Bile Salts at Low pH Cause Dilation of Intercellular Spaces in In Vitro Stratified Primary Esophageal Cells, Possibly by Modulating Wnt Signaling.

BACKGROUND: The presence of dilated intercellular spaces in the stratified squamous lining of the esophagus is the pathognomonic feature of reflux esophagitis secondary to gastroesophageal reflux disease (GERD). In addition to stomach acid, bile salts are major constituents of gastroesophageal refluxate. The aim of our study was to determine the effect of bile salts cocktail at different pHs on epithelial junctions in an in vitro transwell model of stratified esophageal squamous epithelium. DISCUSSION: Human telomerase reverse transcriptase (hTERT) immortalized primary esophageal EPC1 cells were grown on polyester transwell surfaces in calcium-enriched media. The cells exhibited gradual stratification into an 11-layered squamous epithelium over 7 days, together with epithelial barrier function as indicated by increased transepithelial electrical resistance (TEER). This stratified epithelium demonstrated well-formed tight junctions, adherens junctions, and desmosomes as visualized by immunofluorescence and electron microscopy. When exposed to short pulses of bile salts at pH 5, but not either condition alone, there was loss of stratification and decrease in TEER, concomitant with disruption of adherens junctions, tight junctions, and desmosomes, leading to the appearance of dilated intercellular spaces. At the cellular level, bile salts at pH 5 activated the Wnt pathway (indicated by increased ß-catenin Ser552 phosphorylation). CONCLUSION: In conclusion, in our in vitro transwell model bile salts at pH 5, but not bile salts or media at pH 5 alone, modulate Wnt signaling, disrupt different junctional complexes, and cause increased permeability of stratified squamous esophageal epithelium. These changes approximate the appearance of dilated intercellular space similar to that found in GERD patients.

Bile acid at low pH reduces squamous differentiation and activates EGFR signaling in esophageal squamous cells in 3-D culture.

BACKGROUND: Barrett's esophagus is a preneoplastic metaplasia in which the normal squamous epithelium of the esophagus changes to an intestinal, columnar phenotype due to long-term gastro-esophageal reflux. The major components of this reflux are bile and stomach acid. Previous in vitro studies on the effect of bile and acid on esophageal cells have predominantly relied on transformed esophageal squamous cells or cancer cells grown in monolayer culture. DISCUSSION: In this study, we expanded our previous work using an immortalized primary esophageal squamous cell line (EPC1). We demonstrate that EPC1 cells form a multi-layer, stratified epithelium when grown on polyester transwell filters in media supplemented with calcium. When exposed to short pulses of bile and pH 5, but not either condition alone, EPC1 cells demonstrate a reduction in stratification layers and reduced expression of squamous epithelium-specific genes. Bile at pH 5 also causes activation of epidermal growth factor receptor and down-stream pathways. Blocking epidermal growth factor receptor activation partially attenuates the effects of bile acid and pH 5. These results suggest that bile at low pH, but not bile or low pH alone, promotes loss of differentiation status of stratified squamous esophageal epithelium in vitro, possibly by initiating a mucosal repair response through epidermal growth factor activation.

Bile exposure inhibits expression of squamous differentiation genes in human esophageal epithelial cells.

OBJECTIVE: This study aimed to identify pathways and cellular processes that are modulated by exposure of normal esophageal cells to bile and acid. BACKGROUND: Barrett's esophagus most likely develops as a response of esophageal stem cells to the abnormal reflux environment. Although insights into the underlying molecular mechanisms are slowly emerging, much of the metaplastic process remains unknown. METHODS: We performed a global analysis of gene expression in normal squamous esophageal cells in response to bile or acid exposure. Differentially expressed genes were classified into major biological functions using pathway analysis and interaction network software. Array data were verified by quantitative PCR and western blot both in vitro and in human esophageal biopsies. RESULTS: Bile modulated expression of 202 genes, and acid modulated expression of 103 genes. Genes involved in squamous differentiation formed the largest functional group (n = 45) all of which were downregulated by bile exposure. This included genes such as involucrin (IVL), keratinocyte differentiation-associated protein (KRTDAP), grainyhead-like 1 (GRHL1), and desmoglein1 (DSG1) the downregulation of which was confirmed by quantitative PCR and western blot. Bile also caused expression changes in genes involved in cell adhesion, DNA repair, oxidative stress, cell cycle, Wnt signaling, and lipid metabolism. Analysis of human esophageal biopsies demonstrated greatly reduced expression of IVL, KRTDAP, DSG1, and GRHL1 in metaplastic compared to squamous epithelia. CONCLUSIONS: We report for the first time that bile inhibits the squamous differentiation program of esophageal epithelial cells. This, coordinated with induction of genes driving intestinal differentiation, may be required for the development of Barrett's esophagus.

Early G1 cyclin-dependent kinases as prognostic markers and potential therapeutic targets in esophageal adenocarcinoma.

PURPOSE: Chromosomal gain at 7q21 is a frequent event in esophageal adenocarcinoma (EAC). However, this event has not been mapped with fine resolution in a large EAC cohort, and its association with clinical endpoints and functional relevance are unclear. EXPERIMENTAL DESIGN: We used a cohort of 116 patients to fine map the 7q21 amplification using SNP microarrays. Prognostic significance and functional role of 7q21 amplification and its gene expression were explored. RESULTS: Amplification of the 7q21 region was observed in 35% of tumors with a focal, minimal amplicon containing six genes. 7q21 amplification was associated with poor survival and analysis of gene expression identified cyclin-dependent kinase 6 (CDK6) as the only gene in the minimal amplicon whose expression was also associated with poor survival. A low-level amplification (10%) was observed at the 12q13 region containing the CDK6 homologue cyclin-dependent kinase 4 (CDK4). Both amplification and expression of CDK4 correlated with poor survival. A combined model of both CDK6 and CDK4 expressions is a superior predictor of survival than either alone. Specific knockdown of CDK4 and/or CDK6 by siRNAs shows that they are required for proliferation of EAC cells and that their function is additive. PD-0332991 targets the kinase activity of both molecules and suppresses proliferation and anchorage independence of EAC cells through activation of the pRB pathway. CONCLUSIONS: We suggest that CDK6 is the driver of 7q21 amplification and that both CDK4 and CDK6 are prognostic markers and bona fide oncogenes in EAC. Targeting these molecules may constitute a viable new therapy for this disease.

Pathogenesis of Barrett's esophagus: bile acids inhibit the Notch signaling pathway with induction of CDX2 gene expression in human esophageal cells.

BACKGROUND: Barrett's esophagus (BE) is the predominant risk factor for the development of esophageal adenocarcinoma. BE is characterized by intestinal metaplasia with goblet cells. Reflux of bile acids is known to induce intestinal metaplasia, but the mechanisms are unclear. Inhibition of Notch signaling accompanied by increased Hath1 and induction of caudal homeobox 2 (CDX2) may be involved in development of intestinal goblet cells. METHODS: Esophageal adenocarcinoma cell lines OE19 and OE33 were exposed for up to 8 hours to DCA (100-300 microM), and for up to 24 hours with and without the gamma-secretase inhibitor, DAPT (20 microM). Notch signaling components and CDX2 levels were measured by real-time PCR (for mRNA) and by Western blot analysis (for proteins). RESULTS: DCA induced a time and concentration dependent decrease in Notch pathway components mRNAs in OE33 and in the proteins in both cell lines. CDX2 mRNA and Hath1 protein were increased in OE19 by 3-fold. Inhibition of Notch pathway by DAPT decreased downstream Notch signaling mRNAs and proteins in both cell lines and increased Hath1 and CDX2 proteins only in OE19. CONCLUSION: Bile acid inhibition of Notch signaling in esophageal cells is correlated with an increase in Hath1 and CDX2 and may be one of the key processes contributing to the formation of BE.

Bile acid alone, or in combination with acid, induces CDX2 expression through activation of the epidermal growth factor receptor (EGFR).

OBJECTIVES: Bile acids and acid are implicated in the development of Barrett's esophagus. Evidence suggests that Barrett's esophagus intestinal metaplasia may occur via induction of caudal homeobox gene 2 (CDX2). We hypothesized that induction of CDX2 by bile acids may be due to ligand-dependent transactivation of epidermal growth factor receptor (EGFR). METHODS: Human mucosal epithelial cells (SEG-1) were treated for 0 to 24 h with up to 300 microM deoxycholic acid (DCA) at pH 7 or 5 with or without (w/wo) antibodies against EGFR ligand-binding site (Mab528, 3-5 mug/ml). Treatment with 100 ng/ml EGF served as control. CDX2 mRNA expression was determined by real-time polymerase chain reaction. EGFR activation was analyzed by Westerns of phosphorylated EGFR tyrosines. RESULTS: Acid (pH 5) increased the induction of CDX2 mRNA expression caused by DCA. CDX2 mRNA induction was markedly reduced by EGFR blockade with Mab528. Each treatment (pH 5, DCA or pH 5 plus DCA) activated the EGFR on all tyrosines tested but in different time courses. Phosphorylation by DCA was inhibited by Mab528. Activation of EGFR by DCA at pH 5 resulted in EGFR degradation, while that by DCA alone did not. CONCLUSION: Thus, CDX2 induction by DCA w/wo acid occurs through ligand-dependent transactivation of the EGFR. The variations in EGFR degradation pattern with DCA or DCA at pH 5 indicate differential transactivation pathways. The molecular pathogenesis of Barrett's esophagus may occur via bile-stimulated cell signaling through the EGFR.

In human entrocytes, GLN transport and ASCT2 surface expression induced by short-term EGF are MAPK, PI3K, and Rho-dependent.

Glutamine, a key nutrient for the enterocyte, is transported among other proteins by ASCT2. Epidermal growth factor (EGF) augments intestinal adaptation. We hypothesized that short-term treatment of human enterocytes with EGF enhances glutamine transport by increasing membranal ASCT2. To elucidate EGF-induced mechanisms, monolayers of C2(BBe)1 w/wo siRho transfection were treated w/wo EGF and w/wo tyrphostin AG1478 (AG1478), wortmanin, or PD98059. Total and system-specific (3)H-glutamine transports were determined w/wo 5 mmol/l amino acid inhibitors. Total and membranal ASCT2 proteins were measured by Westerns. EGF doubled glutamine transport by increasing B(0)/ASCT2 and B(0,+) activities. Despite the doubling of membranal ASCT2 protein with EGF treatment, total ASCT2 did not change. The increases in B(0)/ASCT2 activity and ASCT2 protein were eliminated by AG1478, PD98059, wortmanin, and siRho, while transport by B(0,+) was inhibited only by PD98059 and siRho. Thus, differential pathways are involved in EGF-induced increase in B(0)/ASCT2 glutamine transport and membranal ASCT2 compared to those involved in B(0,+) activity.

Epidermal growth factor and/or growth hormone induce differential, side-specific signal transduction protein phosphorylation in enterocytes.

BACKGROUND: Epidermal growth factor (EGF) plus growth hormone (GH) enhances luminal glutamine transport into rabbit and human intestinal cells. Our objective was to screen for activation status of signal proteins in C2(BBe)1 cells (enterocyte-like cell line) in response to side-specific EGF or GH treatment and to investigate the dependence of EGF receptor (EGFR) phosphorylation status on its tyrosine kinase. METHODS: C2(BBe)1 cells on Transwells were treated for 15 minutes on either the basolateral or apical-side with EGF or GH. Lysates underwent Kinetworks phospho site-screen-2.1 analysis (duplicate experiments). In addition, lysates from cells treated as above with or without tyrphostin AG1478 (a specific EGFR tyrosine kinase inhibitor) underwent Western blot analysis for total EGFR and EGFR phosphorylated on tyrosine 1173, 1086 or 1068 (4-7 experiments). RESULTS: Kinetworks phospho-screening demonstrated a broad range of interactions dependent on both side of exposure and protein studied. From this screen, it appears that ErbB2, Met, and insulin receptor (R)/insulin-like growth factor 1 R are not involved in the growth factors signals. For EGFR phosphorylation, basolateral, but not apical, EGF was a strong activator. Synergism was seen, but only with apical EGF plus basolateral GH. All EGFR phosphorylations were EGFR tyrosine kinase dependent. In contradistinction, apical EGF phosphorylated FAK and MAPKs. CONCLUSIONS: Kinetworks phosphoprotein screens can suggest pathways involved in side-specific and synergistic interaction between EGF and GH. For EGFR, synergism by EGF + GH was noticed only with Ap EGF plus Bl GH and was EGFR tyrosine kinase dependent. Adaptive intestinal responses due to enterally administrated EGF might be accelerated by the availability of parenteral GH.

ATB0/ASCT2 expression in residual rabbit bowel is decreased after massive enterectomy and is restored by growth hormone treatment.

Two weeks after 70% enterectomy, glutamine (Gln) transport is downregulated in rabbit residual bowel due to a decrease in system B(0) activity. Providing epidermal growth factor (EGF) and growth hormone (GH) restores Gln transport by increasing systems A and B(0,+) activities. We hypothesized that changes in Na(+)-dependent broad-spectrum neutral amino acid transporter (ATB(0)/ASCT2) protein and mRNA expression correlate with system B(0) activity. New Zealand White rabbits underwent 70% jejunoileal resection or no resection. Resected rabbits immediately received parenteral EGF, GH, both, or neither agent for 2 wk. Tissues harvested from jejunum, ileum, and colon were subjected to Western and Northern blot analyses for ATB(0)/ASCT2 protein and mRNA. In all tissues, ATB(0)/ASCT2 mRNA was reduced by approximately 50% in resected rabbits compared with nonresected controls. Similar reductions in protein amount occurred in the ileum and cecum. None of the growth factor treatments restored ATB(0)/ASCT2 protein, but GH treatment increased ATB(0)/ASCT2 mRNA abundance 250% in the residual ileum. Because changes in the ATB(0)/ASCT2 protein amount paralleled those in the system B(0) activity in this model, it is likely that this is the protein responsible for this transport system. The increase in mRNA abundance in rabbits treated with GH for 2 wk may be a harbinger of subsequent increases in transporter protein and activity. Unlike reported upregulation of transporters in human colon after small bowel resection, ATB(0)/ASCT2 protein and mRNA expression in rabbit colon are decreased, suggesting different regulatory pathways.

Macroarray analysis reveals a strain-induced oxidant response in pulmonary epithelial cells.

Mechanical strain initiates a variety of responses in pulmonary epithelial cells. The signaling pathways and molecular alterations leading to these responses remain unclear To identify novel signal transduction pathways activated by strain, macroarray analysis was performed on strained pulmonary epithelial cells. Glutathione S-transferase (GST) pi, GST mu, and heat shock protein (HSP)-27 were increased by strain. Western blotting verified that increases in cDNA of these redox-related molecules resulted in an increase in protein. Phosphorylation of HSP-27 was increased after strain, further supporting the role of HSP-27 in strain-induced signal transduction. Strain-induced oxidative stress was verified with the oxidant-sensitive dye dichlorodihydrofluorescein diacetate.

Growth hormone and epidermal growth factor together enhance amino acid transport systems B0,+ and A in remnant small intestine after massive enterectomy.

BACKGROUND: Sodium-dependent brush-border nutrient transport is decreased 2 weeks after massive enterectomy. This down-regulation is ameliorated by a 1-week infusion of parenteral growth hormone (GH) and epidermal growth factor (EGF) started 1 week after resection. We hypothesize that glutamine (GLN) transport will be enhanced by earlier and longer growth factor infusion, with differential effects on the Na(+)-dependent GLN transport systems A, B(0,+), and B(0)/ASCT2. MATERIALS AND METHODS: New Zealand White rabbits underwent 70% small bowel resection then immediately received parenteral EGF, GH, both EGF and GH, or neither for 2 weeks. Na(+)-dependent 3H-GLN uptake by jejunal and ileal brush-border membrane vesicles was measured and the contribution of systems A, B(0,+), and B(0) was then determined by competitive inhibition. Data were analyzed using one-way analysis of variance. RESULTS: In nonresected animals, the relative contribution of the systems was similar in jejunum (A 9%, B(0,+) 20%, and B(0) 71%) and ileum (A 13%, B(0,+) 27%, and B(0) 60%). Na(+)-dependent GLN uptake was reduced by one half in resected untreated controls, primarily because of decreased B(0) activity. EGF or GH alone did not affect Na(+)-dependent GLN transport, but, as a combination, there was increased uptake in the residual ileum and jejunum by 144% and 150%, respectively, over resected controls (P < 0.05). This was twice that achieved by delayed and shorter-duration combination treatment. This augmentation was a result of a 6.1-8.2-fold increase in system A as well as a 3.8-3.9-fold enhancement of system B(0,+) activity in remnant ileum and jejunum (P < 0.01). CONCLUSIONS: Parenteral EGF and GH, given in combination for 2 weeks immediately after massive enterectomy, synergistically enhance GLN uptake by systems A and B(0,+).

Growth hormone and epidermal growth factor together enhance amino acid transport systems B(0,+) and A in remnant small intestine after massive enterectomy.

BACKGROUND: Sodium-dependent brush border nutrient transport is decreased 2 weeks after massive enterectomy. This downregulation is ameliorated by a 1-week infusion of parenteral growth hormone (GH) and epidermal growth factor (EGF) started 1 week after resection. We hypothesized that glutamine (GLN) transport would be enhanced by earlier and longer growth factor infusion, with differential effects on the Na(+)-dependent GLN transport systems A, B(0,+), and B0/ASCT2. MATERIALS AND METHODS: New Zealand White rabbits underwent 70% small bowel resection then immediately received parenteral EGF, GH, both, or neither for 2 weeks. Na(+)-dependent 3H-GLN uptake by jejunal and ileal brush-border membrane vesicles was measured and the contribution of systems A, B(0,+), and B0 then determined by competitive inhibition. Data were analyzed using one-way analysis of variance. RESULTS: In nonresected animals, the relative contribution of the systems was similar in jejunum (A, 9%, B(0,+), 20%; and B0, 71%) and ileum (A, 13%; B(0,+), 27%; and B0, 60%). Na(+)-dependent GLN uptake was reduced by half in resected, untreated controls, primarily because of decreased B(0) activity. EGF or GH alone did not affect Na(+)-dependent GLN transport, but as a combination, increased uptake in the residual ileum and jejunum by 144% and 150%, respectively, over resected controls (P<0.05). This was twice that achieved by delayed and shorter-duration combination treatment. This augmentation was due to a 6.1- to 8.2-fold increase in system A as well as a 3.8- to 3.9-fold enhancement of system B(0,+) activity in remnant ileum and jejunum (P<0.01). CONCLUSIONS: Parenteral EGF and GH, given in combination for 2 weeks immediately after massive enterectomy, synergistically enhance GLN uptake by systems A and B(0,+).