Debra G. Silberg

Expression of intestine-specific transcription factors, CDX1 and CDX2, in intestinal metaplasia and gastric carcinomas.

Intestinal metaplasia (IM) is part of a stepwise sequence of alterations of the gastric mucosa, leading ultimately to gastric cancer, and is strongly associated with chronic Helicobacter pylori infection. The molecular mechanisms underlying the onset of IM remain elusive. The aim of this study was to assess the putative involvement of two intestine‐specific transcription factors, CDX1 and CDX2, in the pathogenesis of gastric IM and gastric carcinoma. Eighteen foci of IM and 46 cases of gastric carcinoma were evaluated by immunohistochemistry for CDX1 and CDX2 expression. CDX1 was expressed in all foci of IM and in 41% of gastric carcinomas; CDX2 was expressed in 17/18 foci of IM and in 54% of gastric carcinomas. In gastric carcinomas, a strong association was observed between the expression of CDX1 and CDX2, as well as between the intestinal mucin MUC2 and CDX1 and CDX2. No association was observed between the expression of CDX1 and CDX2 and the histological type of gastric carcinoma. In conclusion, these results show that aberrant expression of CDX1 and CDX2 is consistently observed in IM and in a subset of gastric carcinomas. The association of CDX1 and CDX2 with expression of the intestinal mucin MUC2, both in IM and in gastric carcinoma, indirectly implies that CDX1 and CDX2 may be involved in intestinal differentiation along the gastric carcinogenesis pathway. Copyright

Downregulated in adenoma gene encodes a chloride transporter defective in congenital chloride diarrhea

Congenital chloride diarrhea (CLD) is a recessively inherited disorder characterized by massive loss of chloride in stool. We previously identified mutations in the downregulated in adenoma (DRA) gene in patients with CLD and demonstrated that DRA encodes an intestine-specific sulfate transporter. To determine whether DRA is an intestinal chloride transporter and how mutations affect transport, Xenopus oocytes were injected with wild-type and mutagenized DRA cRNA and uptake of Cl- and SO2-4 was assayed. Both Cl- and SO2-4 were transported by wild-type DRA and an outwardly directed pH gradient stimulated Cl- uptake, consistent with Cl-/OH- exchange. Among three mutants, C307W transported both anions as effectively as wild-type, whereas transport activity was lost in V317del and the double mutant identified in 32 of 32 Finnish CLD patients. We conclude that DRA is a chloride transporter defective in CLD and that V317del is a functional mutation and C307W a silent polymorphism.Congenital chloride diarrhea (CLD) is a recessively inherited disorder characterized by massive loss of chloride in stool. We previously identified mutations in the downregulated in adenoma ( DRA) gene in patients with CLD and demonstrated that DRA encodes an intestine-specific sulfate transporter. To determine whether DRA is an intestinal chloride transporter and how mutations affect transport, Xenopus oocytes were injected with wild-type and mutagenized DRA cRNA and uptake of Cl- and[Formula: see text] was assayed. Both Cl- and[Formula: see text] were transported by wild-type DRA and an outwardly directed pH gradient stimulated Cl- uptake, consistent with Cl-/OH-exchange. Among three mutants, C307W transported both anions as effectively as wild-type, whereas transport activity was lost in V317del and the double mutant identified in 32 of 32 Finnish CLD patients. We conclude that DRA is a chloride transporter defective in CLD and that V317del is a functional mutation and C307W a silent polymorphism.

Human MUC2 Mucin Gene Is Transcriptionally Regulated by Cdx Homeodomain Proteins in Gastrointestinal Carcinoma Cell Lines

In intestinal metaplasia and 30% of gastric carcinomas, MUC2 intestinal mucin and the intestine-specific transcription factors Cdx-1 and Cdx-2 are aberrantly expressed. The involvement of Cdx-1 and Cdx-2 in the intestinal development and their role in transcription of several intestinal genes support the hypothesis that Cdx-1 and/or Cdx-2 play important roles in the aberrant intestinal differentiation program of intestinal metaplasia and gastric carcinoma. To clarify the mechanisms of transcriptional regulation of the MUC2 mucin gene in gastric cells, pGL3 deletion constructs covering 2.6 kb of the human MUC2 promoter were used in transient transfection assays, enabling us to identify a relevant region for MUC2 transcription in all gastric cell lines. To evaluate the role of Cdx-1 and Cdx-2 in MUC2 transcription we performed co-transfection experiments with expression vectors encoding Cdx-1 and Cdx-2. In two of the four gastric carcinoma cell lines and in all colon carcinoma cell lines we observed transactivation of the MUC2 promoter by Cdx-2. Using gel shift assays we identified two Cdx-2 binding sites at –177/–171 and –191/–187. Only simultaneous mutation of the two sites resulted in inhibition of Cdx-2-mediated transactivation of MUC2 promoter, implying that both Cdx-2 sites are active. Finally, stable expression of Cdx-2 in a gastric cell line initially not expressing Cdx-2, led to induction of MUC2 expression. In conclusion, this work demonstrates that Cdx-2 activates the expression of MUC2 mucin gene in gastric cells, inducing an intestinal transdifferentiation phenotype that parallels what is observed both in intestinal metaplasia and some gastric carcinomas.

Intestinal inflammation reduces expression of DRA, a transporter responsible for congenital chloride diarrhea

The pathogenesis of diarrhea in intestinal inflammatory states is a multifactorial process involving the effects of inflammatory mediators on epithelial transport function. The effect of colonic inflammation on the gene expression of DRA (downregulated in adenoma), a chloride-sulfate anion transporter that is mutated in patients with congenital chloridorrhea, was examined in vivo as well as in an intestinal epithelial cell line. DRA mRNA expression was diminished five- to sevenfold in the HLA-B27/β2m transgenic rat compared with control. In situ hybridization showed that DRA, which is normally expressed in the upper crypt and surface epithelium of the colon, was dramatically reduced in the surface epithelium of the HLA-B27/β2m transgenic rat, the interleukin-10 (IL-10) knockout mouse with spontaneous colitis, and in patients with ulcerative colitis. Immunohistochemistry demonstrated that mRNA expression of DRA reflected that of protein expression in vivo. IL-1β reduced DRA mRNA expression in vitro by inhibiting gene transcription. The loss of transport function in the surface epithelium of the colon by attenuation of transporter gene expression, perhaps inhibited at the level of gene transcription by proinflammatory cytokines, may play a role in the pathogenesis of diarrhea in colitis.

Cdx1 and c-Myc Foster the Initiation of Transdifferentiation of the Normal Esophageal Squamous Epithelium toward Barrett's Esophagus

Background Barretts esophagus is a premalignant condition whereby the normal stratified squamous esophageal epithelium undergoes a transdifferentiation program resulting in a simple columnar epithelium reminiscent of the small intestine. These changes are typically associated with the stratified squamous epithelium chronically exposed to acid and bile salts as a result of gastroesophageal reflux disease (GERD). Despite this well-defined epidemiologic association between acid reflux and Barretts esophagus, the genetic changes that induce this transdifferentiation process in esophageal keratinocytes have remained undefined. Methodology/Principal Findings To begin to identify the genetic changes responsible for transdifferentiaiton in Barretts esophagus, we performed a microarray analysis of normal esophageal, Barretts esophagus and small intestinal biopsy specimens to identify candidate signaling pathways and transcription factors that may be involved. Through this screen we identified the Cdx1 homeodomain transcription factor and the c-myc pathway as possible candidates. Cdx1 and c-myc were then tested for their ability to induce transdifferentiation in immortalized human esophageal keratinocytes using organotypic culturing methods. Analyses of these cultures reveal that c-myc and cdx1 cooperate to induce mucin production and changes in keratin expression that are observed in the epithelium of Barretts esophagus. Conclusions/Significance These data demonstrate the ability of Cdx1 and c-myc to initiate the earliest stages of transdifferentiation of esophageal keratinocytes toward a cell fate characteristic of Barretts esophagus.

The Caudal-related Homeodomain Protein Cdx1 Inhibits Proliferation of Intestinal Epithelial Cells by Down-regulation of D-type Cyclins

Cdx1 is a homeodomain transcription factor that regulates intestine-specific gene expression. Experimental evidence suggests that Cdx1 may be involved in cell cycle regulation, but its role is ill defined and the mechanisms have not been explored. We used stable transfection of inducible constructs and transient expression with a replication-deficient adenovirus to induce Cdx1 expression in rat IEC6 cells, a non-transformed intestinal epithelial cell line that does not express Cdx1 protein. Expression of Cdx1 markedly reduced proliferation of IEC6 cells with accumulation of cells in the G0/G1 phase of the cell cycle. Cell cycle arrest was accompanied by an increase in the hypophosphorylated forms of the retinoblastoma protein (pRb) and the pRb-related p130 protein. Protein levels of multiple cyclin-dependent kinase inhibitors were either unchanged (p16, p18, p21, p27, and p57) or were not detected (p15 and p19). Most significantly, levels of cyclins D1 and D2 were markedly diminished with Cdx1 expression, but not cyclins D3, E, or the G1 kinases. Additionally, cyclin-dependent kinase-4 activity was decreased in association with decreased cyclin D protein. We conclude that Cdx1 regulates intestinal epithelial cell proliferation by inhibiting progression through G0/G1, most likely via modulation of cyclin D1 and D2 protein levels.

Expression of an intestine-specific transcription factor (CDX1) in intestinal metaplasia and in subsequently developed intestinal type of cholangiocarcinoma in rat liver.

CDX1 is a caudal-type homeobox intestine-specific transcription factor that has been shown to be selectively expressed in epithelial cells in intestinal metaplasia of the human stomach and esophagus and variably expressed in human gastric and esophageal adenocarcinomas (Silberg DG, Furth EE, Taylor JK, Schuck T, Chiou T, Traber PG: Gastroenterology 1997, 113: 478-486). Through the use of immunohistochemistry and Western blotting, we investigated whether CDX1 is also uniquely associated with the intestinal metaplasia associated with putative precancerous cholangiofibrosis induced in rat liver during furan cholangiocarcinogenesis, as well as expressed in neoplastic glands in a subsequently developed intestinal type of cholangiocarcinoma. In normal, control adult rat small intestine, specific nuclear immunoreactivity for CDX1 was most prominent in enterocytes lining the crypts. In comparison, epithelium from intestinal metaplastic glands within furan-induced hepatic cholangiofibrosis and neoplastic epithelium from later developed primary intestinal-type cholangiocarcinoma each demonstrated strong nuclear immunoreactivity for CDX1. CDX1-positive cells were detected in hepatic cholangiofibrotic tissue as early as 3 weeks after the start of chronic furan treatment. We further determined that the percentages of CDX1-positive neoplastic glands and glandular nuclei are significantly higher in primary tumors than in a derived, transplantable cholangiocarcinoma serially-propagated in vivo. Western blotting confirmed our immunohistochemical results, and no CDX1 immunoreactivity was detected in normal adult rat liver or in hyperplastic biliary epithelial cells. These findings indicate that CDX1 is specifically associated with early intestinal metaplasia and a later developed intestinal-type of cholangiocarcinoma induced in the liver of furan-treated rats.

Induction of intestinalization in human esophageal keratinocytes is a multistep process

Barretts esophagus (BE) is the replacement of normal squamous esophageal mucosa with an intestinalized columnar epithelium. The molecular mechanisms underlying its development are not understood. Cdx2 is an intestine-specific transcription factor that is ectopically expressed in BE, but its role in this process is unclear. Herein, we describe a novel cell culture model for BE. Retroviral-mediated Cdx2 expression in immortalized human esophageal keratinocytes [EPC-human telomerase reverse transcriptase (hTERT)] could transiently be established but not maintained and was associated with a reduction in cell proliferation. Coexpression of cyclin D1, but not a dominant-negative p53, rescued proliferation in the Cdx2-expressing cells. Cdx2 expression in the EPC-hTERT.D1 cells decreased cell proliferation but did not induce intestinalization. We investigated for other treatments to enhance intestinalization and found that acidic culture conditions uniformly killed EPC-hTERT.D1.Cdx2 cells. However, treatment with 5-aza-2-deoxycytidine (5-AzaC) to demethylate epigenetically silenced genes did appear to be tolerated. Multiple Cdx2 target genes, markers of intestinal differentiation and markers of BE, were induced by this 5-AzaC treatment. More interestingly, the expression level of several of these genes was enhanced only in the EPC-hTERT.D1-Cdx2 cells treated with 5-AzaC. Two of these, SLC26a3/DRA (downregulated in adenoma) and Na+/H+ exchanger 2 (NHE2), were not previously known to be elevated in BE; however, we confirmed their elevation in BE tissue samples. 5-AzaC treatment also induced cell senescence, even at low doses. We conclude that ectopic proliferation signals, alterations in epigenetic gene regulation and the inhibition of tumor suppressor mechanisms are required for Cdx2-mediated intestinalization of human esophageal keratinocytes in BE.

A novel colonic repressor element regulates intestinal gene expression by interacting with Cux/CDP.

ABSTRACT Intestinal gene regulation involves mechanisms that direct temporal expression along the vertical and horizontal axes of the alimentary tract. Sucrase-isomaltase (SI), the product of an enterocyte-specific gene, exhibits a complex pattern of expression. Generation of transgenic mice with a mutated SI transgene showed involvement of an overlapping CDP (CCAAT displacement protein)-GATA element in colonic repression of SI throughout postnatal intestinal development. We define this element as CRESIP (colon-repressive element of the SI promoter). Cux/CDP interacts with SI and represses SI promoter activity in a CRESIP-dependent manner. Cux/CDP homozygous mutant mice displayed increased expression of SI mRNA during early postnatal development. Our results demonstrate that an intestinal gene can be repressed in the distal gut and identify Cux/CDP as a regulator of this repression during development.

Obesity does not affect treatment outcomes with proton pump inhibitors.

Background: Obesity is associated with increased risk of gastroesophageal reflux disease (GERD). Goal: To evaluate the effect of obesity on symptom resolution in patients with nonerosive reflux disease (NERD) and healing rates in patients with erosive esophagitis (EE). Methods: Two post hoc analyses were performed. Analyses included pooled data from randomized, double-blind, multicenter studies of proton pump inhibitors (PPIs) in GERD patients. Results: Analysis 1 included 704 patients with NERD receiving esomeprazole 20 mg, esomeprazole 40 mg, or placebo. Analysis 2 included 11,027 patients with EE receiving esomeprazole 40 mg, omeprazole 20 mg, or lansoprazole 30 mg. For NERD patients, no significant association between baseline heartburn severity and body mass index (BMI) was observed. In EE patients, overweight (BMI 25 to <35 kg/m2) and obese (BMI ≥35 kg/m2) patients had significantly higher rates of Los Angeles (LA) grade C or D EE than patients with BMI <25 kg/m2 (P<0.0001). Percentages of PPI-treated patients who achieved heartburn resolution or EE healing within a given LA grade were similar across BMI categories. Heartburn resolution was significantly associated with treatment (esomeprazole vs. placebo), increasing age, and for men versus women (all P⩽0.0284). EE healing was significantly associated with PPI treatment (esomeprazole and lansoprazole vs. omeprazole), increasing age, race, presence of a hiatal hernia, and lower LA grade at baseline (all P⩽0.0183). Conclusions: In patients with GERD, high BMI was associated with more severe EE at baseline. However, during PPI treatment, BMI is not a significant independent predictor of heartburn resolution or EE healing.