Jianping Kong

The intestine-specific transcription factor Cdx2 inhibits β-catenin/TCF transcriptional activity by disrupting the β-catenin–TCF protein complex

Cdx2 is an intestine-specific transcription factor known to regulate proliferation and differentiation. We have reported previously that Cdx2 limits the proliferation of human colon cancer cells by inhibiting the transcriptional activity of the beta-catenin-T-cell factor (TCF) bipartite complex. Herein we further elucidate this mechanism. Studies with a classic Cdx2 target gene and a canonical Wnt/beta-catenin/TCF reporter suggest that Cdx2 regulates these promoters by distinctly different processes. Specifically, inhibition of beta-catenin/TCF activity by Cdx2 does not require Cdx2 transcriptional activity. Instead, Cdx2 binds beta-catenin and disrupts its interaction with the DNA-binding TCF factors, thereby silencing beta-catenin/TCF target gene expression. Using Cdx2 mutants, we map the Cdx2 domains required for the inhibition of beta-catenin/TCF activity. We identify a subdomain in the N-terminus that is highly conserved and when mutated significantly reduces Cdx2 inhibition of beta-catenin/TCF transcriptional activity. Mutation of this subdomain also abrogates Cdx2s anti-proliferative effects in colon cancer cells. In summary, we conclude that Cdx2 binds beta-catenin and disrupts the beta-catenin-TCF complex. Considering the pivotal role of beta-catenin/TCF activity in driving proliferation of normal intestinal epithelial and colon cancer cells, our findings suggest a novel mechanism for Cdx2-mediated regulation of Wnt/beta-catenin signaling and cell proliferation.

Ectopic Cdx2 expression in murine esophagus models an intermediate stage in the emergence of Barrett's esophagus.

Barretts esophagus (BE) is an intestinal metaplasia that occurs in the setting of chronic acid and bile reflux and is associated with a risk for adenocarcinoma. Expression of intestine-specific transcription factors in the esophagus likely contributes to metaplasia development. Our objective was to explore the effects of an intestine-specific transcription factor when expressed in the mouse esophageal epithelium. Transgenic mice were derived in which the transcription factor Cdx2 is expressed in squamous epithelium using the murine Keratin-14 gene promoter. Effects of the transgene upon cell proliferation and differentiation, gene expression, and barrier integrity were explored. K14-Cdx2 mice express the Cdx2 transgene in esophageal squamous tissues. Cdx2 expression was associated with reduced basal epithelial cell proliferation and altered cell morphology. Ultrastructurally two changes were noted. Cdx2 expression was associated with dilated space between the basal cells and diminished cell-cell adhesion caused by reduced Desmocollin-3 mRNA and protein expression. This compromised epithelial barrier function, as the measured trans-epithelial electrical resistance (TEER) of the K14-Cdx2 epithelium was significantly reduced compared to controls (1189 Ohm*cm2 ±343.5 to 508 Ohm*cm2±92.48, p = 0.0532). Secondly, basal cells with features of a transitional cell type, intermediate between keratinocytes and columnar Barretts epithelial cells, were observed. These cells had reduced keratin bundles and increased endoplasmic reticulum levels, suggesting the adoption of secretory-cell features. Moreover, at the ultrastructural level they resembled “Distinctive” cells associated with multilayered epithelium. Treatment of the K14-Cdx2 mice with 5′-Azacytidine elicited expression of BE-associated genes including Cdx1, Krt18, and Slc26a3/Dra, suggesting the phenotype could be advanced under certain conditions. We conclude that ectopic Cdx2 expression in keratinocytes alters cell proliferation, barrier function, and differentiation. These altered cells represent a transitional cell type between normal squamous and columnar BE cells. The K14-Cdx2 mice represent a useful model to study progression from squamous epithelium to BE.

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.

Cdx2 Levels Modulate Intestinal Epithelium Maturity and Paneth Cell Development

BACKGROUND & AIMS Caudal-related homeobox protein 2 (Cdx2) is an intestine-specific transcription factor that is important for intestinal development and intestine-specific gene expression. Cdx2 regulates intestinal cell-cell adhesion, proliferation, and the transcriptional activities of Wnt and β-catenin in cell culture systems. We generated transgenic mice that overexpress Cdx2 in the small intestinal and colonic epithelium to investigate the role of Cdx2 in differentiation and function of the intestinal epithelium. METHODS We established 4 different lines of villin-Cdx2 transgenic mice. Intestines were collected from infant, 3-month old, and wild-type mice. Genes of interest and cell lineage markers were examined by polymerase chain reaction and immunohistochemistry. RESULTS Villin-Cdx2 transgenic mice had complex phenotypes that were associated with transgene expression levels. The 2 lines that had the greatest levels of transgene expression had significant, preweaning failure to grow and death; these were the result of early epithelial maturation and alterations in nutrient digestion and absorption. Fat malabsorption was a prominent feature. Other effects associated with the transgene expression included loss of Paneth cell markers, increases in goblet cells, and migration of proliferating, EphB2-expressing cells to the crypt base. Loss of Paneth cell markers was associated with reduced nuclear localization of β-catenin but not homeotic posteriorization of the epithelium by Cdx2. CONCLUSIONS Overexpression of Cdx2 in the small intestine is associated with reduced post-natal growth, early epithelial maturation, alterations in crypt base organization, and changes in Paneth and goblet cell lineages. Cdx2 is a critical regulator not only of intestine-specific genes, but also processes that determine epithelial maturity and function.

Cdx genes, inflammation, and the pathogenesis of intestinal metaplasia.

Intestinal metaplasia (IM) is a biologically interesting and clinically relevant condition in which one differentiated type of epithelium is replaced by another that is morphologically similar to normal intestinal epithelium. Two classic examples of this are gastric IM and Barretts esophagus (BE). In both, a chronic inflammatory microenvironment, provoked either by Helicobacter pylori infection of the stomach or acid and bile reflux into the esophagus, precedes the metaplasia. The Caudal-related homeodomain transcription factors Cdx1 and Cdx2 are critical regulators of the normal intestinal epithelial cell phenotype. Ectopic expression of Cdx1 and Cdx2 occurs in both gastric IM as well as in BE. This expression precedes the onset of the metaplasia and implies a causal role for these factors in this process. We review the observations regarding the role of chronic inflammation and the Cdx transcription factors in the pathogenesis of gastric IM and BE.

Repression of the Desmocollin 2 Gene Expression in Human Colon Cancer Cells Is Relieved by the Homeodomain Transcription Factors Cdx1 and Cdx2

Desmosomes are intracellular junctions that provide strong cell-cell adhesion in epithelia and cardiac muscle. Their disruption causes several human diseases and contributes to the epithelial-to-mesenchymal transition observed in cancer. Desmocollin 2 (DSC2) is a cadherin superfamily member and a critical component of desmosomes found in intestinal epithelium. However, the mechanism regulating DSC2 gene expression in intestinal cells is not known. Cdx1 and Cdx2 are homeodomain transcription factors that regulate intestine-specific gene expression. Cdx expression in the past has been associated with the induction of desmosomes. We now show that the DSC2 gene is a transcriptional target for Cdx1 and Cdx2. Colon cancer cell lines retaining Cdx2 expression typically express DSC2. Restoration of Cdx expression in Colo 205 cells induced DSC2 mRNA and protein and the formation of desmosomes. The 5′-flanking region of the DSC2 promoter contains two consensus Cdx-binding sites. Electrophoretic mobility shift assays show that Cdx1 and Cdx2 bind these sites in vitro, and chromatin immunoprecipitation confirmed Cdx2 binding in vivo. DSC2 promoter truncations established that these regions are Cdx responsive. The truncations also identify a region of the promoter in which potent transcriptional repressors act. This repressor activity is relieved by Cdx binding. We conclude that the homeodomain transcription factors Cdx1 and Cdx2 regulate DSC2 gene expression in intestinal epithelia by reversing the actions of a transcriptional repressor. The regulation of desmosomal junctions by Cdx contributes to normal intestinal epithelial columnar morphology and likely antagonizes the epithelial-to-mesenchymal transition necessary for the metastasis of colon cancer cells in humans. (Mol Cancer Res 2008;6(9):1478–90)

Intestine-specific transcription factor Cdx2 induces E-cadherin function by enhancing the trafficking of E-cadherin to the cell membrane

Cdx2 is an intestine-specific transcription factor required for normal intestinal epithelium development. Cdx2 regulates the expression of intestine-specific genes and induces cell adhesion and columnar morphogenesis. Cdx2 also has tumor-suppressor properties, including the reduction of colon cancer cell proliferation and cell invasion, the latter due to its effects on cell adhesion. E-cadherin is a cell adhesion protein required for adherens junction formation and the establishment of intestinal cell polarity. The objective of this study was to elucidate the mechanism by which Cdx2 regulates E-cadherin function. Two colon cancer cell lines were identified in which Cdx2 expression was associated with increased cell-cell adhesion and diminished cell migration. In both cell lines, Cdx2 did not directly alter E-cadherin levels but increased its trafficking to the cell membrane compartment. Cdx2 enhanced this trafficking by altering receptor tyrosine kinase (RTK) activity. Cdx2 expression diminished phosphorylated Abl and phosphorylated Rac levels, which are downstream effectors of RTKs. Specific chemical inhibition or short interfering RNA (shRNA) knockdown of c-Abl kinase phenocopied Cdx2s cell-cell adhesion effects. In Colo 205 cells, Cdx2 reduced PDGF receptor and IGF-I receptor activation. This was mediated by caveolin-1, which was induced by Cdx2. Targeted shRNA knockdown of caveolin-1 restored PDGF receptor and reversed E-cadherin membrane trafficking, despite Cdx2 expression. We conclude that Cdx2 regulates E-cadherin function indirectly by disrupting RTK activity and enhancing E-cadherin trafficking to the cell membrane compartment. This novel mechanism advances Cdx2s prodifferentiation and antitumor properties and suggests that Cdx2 may broadly regulate RTK activity in normal intestinal epithelium by modulating membrane trafficking of proteins.

Autophagy levels are elevated in barrett's esophagus and promote cell survival from acid and oxidative stress

Autophagy is a highly conserved mechanism that is activated during cellular stress. We hypothesized that autophagy may be induced by acid reflux, which causes injury, and inflammation, and therefore, contributes to the pathogenesis of Barretts esophagus (BE) and esophageal adenocarcinoma (EAC). Currently, the role of autophagy in BE and EAC is poorly studied. We quantitatively define autophagy levels in human BE cell lines, a transgenic mouse model of BE, and human BE, and EAC biopsies. Human non‐dysplastic BE had the highest basal number of autophagic vesicles (AVs), while AVs were reduced in normal squamous cells and dysplastic BE cells, and nearly absent in EAC. To demonstrate a functional role for autophagy in BE pathogenesis, normal squamous (STR), non‐dysplastic BE (CPA), dysplastic BE (CPD), and EAC (OE19) cell lines were exposed to an acid pulse (pH 3.5) followed by incubation in the presence or absence of chloroquine, an autophagy inhibitor. Acid exposure increased reactive oxygen species (ROS) levels in STR and CPA cells. Chloroquine alone had a small impact on intracellular ROS or cell survival. However, combination of chloroquine with the acid pulse resulted in a significant increase in ROS levels at 6 h in STR and CPA cells, and increased cell death in all cell lines. These findings establish increased numbers of AVs in human BE compared to normal squamous or EAC, and suggest that autophagy functions to improve cell survival after acid reflux injury. Autophagy may thus play a critical role in BE pathogenesis and progression.

Molecular mechanisms of Barrett's esophagus and adenocarcinoma.

The following on molecular mechanisms of Barretts esophagus and adenocarcinoma contains commentaries on the mechanism of bile and gastric acid induced damage; the roles of BMP‐4 and CDX‐2 in the development of intestinal metaplasia; the transcription factors driving intestinalization in Barretts esophagus; the contribution of bone marrow to metaplasia and adenocarcinoma; activation and inactivation of transcription factors; and a novel study design targeting molecular pathways in Barretts esophagus.

Math1/Atoh1 contributes to intestinalization of esophageal keratinocytes by inducing the expression of Muc2 and Keratin-20.

BackgroundEsophageal intestinal metaplasia, also known as Barrett’s esophagus, is the replacement of the normal epithelium with one that resembles the intestine morphologically. Generally, this includes intestinal mucin-secreting goblet cells. Barrett’s esophagus is an important risk factor for adenocarcinoma development. In-vitro models for Barrett’s esophagus have not, to date, focused on the induction of goblet cells in Barrett’s epithelium.AimsTo explore the contribution of Math1/Atoh1 to induction of Barrett’s esophagus and intestinal mucin-secreting goblet cells from normal human esophageal epithelium.MethodsWe explored the level and pattern of Math1/Atoh1 mRNA and protein expression in human Barrett’s esophagus. Then, using retroviral-mediated gene expression, we induced Math1 mRNA and protein expression in a human esophageal keratinocyte cell line. We evaluated the effects of this ectopic Math1 expression on cell proliferation and gene expression patterns in cells cultured under two-dimensional and three-dimensional tissue-engineering conditions.ResultsMath1/Atoh1 mRNA and protein are detected in human Barrett’s esophagus specimens, but the mRNA levels vary substantially. In the keratinocyte expression studies, we observed that Math1/Atoh1 ectopic expression significantly reduced cell proliferation and altered cell morphology. Moreover, Math1/Atoh1 expression is associated with a more intestinalized gene expression pattern that is distinct from that reported in after studies using other intestinal transcription factors. Most significantly, we observe the induction of the Barrett’s esophagus markers Mucin-2 and Keratin-20 with Math1/Atoh1 expression.ConclusionsWe conclude that ectopic Math1/Atoh1 expression makes unique contributions to intestinalization of the esophageal epithelium in Barrett’s esophagus.