1. Volume 130, Issue 4, Pages 1191-1206 (April 2006)
Bile Acids Induce Ectopic Expression of Intestinal Guanylyl Cyclase C Through Nuclear Factor-κB and Cdx2 in Human Esophageal Cells 
Philip R. Debruyne, Matthew Witek, Li Gong, Ruth Birbe, Inna Chervoneva, Tianru Jin, Claire Domon–Cell, Juan P. Palazzo, Jean–Noel Freund, Peng Li, Giovanni M. Pitari, Stephanie Schulz, Scott A. Waldman 
Gastroenterology 
Volume 130, Issue 4, Pages (April 2006)
DOI: /j.gastro
Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

2. Figure 1
GC-C expression in normal mucosae and adenocarcinoma from esophagus and stomach. GC-C mRNA expression, quantified by RT-PCR, in (A) normal mucosae, squamous cancer (SCE), and adenocarcinoma (ACE) of the esophagus and (B) normal mucosae and adenocarcinoma (ACS) of the stomach. For comparison, GC-C mRNA expression also was quantified in adenocarcinoma of the colon (Colon Ca; B). Plots represent copy numbers and medians (log scale) and results are the means of at least duplicate determinations. GC-C protein, detected by immunohistochemistry, in (C) normal colon, (D) esophagus, and (E) stomach and in (F) adenocarcinoma of the colon, (G) esophagus, and (H) stomach. Results for each tissue are representative of 5 specimens from different patients (original magnification: C, 10×; D–H, 40×). Arrowheads highlight expression of GC-C in brush border membranes of colonic epithelial cells in panel C.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

3. Figure 2
GC-C expression in human cell lines established from tumors of the upper GI tract. (A) GC-C mRNA expression in cells from colorectal cancer (T84; positive control), SCE (TE12b), ACE, and ACS quantified by RT-PCR. Results (log scale; means ± standard error of the mean [SEM]) represent at least three experiments performed in duplicate. (B) Immunoblot analysis of GC-C expression. (B1) Anti–GC-C antibody recognized multiple proteolytic products of GC-C in T84 cells,37 with a predominant fragment at 31 kDa, and recognition of all bands could be competed by the specific peptide antigen derived from GC-C to which this antibody was raised (GC-C Blocking Peptide) but not by a control peptide. (B2) Immunoblot analysis of the expression of the 31-kDa fragment of GC-C in cell lines from colorectal cancer, SCE, ACE, and ACS. (C) ST (1 μmol/L) induced cGMP accumulation (log % increase) in T84 and OE19 cells. Boxes represent median and interquartile ranges from 1 of at least 3 experiments each performed in duplicate. Whiskers indicate extreme values. Figures represent P values of comparisons between respective groups. (D) ST (1 μmol/L) and the cell-permeant analog of cGMP, 8-Br-cGMP (1 mmol/L), inhibited proliferation in T84 human colon cancer cells, but only 8-Br-cGMP inhibited proliferation of OE19 cells. Cells were exposed to ST or 8-Br-cGMP for 3 h and 3H-thymidine incorporation quantified. **P < .01, ***P < .001 compared with untreated cells.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

4. Figure 3
DCA and acid induce GC-C expression in OE19 cells. (A and B) DCA induced the expression of (A) GC-C mRNA and (B) protein in OE19 cells. In A, OE19 cells were exposed for 8 hours to the indicated concentrations of DCA, total RNA was extracted, and 1 μg subjected to quantitative RT-PCR using GC-C- or β-actin–specific primers and probes. In B, protein was extracted from treated and control cells and 25 μg subjected to immunoblot analysis using antibody against GC-C or β-actin. (A) Results (means ± SEM) obtained by quantitative RT-PCR are representative of at least 3 experiments each performed in duplicate, whereas (B) those obtained by immunoblot analysis are representative of 5 determinations. In immunoblot analyses, relative intensities were quantified by densitometry. (C) DCA (100 μmol/L for 8 hours) increased the accumulation of cGMP in OE19 cells in response to ST. Boxes represent median and interquartile ranges and whiskers indicate extreme values of at least 3 experiments performed in triplicate. Figures indicate P values of comparisons between respective groups. (D) Expression of GC-C mRNA (D1), quantified by RT-PCR (A), and protein (D2), quantified by immunoblot analysis (B), in OE19 cells was induced by acid. OE19 cells were treated for 8 hours with control (pH 7.4) or acidified (pH 6.5) medium. Similarly, (D3) acid and DCA were additive in their induction of GC-C mRNA expression. (D1) Results obtained by quantitative RT-PCR are the means (± SEM) of 14 determinations, (D2) those obtained by immunoblot analysis are representative of 5 determinations, and (D3) those quantifying GC-C mRNA expression induced by acid and DCA exemplify a single study performed in triplicate which is representative of four experiments. In D3, additivity of DCA is expressed in the context of activity in the presence of acid (100%). *P < .05; **P < .01.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

5. Figure 4
DCA and acid increase GC-C in OE19 cells by inducing Cdx2 expression. (A) DCA-induced GUCY2C promoter activity in OE19 cells. OE19 cells (2 × 105 cells) were transfected with the GUCY2C promoter-luciferase reporter plasmid (0.5 μg) together with a Renilla luciferase reporter construct (0.005 μg) for normalization. After 48 hours, cells were treated for 4 hours with DCA at the indicated concentrations. Promoter activity, quantified as the mean normalized GC-C firefly/Renilla luciferase ratios, represents 1 of at least 3 experiments performed with 7 replicates. (B) Deletion of the Cdx2-binding site nearly eliminated the ability of DCA (100 μmol/L, 4 hours) to stimulate GUCY2C promoter activity. Promoter activity, quantified as the mean (± SEM) normalized GC-C firefly/Renilla luciferase ratios, represents 1 of at least 2 experiments each performed with 6 replicates of the wild-type vector (full length) and the reporter in which the Cdx2-binding site is mutated (Cdx2MUT). Arrows represent DCA-stimulated promoter activity and figures indicate P values. (C) DCA or acid induce expression of Cdx2 mRNA, quantified by RT-PCR, in OE19 cells. OE19 cells were exposed for 8 hours to the indicated concentrations of (C1) DCA or (C2) acidified (pH 6.5) medium, total RNA was extracted, and 1 μg subjected to quantitative RT-PCR using GC-C- or β-actin–specific primers and probes. Results obtained by quantitative RT-PCR are the means (± SEM) of 5 determinations. (D) DCA and acid increase Cdx2 protein expression in the cytoplasm and nucleus of OE19 cells. Nuclear (50 μg) and cytoplasmic (25 μg) extracts of OE19 cells treated for 8 hours with the indicated concentrations of DCA at pH 7.4 or 6.5 were subjected to immunoblot analysis employing a polyclonal antibody generated to Cdx2. Relative intensities of immunostained proteins compared with 0 μmol/L DCA at pH 7.4 (control) were quantified by densitometry. Data are representative of at least 3 experiments. *P < .05; **P < .01.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

6. Figure 5
NF-κB binding sites in the promoter mediate induction of Cdx2 expression by DCA in OE19 cells. (A) Schema of the luciferase reporter constructs employed. CRE, cAMP response element; Oct-1, octamer-binding motif; TATA, putative TATA box. In these studies, OE19 cells (2 × 105 cells) were transfected with Cdx2 promoter-luciferase reporter plasmids (0.5 μg) together with a Renilla luciferase reporter construct (0.005 μg) for normalization. After 48 hours, cells were treated for 4 hours with 100 μmol/L DCA and expression of Cdx2 reporter constructs examined. (B) Mutation of the NF-κB–binding sites nearly eliminated induction of the Cdx2 promoter by DCA. The effect of DCA on full-length Cdx2 promoter activity (−908/+119; WT) was compared with that on the promoter containing mutations of the NF-κB–binding sites (NF-κBMUT). In contrast, mutation of the (C) AP-1– or (D) OCT-1–binding sites did not alter the ability of DCA to induce Cdx2 promoter activity. The role of the AP-1–binding site was examined by comparing the effects of DCA on the wild-type construct AP1WT (−195 /+119) with those on the AP1MUT construct containing a mutation in the AP-1–binding site. The role of the OCT-1–binding site was examined by comparing the effects of DCA on the wild-type construct OCTWT (−138 to +137) with those on OCTMUT containing a mutation in the Oct-1–binding site. (E) Regulatory sites upstream of Oct-1 are required for maximum basal and DCA-stimulated Cdx2 promoter activity. The role of sites upstream from OCT-1 were examined by comparing basal and DCA-stimulated activities of the full-length Cdx2 promoter (−908/+119, WT) and the truncated construct (−195 /+119, AP1WT). Results (means ± SEM) reflect the normalized firefly/Renilla luciferase ratios of the control vectors and the reporters with mutated sites from 6 replicates representative of at least 2 independent experiments. Figures indicate P values.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

7. Figure 6
DCA induced Cdx2 expression by increasing nuclear NF-κB–dependent transcriptional activity. (A) DCA increased the activity of an NF-κB–specific reporter construct in OE19 cells. OE19 cells (2 × 105 cells) were transfected with the NF-κB–specific promoter-luciferase reporter plasmids (0.5 μg) together with a Renilla luciferase reporter construct (0.005 μg) for normalization. After 48 hours, cells were treated for 4 hours with 100 μmol/L DCA and expression of the reporter constructs examined. Results represent the mean (± SEM) normalized firefly/Renilla luciferase ratios from at least 2 independent experiments with 6 replicates. (B) OE19 cells express the p50 and p65 subunits of NF-κB, but only the p50 subunit is translocated into the nucleus. Nuclear (50 μg) and cytoplasmic (50 μg) extracts from OE19 cells treated with 100 μmol/L DCA for 4 hours were subjected to immunoblot analysis using polyclonal antibodies specific for the p50 and p65 subunits of NF-κB. (C) Intensities of immunostained proteins, quantified by densitometry, were used to calculate the relative distribution of p50 in the cytoplasm and nucleus to compare that distribution in control OE19 cells and cells treated with DCA. Results in B and C are representative of 3 independent experiments. (D) OE19 cells contain functional p50 subunits of NF-κB in the nucleus. Nuclear proteins were extracted from OE19 cells and 10 μg incubated with labeled oligonucleotides corresponding to NF-κB–binding sites 1 and 2 in the Cdx2 promoter (lanes 1 and 4). The presence of NF-κB in the DNA/protein complex was assessed by supershift analysis using a polyclonal antibody specific for p50 (lanes 2 and 5). Similarly, the specificity of the p50-containing DNA/protein complex was assessed by competition analysis by using a 100-fold molar excess of unlabeled consensus NF-κB–binding oligonucleotide (lanes 3 and 6). (E) DCA increases p50-specific DNA-binding activity in the nucleus of OE19 cells. Nuclear proteins were extracted from control OE19 cells and cells treated with 100 μmol/L DCA for 4 hours and 10 μg incubated with labeled oligonucleotides corresponding to NF-κB–binding sites 1 and 2 in the Cdx2 promoter (lanes 1 and 5). Protein interaction with sites 1 and 2 each produced a single complex that was eliminated by using a polyclonal antibody specific for p50 (lanes 2 and 6). DCA treatment increased the intensity of this DNA-protein complex (lanes 3 and 7), which, again, was eliminated by using a polyclonal antibody specific for p50 (lanes 4 and 8). (F and G) As outlined in Figure 5, OE19 cells (2 × 105 cells) infected with the control vector MSCV or that vector incorporating the mutant pIκBαS32/36A were transfected with the full-length Cdx2 promoter-luciferase reporter plasmid (WT, 0.5 μg) together with a Renilla luciferase reporter construct (0.005 μg) for normalization. After 48 hours, cells were treated for 4 hours with 100 μmol/L DCA. Stable expression of pIκBαS32/36A prevented translocation of p50 from the cytoplasm (C) to the nucleus (N) induced by DCA, quantified by immunoblot analysis (F). Moreover, expression of pIκBαS32/36A prevented induction of Cdx2 promoter activity by DCA, quantified by luciferase analysis (G). All results are representative of at least 3 independent experiments. *P < .05.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions