CDX2 mRNA Research Articles

Sox2 expression is maintained while gastric phenotype is completely lost in Cdx2-induced intestinal metaplastic mucosa

Sox2 is closely related to the gastric phenotype. Sox2 plays a pivotal role in gastric epithelial differentiation in the adult. Sox2 expression is reduced in Helicobacter pylori-associated intestinal metaplastic change of the gastric epithelium. The gastric mucosa is replaced by intestinal metaplastic mucosa in the stomach of caudal type homeobox 2 (Cdx2)-transgenic mice. The aim of this study was to use Cdx2-transgenic mice to investigate: (i) Sox2 expression in the intestinal metaplastic mucosa of the Cdx2-transgenic mouse stomach; and (ii) the relationship between Sox2 and Cdx2. Quantitative real-time PCR was performed to determine Sox2, Cdx2, Muc5Ac, and alkaline phosphatase mRNA expression levels and single- or double-label immunohistochemistry was used to evaluate the localization of Sox2, Cdx2, gastric mucin and alkaline phosphatase activity. We determined that Sox2 mRNA in the intestinal metaplastic mucosa of the Cdx2-transgenic mouse stomach was expressed 3.5-fold compared to the normal mouse stomach. Immunohistochemical analysis showed that the same cells in the intestinal metaplastic mucosa expressed both Cdx2 and Sox2. Gastric mucin was not expressed while alkaline phosphatase activity was recognized in the intestinal metaplastic mucosa in spite of the Sox2 expression. Cdx2 increased the transcriptional activity of the Sox2 gene, and Sox2 increased the transcriptional activity of the Muc5Ac gene, which was reduced by cotransfecion of Cdx2 together with Sox2 in the human gastric carcinoma cell line AGS. In conclusion, Sox2 expression is maintained while gastric phenotype is completely lost in the intestinal metaplastic mucosa of Cdx2-transgenic mouse stomach.

Hydrogen peroxide stimulates nuclear import of the POU homeodomain protein Oct-1 and its repressive effect on the expression of Cdx-2

BackgroundThe ubiquitously expressed POU homeodomain protein Oct-1 serves as a sensor for stress induced by irradiation. We found recently that in pancreatic and intestinal endocrine cells, Oct-1 also functions as a sensor for cyclic AMP (cAMP). The caudal homeobox gene Cdx-2 is a transactivator of proglucagon (gcg) and pro-insulin genes. Oct-1 binds to Cdx-2 promoter and represses its expression. cAMP elevation leads to increased nuclear exclusion of Oct-1, associated with reduced recruitment of nuclear co-repressors to the Cdx-2 promoter and increased Cdx-2 expression.ResultsWe show in this study that inducing oxidative stress by hydrogen peroxide (H2O2) increased nuclear Oct-1 content in both pancreatic α and β cell lines, as well as in a battery of other cells. This increase was then attributed to accelerated nuclear import of Oct-1, assessed by Fluorescence Recovery After Photobleaching (FRAP) using green fluorescence protein (EGFP) tagged Oct-1 molecule. H2O2 treatment was then shown to stimulate the activities of DNA-dependent protein kinase (DNA-PK) and c-jun N-terminal kinase (JNK). Finally, increased Oct-1 nuclear content upon H2O2 treatment in a pancreatic α cell line was associated with reduced Cdx-2 and gcg mRNA expression.ConclusionThese observations suggest that Oct-1 functions as a sensor for both metabolic and stress/survival signaling pathways via altering its nuclear-cytoplasmic shuttling.

Significance of IL-1beta-induced ectopic expression of CDX2 in the intestinal metaplasia of gastric epithelium

To evaluate the effect of IL-1beta on the expression of CDX2 in human gastric epithelial cell line GES-1 and its role in the intestinal metaplasia. GES-1 cells were treated with IL-1beta in different concentrations and the expressions of CDX2 mRNA and protein were detected by real-time PCR, immunocytochemistry and Western blot at different time points. GES-1 cells were then pre-treated with NF-KappaB pathway inhibitor PDTC, and the expression of CDX2 mRNA and protein induced by IL-1beta were detected. The cell ultra-structure of GES-1 cells was observed by electronic microscope after GES-1 being treated with IL-1beta for 25 days. Levels of CDX2 mRNA and protein were 0.0749 + or - 0.0021 and 0.56 + or - 0.04 in the cells treated with 1 microg/L IL-1beta(P<0.05). After pre-treatment with PDTC, levels of CDX2 mRNA and protein were 0.0006 + or - 0.0002 and 0.40 + or - 0.06(P<0.05). Some changes in the cell ultra-structure of GES-1 were found by electronic microscope when GES-1 was treated with IL-1beta for 25 days. IL-1beta can stimulate CDX2 mRNA and protein expression in GES-1 cells through the NF-KappaB signal pathway, indicating that IL-1beta plays an important role in the intestinal metaplasia.

Maternally and zygotically provided Cdx2 have novel and critical roles for early development of the mouse embryo

Divisions of polarised blastomeres that allocate polar cells to outer and apolar cells to inner positions initiate the first cell fate decision in the mouse embryo. Subsequently, outer cells differentiate into trophectoderm while inner cells retain pluripotency to become inner cell mass (ICM) of the blastocyst. Elimination of zygotic expression of trophectoderm-specific transcription factor Cdx2 leads to defects in the maintenance of the blastocyst cavity, suggesting that it participates only in the late stage of trophectoderm formation. However, we now find that mouse embryos also have a maternally provided pool of Cdx2 mRNA. Moreover, depletion of both maternal and zygotic Cdx2 from immediately after fertilization by three independent approaches, dsRNAi, siRNAi and morpholino oligonucleotides, leads to developmental arrest at much earlier stages than expected from elimination of only zygotic Cdx2. This developmental arrest is associated with defects in cell polarisation, reflected by expression and localisation of cell polarity molecules such as Par3 and aPKC and cell compaction at the 8- and 16-cell stages. Cells deprived of Cdx2 show delayed development with increased cell cycle length, irregular cell division and increased incidence of apoptosis. Although some Cdx2-depleted embryos initiate cavitation, the cavity cannot be maintained. Furthermore, expression of trophectoderm-specific genes, Gata3 and Eomes, and also the trophectoderm-specific cytokeratin intermediate filament, recognised by Troma1, are greatly reduced or undetectable. Taken together, our results indicate that Cdx2 participates in two steps leading to trophectoderm specification: appropriate polarisation of blastomeres at the 8- and 16-cell stage and then the maintenance of trophectoderm lineage-specific differentiation.

Acid and Bile Salt–Induced CDX2 Expression Differs in Esophageal Squamous Cells From Patients With and Without Barrett's Esophagus

It is not clear why only a minority of patients with gastroesophageal reflux disease (GERD) develop Barrett's esophagus. We hypothesized that differences among individuals in molecular pathways activated when esophageal squamous epithelium is exposed to reflux underlie the development of Barrett's metaplasia. We used esophageal squamous cell lines from patients who had GERD with Barrett's esophagus (normal esophageal squamous [NES]-B3T and NES-B10T) and without Barrett's esophagus (NES-G2T and NES-G4T) to study effects of acid and bile salts on expression of the CDX2 gene. Bay 11-705, Ad5 inhibitor kappaB(IkappaB)alpha-SR, and site-directed mutagenesis were used to explore effects of nuclear factor-kappaB (NF-kappaB) inhibition on CDX2 promoter activity; DNA binding of the NF-kappaB subunits p50 and p65 was assessed by chromatin immune-precipitation. Acid and bile salts increased CDX2 messenger RNA (mRNA), protein, and promoter activity in NES-B3T and NES-B10T cells, but not in NES-G2T or NES-G4T cells. Inhibition of NF-kappaB abolished the increase in CDX2 promoter activity. Increased CDX2 promoter activity was associated with nuclear translocation of p50, which bound to the promoter. We found CDX2 mRNA in 7 of 10 esophageal squamous biopsy specimens from patients with Barrett's esophagus, but in only 1 of 10 such specimens from patients who had GERD without Barrett's esophagus. Acid and bile salts induce CDX2 mRNA and protein expression in esophageal squamous cells from patients with Barrett's esophagus, but not from GERD patients without Barrett's esophagus. We speculate that these differences in acid- and bile salt-induced activation of molecular pathways may underlie the development of Barrett's metaplasia.

Helicobacter pylori Eradication Prevents Extension of Intestinalization Even in the High-Risk Group for Gastric Cancer

Background/Aims: CDX2 is associated with the intestinal phenotype in the gastrointestinal tracts and is expressed in the intestinal type of gastric cancer. Helicobacter pylori-associated atrophic gastritis is characterized by aberrant expression of CDX2. The aim was to investigate the effects of eradication to the expression of genes related to the gastric and intestinal phenotype including CDX2. We compared the effect of eradication between the patients at high risk for gastric cancer and controls. Methods: 20 patients with endoscopic resection for early gastric cancer and 12 sex- and age-matched controls were studied. CDX2 and mucin mRNA expressions were examined using whole biopsy specimens and microdissected gastric glands taken from corpus lesser and greater curves before and 1 year after eradication. Results:CDX2 and MUC2 expressions in the cancer group were significantly higher than in the controls and were significantly decreased after eradication. MUC5AC (p = 0.01) and MUC6 (p = 0.02) expression significantly increased in the control group; the difference between the two groups became significant after eradication. CDX2 expression in the glands without goblet cells was detectable and disappeared after eradication. Conclusion:H. pylori eradication can reverse gastric phenotype and diminish aberrant CDX2 expression in the early stage of intestinalization.

Transgenic Cdx2 induces endogenous Cdx1 in intestinal metaplasia of Cdx2‐transgenic mouse stomach

Cdx1 and Cdx2, which are transcription factors regulating normal intestinal development, have been studied as potential key molecules in the pathogenesis of the precancerous intestinal metaplasia of the human stomach. However, the regulation of Cdx1 expression in the intestinal metaplasia is poorly understood. Cdx2-expressing gastric mucosa of Cdx2-transgenic mouse stomach was replaced by intestinal metaplastic mucosa. The aim of this study was to investigate the following: (a) Cdx1 expression in the intestinal metaplastic mucosa of the Cdx2-transgenic mouse stomach; and (b) the relationship between Cdx1 and Cdx2. A mouse model of intestinal metaplasia, the Cdx2-transgenic mouse, was used to investigate Cdx1 gene expression by RT-PCR. DNA methylation profile analysis was performed by bisulfite sequencing, and the interaction of Cdx2 with the Cdx1 promoter was examined by chromatin immunoprecipitation assay, electrophoretic mobility shift assay, and luciferase reporter assays. Cdx2 mRNA was expressed in the Cdx2-transgenic mouse stomach. However, endogenous Cdx2 mRNA was not expressed in the intestinal metaplasia of the Cdx2-transgenic mouse stomach. On the other hand, endogenous Cdx1 mRNA and protein were expressed in the intestinal metaplasia of the Cdx2-transgenic mouse stomach. The Cdx1 promoter was unmethylated in the intestinal metaplasia of the Cdx2-transgenic mouse stomach. Chromatin immunoprecipitation assay and electrophoretic mobility shift assay showed that Cdx2 was bound to the Cdx1 promoter region in the intestinal metaplasia and the normal intestine. Cdx2 upregulated and siRNA-Cdx2 downregulated the transcriptional activity of the Cdx1 gene in the human gastric carcinoma cell lines AGS, MKN45, and MKN74. In conclusion, transgenic Cdx2 induced endogenous Cdx1 through the binding of Cdx2 to the unmethylated Cdx1 promoter region in the intestinal metaplasia of the Cdx2-transgenic mouse stomach.

CDX2 in the formation of the trophectoderm lineage in primate embryos

The first lineage decision during mammalian development is the establishment of the trophectoderm (TE) and the inner cell mass (ICM). The caudal-type homeodomain protein Cdx2 is implicated in the formation and maintenance of the TE in the mouse. However, the role of CDX2 during early embryonic development in primates is unknown. Here, we demonstrated that CDX2 mRNA levels were detectable in rhesus monkey oocytes, significantly upregulated in pronuclear stage zygotes, diminished in early cleaving embryos but restored again in compact morula and blastocyst stages. CDX2 protein was localized to the nucleus of TE cells but absent altogether in the ICM. Knockdown of CDX2 in monkey oocytes resulted in formation of early blastocyst-like embryos that failed to expand and ceased development. However, the ICM lineage of CDX2-deficient embryos supported the isolation of functional embryonic stem cells. These results provide evidence that CDX2 plays an essential role in functional TE formation during primate embryonic development.

Involvement of SMAD Signaling in Bovine Early Embryonic Development.

We have previously demonstrated a positive functional role for maternal (oocyte-derived) follistatin in control of time to first cleavage of bovine embryos, subsequent development to the blastocyst stage and in blastocyst cell allocation to the trophectoderm lineage. Follistatin binds to and inhibits activity of members of the transforming growth factor superfamily, whose signals are mediated intracellularly primarily through activation of receptor associated SMADs (rSMADs) including SMAD 2/3 (activin, TGF-beta) and SMAD 1/5/8 (BMPs). Thus, we hypothesized that follistatin's positive actions on early embryogenesis may be mediated via inhibition of rSMAD signaling. To address this hypothesis we determined the temporal expression and effects on bovine early embryogenesis of siRNA mediated knockdown of SMAD 4, a common component of above signaling pathways which complexes with respective rSMADs. Temporal changes in SMAD 4 mRNA during early embryogenesis were determined by real time PCR analysis of embryos collected at pronuclear, 2-, 4-, 8- and 16-cell and morula and blastocyst stages. SMAD 4 mRNA abundance was highest in 2-cell embryos and decreased dramatically to very low levels between the 8- to 16-cell stages, suggesting that SMAD 4 transcripts in early embryos are oocyte-derived. The maternal origin of SMAD 4 transcripts was confirmed by real time PCR analysis of embryos cultured to the 8-cell stage in the presence of the transcription inhibitor alpha-amanitin. Furthermore, SMAD 4 mRNA was higher in alpha-amanitin treated embryos, suggesting potential transcriptional dependence of observed decline in maternal SMAD 4 mRNA. To determine the functional role of SMAD 4 in bovine early embryogenesis, multiple siRNA species targeting bovine SMAD 4 were synthesized and evaluated for efficacy in reducing SMAD 4 mRNA levels (in 4-cell embryos) following microinjection into presumptive zygotes. Two of the validated SMAD 4 siRNA species with > 90 % efficiency in targeting SMAD 4 mRNA were combined and the efficacy of this siRNA cocktail (utilized in all subsequent studies) in reducing SMAD 4 mRNA and protein in early embryos verified. Presumptive zygotes were then injected with SMAD 4 siRNA, a negative control siRNA or served as uninjected controls. Effects of SMAD 4 knockdown on proportion of embryos cleaving early (within 30 h post insemination; hpi), total cleavage rates (determined 48 hpi), proportion of embryos developing to 8-16 cell (72 hpi) and blastocyst stages (d 7) and on blastocyst CDX2 mRNA abundance (index of trophectoderm cell allocation) were determined. SMAD 4 knockdown significantly reduced the proportion of embryos that cleaved early and embryonic development to 8- to 16-cell and blastocyst stages relative to uninjected and negative control siRNA injected embryos. Such results support a functional role for SMAD signaling in control of time to first cleavage and subsequent development to the blastocyst stage. However, effects of SMAD 4 knockdown on abundance of CDX2 mRNA in resulting blastocysts were not observed. Collectively, results support a requirement for the common SMAD (SMAD 4) of maternal origin in promoting bovine early embryogenesis, but do not suggest that previously observed stimulatory effects of follistatin on embryonic development are likely mediated via blocking stimulation of classical SMAD 4 dependent rSMAD signaling via endogenous TGF-beta superfamily members. (poster)

Major Changes in Cdx2 mRNA and Protein Expression Occur Around Compaction in Pre-Implantation Mouse Embryos.

Major Changes in Cdx2 mRNA and Protein Expression Occur Around Compaction in Pre-Implantation Mouse Embryos.

CDX2 promoter methylation is not associated with mRNA expression

Dear Sir, Yuasa et al. published that specific lifestyle factors, described to be potentially preventive of gastric cancer on epidemiological basis, may directly influence the carcinogenic process by affecting demethylation or maintenance of unmethylated status of selected genes. Specifically, it was shown that CDX2 was methylated in 23.6% of primary gastric carcinomas, whereas no methylation was seen in adjacent noncancerous gastric tissue of some of the cancer patients studied. They also demonstrated a significant inverse association between intake of green tea (more than 7 cups/day) and the methylation status of CDX2 in gastric cancer patients. However, in the same article, the authors did not show that the methylation status was correlated with CDX2 expression. In our study, we investigated if methylation of the CDX2 promoter could be a mechanism involved in the regulation of CDX2 expression in gastric carcinomas using gastric carcinoma cell lines as models and also in intestinal metaplasia (IM) of the stomach. CDX2 is a transcription factor that plays a major role in intestinal differentiation both in the intestine and in aberrant locations, such as in IM of the stomach. We show that there is no association between CDX2 promoter methylation and CDX2 expression, suggesting that methylation does not constitute a primary mechanism regulating CDX2 expression both in gastric carcinoma cell lines and in gastric preneoplastic lesions. Thus, our findings do not support the conclusions taken by Yuasa et al. We used the bioinformatic tool CpGPlot to search for potential CpG islands in the proximal region of the CDX2 promoter and identified two regions that fulfil the criteria (Fig. 1a). Then we studied the basal CDX2 mRNA expression and the methylation status of the CDX2 promoter in a panel of four human gastric carcinoma cell lines (AGS, GP202, IPA220 and MKN45). CDX2 mRNA expression analysis in the gastric carcinoma cell lines showed that AGS and IPA220 strongly express CDX2, while GP202 and MKN45 present equally low levels of CDX2 expression (Fig. 1b). The methylation of the CDX2 promoter in these cell lines was determined using the bisulfite-genome sequencing method (Fig. 1c). CpG island 1 was methylated in all cell lines, whereas CpG island 2 was methylated in GP202 and AGS cell lines and unmethylated in IPA220 and MKN45 cell lines. These results did not correlate with CDX2 mRNA expression. To further confirm if methylation could be in any way involved in CDX2 regulation, we treated these cell lines with the demethylating agent 5-aza-2’deoxycytidine (Fig. 1d). Once more, the results obtained suggest that methylation is not directly regulating CDX2 expression since cell lines with similar basal methylation status, AGS and GP202, react differently to the treatment with a demethylating agent. Although there is an increase of expression in GP202 after treatment, CDX2 remains unchanged in AGS. Strikingly, MKN45 which shows unmethylated CDX2 promoter also reacts to the treatment with an increased expression. Furthermore, we examined CDX2 expression and the methylation status of 42 CpG sites, contained in CpG island 2 of the CDX2 promoter, in two specimens of normal gastric mucosa, adjacent IM foci and in two normal colonic mucosa samples. As expected no CDX2 expression was observed in normal gastric mucosa in contrast to IM and colonic tissue (data not shown). The methylation pattern was inconsistent and no correlation was found with CDX2 expression in all samples (Fig. 2). In summary, and although previous studies show that methylation might be involved in CDX2 regulation, our in vitro and in vivo results demonstrate that the pattern of methylation at the CDX2 promoter is random and unrelated with its expression. The increase in CDX2 expression observed in some of the cell lines studied is more likely due to the effect of the demethylating agent over epigenetically modified regulators, such as silenced transcription factors or modified histones, and not directly over CDX2. Yours sincerely, Bruno PEREIRA, Carla OLIVEIRA, Leonor DAVID, Raquel ALMEIDA*

Breaking the seals: Efficient mRNA detection from human archival paraffin-embedded tissue

During our study on HOXA13, HOXD12, and HOXD13 mRNA expression in human adult and embryonic tissues, we were confronted with the fact that, within our specimen collection, as in other University Departments in Europe, <20% of all samples yielded reliable labeling, while most samples were resistant to hybridization by standard protocols due to over-fixation. Fixation is essential for specimen stability, especially when samples are stored at room temperature and used for histology, and people tend to be more worried about under- than over-fixation. On the other hand fixation inhibits penetration by the probe and may also trap mRNA within ribosomes. Therefore, we developed a nonradioactive in situ hybridization technique, which allows detection of mRNA expressed on low levels from a variety of differentially fixed tissues while maintaining tissue integrity. This was achieved by improving target retrieval and probe detection. In contrast with others, our method allows reliable staining from tissues that are fixed in paraformaldehyde from four hours to over one week, and archived samples that were stored at room temperature for several years (17-19 yr in some cases) and exceeds detection limits of purely fluorescent methods. Our protocol is highly suitable for detecting CDX-2 mRNA in carcinoma specimens, but especially designed to investigate mRNAs in nonpathological adult and embryonic tissues. Due to the use of standardized probes, we do not expect problems in detecting other mRNAs expressed in suitable amounts.

Cdx1 and Cdx2 are functionally equivalent in vertebral patterning

The Cdx transcription factors regulate anterior–posterior (AP) vertebral patterning, at least in part, through direct regulation of Hox gene expression. Analysis of allelic series of Cdx mutant mice suggests functional overlap between these family members. However, the lack of a Cdx2 null mutant makes these analyses incomplete. Moreover, Hox proteins are sometimes redundant, making it difficult to discern whether Cdx members regulate identical Hox target genes in a redundant manner, or whether they regulate separate Hox genes which then converge on events related to vertebral patterning. To more directly assess this question, we developed a “knock in” model whereby Cdx2 was substituted for Cdx1. Consistent with functional redundancy Cdx2 “knock-in” mice exhibited perfect complementation of the Cdx1-null phenotype, as evidenced by the lack of skeletal defects or altered expression of Hox genes typically impacted by Cdx1 loss-of-function.It has been proposed that vertebral AP patterning is reliant on a gradient of the sum total of Cdx proteins, a posit that is consistent with functional redundancy between Cdx family members. To further assess this, we generated a gain-of-function model using BAC trangenesis to alter Cdx1 dosage. Cdx1 BAC transgenic mice overexpressed Cdx1 mRNA and protein, and fully complemented the Cdx1 null allele. However, gain of Cdx1 dosage via this BAC transgene in an otherwise wild type background had no discernible effects on vertebral patterning or Hox gene expression, suggesting that a moderate alteration in the Cdx protein gradient is of no consequence.

Induction of Endogenous Interferon Tau Gene Transcription by CDX2 and High Acetylation in Bovine Nontrophoblast Cells1

Interferon tau gene (IFNT) is expressed only by mononuclear trophectoderm cells in ruminant ungulates. To our knowledge, its epigenetic regulation and interaction with trophectoderm lineage-specific caudal-related homeobox 2 transcription factor (CDX2) have not been characterized. Herein, we studied differences in chromatin structures and transcription of endogenous bovine IFNT in bovine trophoblast BT-1 and CT-1 cells and in nontrophoblast MDBK cells. Transcripts from endogenous IFNT and CDX2 genes were found in BT-1 and CT-1 cells but not in MDBK cells. Chromatin immunoprecipitation study revealed that CDX2 binding sites exist in proximal upstream regions of IFNT (IFN-tau-c1). Endogenous IFNT transcription in BT-1 cells was increased with CDX2 overexpression but was reduced with short interfering RNA specific for the CDX2 transcript. In chromatin immunoprecipitation studies, histone H3K18 acetylation of IFNT was higher in CT-1 cells than in MDBK cells, while histone H3K9 methylation was lower in CT-1 cells than in nontrophoblast cells. In MDBK cells (but not in CT-1 cells), histone deacetylases were bound to IFNT, which was reversed with trichostatin A treatment; treatment with trichostatin A and CDX2 then increased IFNT mRNA levels that resulted from abundant CDX2 mRNA expression. These data provide evidence that significant increase in endogenous IFNT transcription in MDBK cells (which do not normally express IFNT) can be induced through CDX2 overexpression and high H3K18 acetylation, but lowering of H3K9 methylation could also be required for the degree of IFNT transcription seen in trophoblast cells.

Gastrointestinal differentiation marker Cytokeratin 20 is regulated by homeobox gene CDX1

CDX1 is a transcription factor that plays a key role in intestinal development and differentiation. However, the downstream targets of CDX1 are less well defined than those of its close homologue, CDX2. We report here the identification of downstream targets of CDX1 using microarray gene-expression analysis and other approaches. Keratin 20 (KRT20), a member of the intermediate filament and a well-known marker of intestinal differentiation, was initially identified as one of the genes likely to be directly regulated by CDX1. CDX1 and KRT20 mRNA expression were significantly correlated in a panel of 38 colorectal cancer cell lines. Deletion and mutation analysis of the KRT20 promoter showed that the minimum regulatory region for the control of KRT20 expression by CDX1 is within 246 bp upstream of the KRT20 transcription start site. ChIP analysis confirmed that CDX1 binds to the predicted CDX elements in this region of the KRT20 promoter in vivo. In addition, immunohistochemistry showed expression of CDX1 parallels that of KRT20 in the normal crypt, which further supports their close relationship. In summary, our observations strongly imply that KRT20 is directly regulated by CDX1, and therefore suggest a role for CDX1 in maintaining differentiation in intestinal epithelial cells. Because a key feature of the development of a cancer is an unbalanced program of proliferation and differentiation, dysregulation of CDX1 may be an advantage for the development of a colorectal carcinoma. This could, therefore, explain the relatively frequent down regulation of CDX1 in colorectal carcinomas by hypermethylation.

Bile Acids Induce Cdx2 Expression Through the Farnesoid X Receptor in Gastric Epithelial Cells

Clinical and experimental studies showed that the reflux of bile into the stomach contributes to the induction of intestinal metaplasia of the stomach and gastric carcinogenesis. Caudal-type homeobox 2 (Cdx2) plays a key role in the exhibition of intestinal phenotypes by regulating the expression of intestine-specific genes such as goblet-specific gene mucin 2 (MUC2). We investigated the involvement of the farnesoid X receptor (FXR), a nuclear receptor for bile acids, in the chenodeoxycholic acid (CDCA)-induced expression of Cdx2 and MUC2 in normal rat gastric epithelial cells (RGM-1 cells). RGM-1 cells were treated with CDCA or GW4064, an FXR agonist, in the presence or absence of guggulsterone, an FXR antagonist. CDCA induced dose-dependent expression of Cdx2 and MUC2 at both the mRNA and protein levels. The maximum stimulation of Cdx2 and MUC2 mRNA induced by CDCA was observed at 3 h and by 6 h, respectively. GW4064 also induced expression of these molecules. The effects of CDCA and GW4064 on expression of Cdx2 and MUC2 were abolished by guggulsterone. These findings suggest that bile acids may induce gastric intestinal metaplasia and carcinogenesis through the FXR.

Role of Cdx2 and cell polarity in cell allocation and specification of trophectoderm and inner cell mass in the mouse embryo

Genesis of the trophectoderm and inner cell mass (ICM) lineages occurs in two stages. It is initiated via asymmetric divisions of eight- and 16-cell blastomeres that allocate cells to inner and outer positions, each with different developmental fates. Outside cells become committed to the trophectoderm at the blastocyst stage through Cdx2 activity, but here we show that Cdx2 can also act earlier to influence cell allocation. Increasing Cdx2 levels in individual blastomeres promotes symmetric divisions, thereby allocating more cells to the trophectoderm, whereas reducing Cdx2 promotes asymmetric divisions and consequently contribution to the ICM. Furthermore, both Cdx2 mRNA and protein levels are heterogeneous at the eight-cell stage. This heterogeneity depends on cell origin and has developmental consequences. Cdx2 expression is minimal in cells with unrestricted developmental potential that contribute preferentially to the ICM and is maximal in cells with reduced potential that contribute more to the trophectoderm. Finally, we describe a mutually reinforcing relationship between cellular polarity and Cdx2: Cdx2 influences cell polarity by up-regulating aPKC, but cell polarity also influences Cdx2 through asymmetric distribution of Cdx2 mRNA in polarized blastomeres. Thus, divisions generating inside and outside cells are truly asymmetric with respect to cell fate instructions. These two interacting effects ensure the generation of a stable outer epithelium by the blastocyst stage.

Tumor necrosis factor‐α induces the aberrant expression of mucus core protein‐2 in non‐neoplastic biliary epithelial cells via the upregulation of CDX2 in chronic cholangitis

Chronic cholangitis, such as hepatolithiasis, is frequently associated with goblet cell metaplasia and the aberrant expression of mucus core protein-2 (MUC2). In this study, we clarified the role of inflammatory cytokines in the expression of MUC2 in lining biliary epithelial cells (BEC) in chronic cholangitis with an emphasis on CDX2, an intestine-specific transcription factor. We used human hepatolithiatic livers and polycystic kidney (PCK) rats, an animal model of Caroli's disease, and cultured BEC from PCK rats. As a control, extrahepatic biliary obstruction and histologically normal livers and Crj:CD rats were used. Immunohistochemically, tumor necrosis factor-alpha (TNF-alpha) was expressed in periductal inflammatory cells and BEC of the affected intrahepatic bile ducts with an aberrant expression of MUC2 and CDX2 in hepatolithiasis and the PCK rats. In cultured BEC, TNF-alpha, interleukin (IL)-1beta, IL-6, and interferon-gamma induced the expression of CDX2 mRNA, though only TNF-alpha additionally induced the expression of MUC2 mRNA. The expression of CDX2 mRNA and the MUC2 protein induced in BEC by TNF-alpha were abolished by pretreatment of nuclear factor-kappaB inhibitors. The aberrant expression of CDX2 and MUC2 in the affected bile ducts showing goblet cell metaplasia was closely associated with TNF-alpha expressed in periductal infiltrating inflammatory cells and BEC. TNF-alpha induced the expression of CDX2 and MUC2 in cultured BEC. Taken together, it seems likely that TNF-alpha plays a role in MUC2 expression via CDX2 upregulation in the bile ducts with chronic cholangitis and goblet cell metaplasia.

Trophoblast Differentiation in the Ovine Placenta: Insights into Transcription Factors and Regulatory Pathways.

Placental morphogenesis in ruminants, humans and rodents involves the growth, development and differentiation of mononuclear trophectoderm cells (MTC) into giant binucleate cells (BNC) as well as multinucleated syncytiotrophoblasts (STB). For example in sheep, MTC express IFNT only from Days 10 to 25, while BNC, which begin to differentiate on Day 14, and STB express CSH1 but not IFNT. In rodents, trophoblast differentiation and gene expression is governed by temporal and cell-specific changes in transcription factors, but little is known about this process in ruminants. In Study One, candidate transcription factors identified previously in humans, rodents and cattle (CDX2, EED, ETS2, ID2, GCM1, TEAD3, TCF12, TFAP2A, TFAP2B, TFAP2C) as well as IFNT and CSH1 were studied in the Day (D) 13-20 conceptus and D40, D80 and D120 placenta (intercotyledonary and cotyledonary). IFNT mRNA was observed in most MTC in D13-20 conceptuses but not in MTC of the D40-120 placenta; CSH1 was first observed in BNC of D16 conceptuses and in BNC and STB of the D18-120 placenta. EED, ID2 and TFAP2B were not detected in the trophectoderm, but EED and ID2 were observed in the endometrial epithelia. ETS2, TCF12 and TEAD3 were observed in both the conceptus trophectoderm and endometrium. CDX2, GCM1, TFAP2A and TFAP2C appeared trophectoderm-specific. CDX2 and GCM1 mRNAs were observed specifically in the BNC and STB, whereas TFAP2A and TFAP2C were in MTC, BNC, and STB. Thus, CDX2, GCM1, TFAP2A and TFAP2C may play a critical role in trophoblast growth and differentiation in the ovine conceptus. In Study Two, microarray analysis was conducted using total RNA isolated from D16 conceptuses, D45 intercotyledonary placenta, and D45 placental cotyledons (n=4 ewes/day/tissue) and a comprehensive 24K bovine oligonucleotide array. As expected, IFNT was 132-fold higher (P<0.05) in the D16 conceptus than D45 placental tissues, and CSH1 was 70-fold higher (P<0.05) in the D45 placenta. Gene ontology analysis found that transcriptional regulators (CDCA7, EGR1, HTATIP2, KLF5, MAZI, MYBL2, PPP1R13B, TP53BP2), cyclins (CCNC, CCNY), and chromatin modification factors (CUL4A, MYST4, JARID1B, PHF12, SSRP1, SUDS3) were higher in D16 conceptuses than D45 placental tissues. TGFB superfamily-related, IGF-related genes, and Eph/Ephrin-related genes were also higher in the D16 conceptus. EGR1 and KLF5 interact with TP53 that is key factor in the cell cycle, and TP53BP2, PPP1R13B and SSRP1 enhance TP53 activity. The Eph/Ephrin family is regulated by TP53 and plays a critical role in vascular development and trophoblast differentiation in rodents. In D45 placenta, vasculogenesis/angiogenesis-related genes (AGTR1, AGTR2, ANGPT1, CTGF FGF2, SPP1, TNFRSF12A, TNFSF15, VWF) were higher than D16 conceptus. TGFB superfamily-related genes (BMP1, BMP5, BMP7, FSTL1, KLF11, LTBP1, LTBP2, LTBP4, TGFB1) were also higher in D45 placenta. In contrast to Day 16 conceptus, transcription factor AP1-related genes (JUNB, JUN, JUND, FOSL1) were higher in Day 45 placenta. AP1 regulates the transcription of many genes including TGFB1. These results suggest that TGFB superfamily signaling and AP1 regulate placental development. These studies suggest species-specific differences in transcription factor expression during trophoblast differentiation and provide new insights into regulatory networks and pathways governing ovine placental morphogenesis. (Supported by NIH HD052745)

Quantitative analysis of the effect of Helicobacter pylori on the expressions of SOX2, CDX2, MUC2, MUC5AC, MUC6, TFF1, TFF2, and TFF3 mRNAs in human gastric carcinoma cells

Objective. To investigate the phenotypic characters of carcinoma cells and the response of gastric epithelial cells to Helicobacter pylori (H. pyroli) infection using the gastric carcinoma cell lines. Material and methods. Real-time reverse transcription-polymerase chain reaction (RT-PCR) was used to assess the effect of H. pylori infection on mRNA levels of transcription factors (SOX2 and CDX2), mucin core proteins (MUC2, MUC5AC, and MUC6), and trefoil factor family peptides (TFF) (TFF1, TFF2, and TFF3) in gastric carcinoma cells (AGS, MKN45, and KATO III cells). H. pylori ATCC 43504 and its isogenic cag pathogenicity island (PAI) deleted mutant were used. Results. These cell lines expressed mixed gastric and intestinal phenotypes. The intestinal phenotype predominated in AGS cells and gastric phenotypes in MKN45 and KATO III cells. In all three cell lines, H. pylori infection inhibited SOX2 mRNA expression, but induced the three TFFs mRNAs. In AGS cells, H. pylori induced cag PAI-dependent mRNA expression of CDX2, MUC2, MUC5AC, and MUC6. mRNA expressions of CDX2, MUC5AC, and MUC6 were inhibited in KATO III cells, whereas MUC2 mRNA expression was unchanged. In MKN45 cells, H. pylori induced the three MUCs mRNAs but inhibited CDX2 mRNA expression. Conclusions. This study provides a useful platform for selecting appropriate cell lines to model H. pylori-related changes in the gastric epithelium that mirror the changes seen in vivo. The outcome of H. pylori infection may reflect changes in the mucus gel layer caused by altered expression of mucins and TFF peptides.