inhibition by forskolin and hyperosmolarity. The S719A mutant had reduced NHE3 complex size, reduced expression in lipid rafts, increased BB mobile fraction, and reduced binding to multiple proteins that bind throughout the NHE3 intracellular C-terminus, including calcineurin homologous protein, the NHERF family and SNX27 (related PDZ domains). These studies show that phosphorylation of the NHE3 at a single amino acid in the distal part of the C-terminus affects multiple aspects of NHE3 complex formation and changes the NHE3 lipid raft distribution, which cause changes in specific aspects of basal as well as acutely stimulated and inhibited Na+/H+ exchange activity.

Individual Amino Acids Differentially Regulate Paracellular Calcium Flux across an Intestinal Cell Model

Acute high protein intake results in increased intestinalcalcium absorption in humans and animals. Calcium is absorbed through both passive paracellular and facilitated trans cellular pathways. In rats, the increase in intestinal calcium absorption observed during a high protein dietis mediated at least in part through increased apical intestinal calcium uptake suggesting involvement of the transcellular pathway. However, we previously found that the expression of a paracellular calcium‐permissive tight junction protein, Claudin‐2, is higher in the duodenum of rats consuming a high compared to lowprotein diet. Whether dietary protein affects paracellular calcium flux has not been evaluated. Furthermore, the bioactive amino acids that might underlie this effect have not been identified. The purpose of this study was to evaluate the direct effects of mixed and individual amino acids on paracellular calcium flux across a small intestinal absorptive cell model.MethodsCaco‐2 Bbe cells were used as a model of small intestinal absorptive epithelial cells. Confluent monolayers were grown on snap well inserts and mounted in Ussing chambers for flux studies under a mucosal to serosal calcium gradient to simulatepost‐prandial conditions. Unidirectionalcalcium flux over 90 min was calculated using 44Ca as a tracer with serosal samples collected every 30 min. Treatments were applied acutely to bothmucosal and serosal chambers and included vehicle (no treatment control), 80mMraffinose as a positive control, mixed amino acids, and 2, 5 and 10mMleucine, phenylalanine and lysine. RNA expression of calcium permissive tight junction proteins was measured using qPCR.ResultsLinear regression analysis modeling Ca flux (nmol/cm2/hr) by treatment using vehicle control as the reference group elicited significant increases with 80mM raffinose (β= 66.7, t= 3.2, p&lt; 0.05) and 5mM leucine (β= 67.6, t= 1.9, p≤0.05), whereas 10mM phenylalanine resulted in a significant decrease in Ca flux (β= −89.3, t=−1.9, p&lt; 0.05). No significant changes were observed with mixed amino acidor lysine treatment. The effects of amino acids on Ca flux were not associated with changes in mRNA expression of the Ca permissive tight junction proteins claudins‐2 and 12.ConclusionsThese data demonstrate that amino acids have differential effects on paracellular Ca flux in a model of intestinalabsorptive epithelia. This suggests that specific amino acid constituents of dietary protein may be partially responsible for protein‐mediated increases inintestinal Ca absorption. Gene expression of claudins‐2 and 12 did not explain aminoacid induced changes in Ca flux. Future research will investigate posttranscriptional effects of amino acids on tight junction proteins in anintestinal epithelial model.Support or Funding InformationResearch supported by: USAMRMC. The views expressed in this abstract are those of the authors and do not reflect the official policy of the Department of Army, Department of Defense, or the U.S. Government.

Tu1401 Differential Intracellular Calcium Inhibition of NHE3 Activity

BACKGROUND: Elevation of intracellular calcium ([Ca]i) in intestinal epithelial cells occurs as part of normal digestion resulting in a transient decrease of NaCl absorption and increase in anion secretion. Under pathophysiological conditions, particularly rotavirus (RV) infection, elevation of [Ca]i is responsible for decreased Na+ absorption, increased Clsecretion and diarrhea. We have shown that elevated [Ca]i by either carbachol (CCH) or RV decreases NHE3 activity by stimulating rapid endocytosis; however, the differential signaling mechanisms responsible for the severe water loss due to RV infection are unclear. PURPOSE: Since NHE3 contributes to RV diarrhea, the current goal was to compare how elevated [Ca]i signaling differs under physiological (CCH) and pathophysiological (RV) conditions and determine their effects on NHE3. METHODS: Caco-2/BBe cells were grown on filters and transiently infected with a 3HA-NHE3 adenovirus construct and studied 48 h later. NHE3 activity was measured by fluorimetry using pH-sensitive dye, BCECF, in cells treated with vehicle, BAPTA-AM, latrunculin, methyl-β-cylcodextrin (MβCD), or inhibitors to PLD1 (EVJ), PLD2 (JWJ), Cdc42 (Pirl1), or acid sphingomyelinase (ASM; imipramine), in the presence or absence of CCHor simian rotavirus, RRV.NHE3 trafficking and degradation were determined by surface biotinylation. RESULTS: Both CCH and RV inhibited NHE3 activity by ~60% in a Ca2+ dependent manner. These effects were correlated with >50% decrease in NHE3 surface expression due to increased NHE3 endocytosis. NHE3 exocytosis was not different after CCH compared to control; however, NHE3 exocytosis was greatly reduced after RV infection. In addition, RV, but not CCH, treatment decreased NHE3 halflife. Pretreatment with either Pirl1, MβCD, both PLD inhibitors, imipramine, or latrunculin prevented CCH-mediated inhibition of NHE3 activity. In contrast, only MβCD and JWJ prevented RV inhibition of NHE3 while latrunculin had additive effects on NHE3 (>90% inhibition) after RV infection.CONCLUSIONS: (1) Physiologic and pathophysiologic [Ca]i elevation has differential effects on NHE3 regulation. (2) While both CCH and RV inhibit apical NHE3 activity through changes in trafficking due to elevated [Ca]i, RV also inhibits NHE3 activity by decreasing protein amount possibly due to a block in apical recycling since RV prevented NHE3 exocytosis. (3) These effects involve lipid rafts since cholesterol depletion and ceramide production by ASM prevent NHE3 inhibition; however, actin remodeling involving Cdc42 was not required for RV inhibition of NHE3 suggesting that RV infection stimulates a different endocytosis pathway for NHE3 compared to CCH resulting in increased NHE3 protein degradation. (4) Rotavirus infection is a new model to dissect mechanisms that regulate NHE3 activity in intestinal epithelial cells.

Mechanism of Linaclotide‐induced Trafficking of CFTR and NHE3 in the Human Intestinal Cell‐lines Caco‐2 BBe and T84 cells

Linaclotide (Linzess) is a potent guanylate cyclase‐C (GCC) agonist in clinical use for treatment of chronic constipation and irritable bowel syndrome. Electrophysiological studies indicated that linaclotide modulates CFTR and NHE3 ion transport. Here we investigated linaclotide‐dependent intracellular signaling pathways using human intestinal cell lines resembling crypt (T84) and villus (Caco‐2BBe) enterocytes. PCR and immunoblots were employed to examine GCC‐dependent machinery critical for CFTR and NHE3 trafficking. Linaclotide‐induced phosphorylation of VASPSer239, a substrate for cGKII, the kinase necessary for cGMP dependent trafficking CFTR and NHE3 in the intestine, was examined. Trafficking of CFTR and NHE3 into the apical membrane was examined by surface biotinylation following treatment with cGMP agonists and linaclotide in the absence or presence of H7 (PKA inhibitor) or H8 (PKGII inhibitor). Both cell lines expressed the cGMP‐dependent kinase cGKII. Protein levels of cGKII were higher in the villus resembling Caco‐2BBe cells, consistent with findings in native intestine. Apical treatment of filter‐grown polarized cells with the cGMP analog 8Br‐cGMP (0.2mM,1mM) and linaclotide (1µM, 3.27µM) resulted in phosphorylation of VASPser239, indicating activation of cGKII. Levels of surface‐biotinylated CFTR and NHE3 were consistent with the phosphorylated cGKII responses to 8Br‐cGMP and linaclotide and were reduced by both PKA and PKG inhibitors. These data indicate that linaclotide stimulates trafficking of CFTR and NHE3 into the apical membrane through cGMP dependent PKGII and cross activation of cAMP dependent PKA signaling pathways.

Cyclic GMP Kinase II (cGKII) Inhibits NHE3 by Altering Its Trafficking and Phosphorylating NHE3 at Three Required Sites: IDENTIFICATION OF A MULTIFUNCTIONAL PHOSPHORYLATION SITE

The epithelial brush-border Na(+)/H(+) exchanger NHE3 is acutely inhibited by cGKII/cGMP, but how cGKII inhibits NHE3 is unknown. This study tested the hypothesis that cGMP inhibits NHE3 by phosphorylating it and altering its membrane trafficking. Studies were carried out in PS120/NHERF2 and in Caco-2/Bbe cells overexpressing HA-NHE3 and cGKII, and in mouse ileum. NHE3 activity was measured with 2',7'-bis(carboxyethyl)-S-(and 6)carboxyfluorescein acetoxy methylester/fluorometry. Surface NHE3 was determined by cell surface biotinylation. Identification of NHE3 phosphorylation sites was by iTRAQ/LC-MS/MS with TiO2 enrichment and immunoblotting with specific anti-phospho-NHE3 antibodies. cGMP/cGKII rapidly inhibited NHE3, which was associated with reduced surface NHE3. cGMP/cGKII increased NHE3 phosphorylation at three sites (rabbit Ser(554), Ser(607), and Ser(663), equivalent to mouse Ser(552), Ser(605), and Ser(659)), all of which had to be present at the same time for cGMP to inhibit NHE3. NHE3-Ser(663) phosphorylation was not necessary for cAMP inhibition of NHE3. Dexamethasone (4 h) stimulated wild type NHE3 activity and increased surface expression but failed to stimulate NHE3 activity or increase surface expression when NHE3 was mutated to either S663A or S663D. We conclude that 1) cGMP inhibition of NHE3 is associated with phosphorylation of NHE3 at Ser(554), Ser(607), and Ser(663), all of which are necessary for cGMP/cGKII to inhibit NHE3. 2) Dexamethasone stimulates NHE3 by phosphorylation of a single site, Ser(663). The requirement for three phosphorylation sites in NHE3 for cGKII inhibition, and for phosphorylation of one of these sites for dexamethasone stimulation of NHE3, is a unique example of regulation by phosphorylation.

Competitive regulation of human intestinal β-carotene 15,15′-monooxygenase 1 (BCMO1) gene expression by hepatocyte nuclear factor (HNF)-1α and HNF-4α

AimAmong the pro-vitamin A carotenoids, β-carotene is an excellent source of vitamin A. β-Carotene 15,15′-monooxygenase 1 (BCMO1) is a critical enzyme involved in the conversion of β-carotene into vitamin A (retinal) in the small intestine of many vertebrates. In the present study, we investigated the regulation of human BCMO1 gene expression using human intestinal Caco-2 BBe cells. Main methodsWe performed electrophoretic mobility shift assays and chromatin immunoprecipitation assays to investigate the binding properties of hepatocyte nuclear factor (HNF)-1α and HNF-4α to the proximal promoter of the human BCMO1 gene. Caco-2 BBe cells were also transfected with HNF-1α and HNF-4α siRNAs, and BCMO1 gene expression levels and promoter activity were analyzed by real-time reverse transcription-polymerase chain reaction and luciferase reporter assays, respectively. Key findingsWe identified overlapping binding sites for HNF-1α and HNF-4α in the human BCMO1 gene proximal promoter. Endogenous nuclear HNF-1α and HNF-4α proteins competitively bound these sites in Caco-2 BBe cells. BCMO1 gene expression levels and promoter activity were significantly decreased in HNF-1α siRNA-transfected Caco-2 BBe cells. In contrast, HNF-4α siRNA-transfected cells exhibited a significant increase in BCMO1 gene expression and promoter activity. Mutation of these overlapping binding sites dramatically decreased BCMO1 promoter activity. SignificanceOur study indicates that the competitive actions of HNF-1α and HNF-4α on their overlapping binding sites in the human BCMO1 gene promoter oppositely regulate BCMO1 gene expression in the human small intestine.

CD137 signaling enhances tight junction resistance in intestinal epithelial cells.

Treatment of Caco‐2‐BBe intestinal epithelial cells (BBe) with TNF‐α and lymphotoxin‐β (LT‐β) receptor agonists induced the expression of the TNF receptor superfamily gene TNFRSF9/CD137. In the gut, these cytokines are known to be involved in both inflammatory responses and development of organized lymphoid tissues; thus, it was notable that in CD137‐deficient mice Peyer's patch M cells lacked transcytosis function. To examine the direct effect of CD137 expression on epithelial cell function independent of other cytokine effects including CD137L triggering, we stably transfected BBe cells to express CD137. CD137 was found at the cell surface as well as the cytoplasm, and confocal microscopy suggested that aggregates of CD137 at the lateral and basolateral surface may be associated with cytoplasmic actin filament termini. Many of the CD137 clusters were colocalized with extracellular fibronectin providing a possible alternative ligand for CD137. Interestingly, we found that CD137‐expressing cells showed significantly higher transepithelial electrical resistance (TEER) accompanied by an increase in claudin‐4 and decrease in claudin‐3 protein expression. By contrast, transfection with a truncated CD137 lacking the cytoplasmic signaling domain did not affect TEER. Finally, CD137‐deficient mice showed increased intestinal permeability upon dextran sodium sulfate (DSS) treatment as compared to control mice. Our results suggest that cytokine‐induced expression of CD137 may be important in enhancing epithelial barrier function in the presence of intestinal inflammation as well as influencing cytoskeletal organization.