Intestinal regulation of suppression of tumorigenicity 14 (ST14) and serine peptidase inhibitor, Kunitz type -1 (SPINT1) by transcription factor CDX2.

The type II membrane-anchored serine protease, matriptase, encoded by suppression of tumorgenicity-14 (ST14) regulates the integrity of the intestinal epithelial barrier in concert with its inhibitor, HAI-1 encoded by serine peptidase inhibitor, Kunitz type -1 (SPINT1). The balance of the protease/inhibitor gene expression ratio is vital in preventing the oncogenic potential of matriptase. The intestinal cell lineage is regulated by a transcriptional regulatory network where the tumor suppressor, Caudal homeobox 2 (CDX2) is considered to be an intestinal master transcription factor. In this study, we show that CDX2 has a dual function in regulating both ST14 and SPINT1, gene expression in intestinal cells. We find that CDX2 is not required for the basal ST14 and SPINT1 gene expression; however changes in CDX2 expression affects the ST14/SPINT1 mRNA ratio. Exploring CDX2 ChIP-seq data from intestinal cell lines, we identified genomic CDX2-enriched enhancer elements for both ST14 and SPINT1, which regulate their corresponding gene promoter activity. We show that CDX2 displays both repressive and enhancing regulatory abilities in a cell specific manner. Together, these data reveal new insight into transcriptional mechanisms controlling the intestinal matriptase/inhibitor balance.

In Vitro Functional Analyses of Infrequent Nucleotide Variants in the Lactase Enhancer Reveal Different Molecular Routes to Increased Lactase Promoter Activity and Lactase Persistence.

The genetic trait that allows intestinal lactase to persist into adulthood in some 35% of humans worldwide operates at the level of transcription, the effect being caused by cis-acting nucleotide changes upstream of the lactase gene (LCT). A single nucleotide substitution, -13910 C>T, the first causal variant to be identified, accounts for lactase persistence over most of Europe. Located in a region shown to have enhancer function in vitro, it causes increased activity of the LCT promoter in Caco-2 cells, and altered transcription factor binding. Three other variants in close proximity, -13907 C>G, -13915 T>C and -14010 G>C, were later shown to behave in a similar manner. Here, we study four further candidate functional variants. Two, -14009 T>G and -14011 C>T, adjacent to the well-studied -14010 G>C variant, also have a clear effect on promoter activity upregulation as assessed by transfection assays, but notably are involved in different molecular interactions. The results for the two other variants (-14028 T>C, -13779 G>C) were suggestive of function, -14028*C showing a clear change in transcription factor binding, but no obvious effect in transfections, while -13779*G showed greater effect in transfections but less on transcription factor binding. Each of the four variants arose on independent haplotypic backgrounds with different geographic distribution.

Involvement of CDX2 in the cross talk between TNF-α and Wnt signaling pathway in the colon cancer cell line Caco-2.

Tumor necrosis factor-α (TNF-α) is highly upregulated in inflammation and reduces the expression of the intestinal transcription factor, Caudal-related homeobox transcription factor 2 (CDX2). Wnt/ß-catenin signaling is critical for intestinal cell proliferation, but a decreased CDX2 expression has influence on the Wnt signaling-related genes and progression of colorectal cancer. Although several inflammatory signaling pathways, including TNF-α, have been reported to promote Wnt/ß-catenin activity and development of cancer, the underlying molecular mechanisms remain unclear. The aim was to investigate the signaling pathways involved in the TNF-α-mediated downregulation of CDX2, and its influence on Wnt/ß-catenin signaling components in colon cancer cells. The expression of TNF-α and CDX2 at the invasive front were evaluated by immunohistochemical staining and showed reduced CDX2-positive cells in tumor buddings in areas with TNF-α expression in the surrounding inflammatory cells. In vitro studies revealed that TNF-α treatment showed a dose-dependent decrease of CDX2 messenger RNA (mRNA) and protein expression in Caco-2 cells. Inhibition of nuclear factor-kappaB or p38 pathways showed that these are involved in the TNF-α-dependent downregulation of CDX2. Furthermore, TNF-α-mediated downregulation of CDX2 was found to significantly decrease the mRNA levels of adenomatous polyposis coli (APC), axis inhibition protein 2 (AXIN2) and glycogen synthase kinase-3 beta (GSK3ß), whereas the mRNA levels of Wnt targets were significantly elevated in TNF-α-treated Caco-2 cells. These findings were associated with reduced binding of CDX2 to promoter or enhancer regions of APC, AXIN2 and GSK3ß. In conclusion, it was found that TNF-α induces the expression of Wnt signaling components through a downregulation of the CDX2 expression that might have a tumor-promoting effect on colon cancer cells.

Diversity of lactase persistence alleles in Ethiopia: signature of a soft selective sweep.

The persistent expression of lactase into adulthood in humans is a recent genetic adaptation that allows the consumption of milk from other mammals after weaning. In Europe, a single allele (-13910(∗)T, rs4988235) in an upstream region that acts as an enhancer to the expression of the lactase gene LCT is responsible for lactase persistence and appears to have been under strong directional selection in the last 5,000 years, evidenced by the widespread occurrence of this allele on an extended haplotype. In Africa and the Middle East, the situation is more complicated and at least three other alleles (-13907(∗)G, rs41525747; -13915(∗)G, rs41380347; -14010(∗)C, rs145946881) in the same LCT enhancer region can cause continued lactase expression. Here we examine the LCT enhancer sequence in a large lactose-tolerance-tested Ethiopian cohort of more than 350 individuals. We show that a further SNP, -14009T>G (ss 820486563), is significantly associated with lactose-digester status, and in vitro functional tests confirm that the -14009(∗)G allele also increases expression of an LCT promoter construct. The derived alleles in the LCT enhancer region are spread through several ethnic groups, and we report a greater genetic diversity in lactose digesters than in nondigesters. By examining flanking markers to control for the effects of mutation and demography, we further describe, from empirical evidence, the signature of a soft selective sweep.

Regulation of APC and AXIN2 expression by intestinal tumor suppressor CDX2 in colon cancer cells.

Wnt signaling is often constitutively active in colorectal cancer cells. The expression of the intestinal specific transcription factor CDX2 is found to be transiently decreased in invasive cells at the tumor/stroma interface. A recent ChIP-Seq study has indicated that several Wnt signaling-related genes are regulated by CDX2. The aim was to investigate the role of decreased CDX2 level on the expression of APC, AXIN2 and GSK3ß in migrating colon cancer cells at the invasive front. CDX2-bound promoter and enhancer regions from APC, AXIN2 and GSK3ß were analyzed for gene regulatory activity and the expression pattern of APC and GSK3ß at the invasive front was evaluated by immunohistochemical procedures. Transfection of intestinal and non-intestinal cell lines demonstrated that CDX2 activated APC and AXIN2 promoter activities via intestinal cell-specific enhancer elements. Suppressed CDX2 expression was associated with endogenous downregulation of APC and AXIN2 expression in Caco-2 cells but did not affect GSK3ß expression. Furthermore, elevated levels of nuclear ß-catenin and reduced levels of cytoplasmic APC were correlated to a low CDX2 expression in migrating colon cancer cells in vivo. These results suggest that a low CDX2 level has influence on the Wnt signaling in invasive colon cancer cells possibly promoting cellular migration.

TNF-α-induced down-regulation of CDX2 suppresses MEP1A expression in colitis.

BACKGROUND/AIMS: High levels of pro-inflammatory cytokines are linked to inflammatory bowel disease (IBD). The transcription factor Caudal-related homeobox transcription factor 2 (CDX2) plays a crucial role in differentiation of intestinal epithelium and regulates IBD-susceptibility genes, including meprin 1A (MEP1A). The aim was to investigate the expression of CDX2 and MEP1A in colitis; to assess if they are regulated by tumor necrosis factor-α (TNF-α), and finally to reveal if CDX2 is involved in a TNF-α-induced down-regulation of MEP1A. METHODS: Expression of CDX2 and MEP1A was investigated in colonic biopsies of ulcerative colitis (UC) patients and in dextran sodium sulfate (DSS)-induced colitis. CDX2 protein expression was investigated by immunoblotting and immunohistochemical procedures. CDX2 and MEP1A regulation was examined in TNF-α-treated Caco-2 cells by reverse transcription-polymerase chain reaction and with reporter gene assays, and the effect of anti-TNF-α treatment was assessed using infliximab. Finally, in vivo CDX2-DNA interactions were investigated by chromatin immunoprecipitation. RESULTS: The CDX2 and MEP1A mRNA expression was significantly decreased in active UC patients and in DSS-colitis. Colonic biopsy specimens from active UC showed markedly decreased CDX2 staining. TNF-α treatment diminished the CDX2 and MEP1A mRNA levels, a decrease which, was counteracted by infliximab treatment. Reporter gene assays showed significantly reduced CDX2 and MEP1A activity upon TNF-α stimulation. Finally, TNF-α impaired the ability of CDX2 to interact and activate its own, as well as the MEP1A expression. CONCLUSIONS: The present results indicate that a TNF-α-mediated down-regulation of CDX2 can be related to suppressed expression of MEP1A during intestinal inflammation.

Current and emerging approaches to define intestinal epithelium-specific transcriptional networks.

Upon developmental or environmental cues, the composition of transcription factors in a transcriptional regulatory network is deeply implicated in controlling the signature of the gene expression and thereby specifies the cell or tissue type. Novel methods including ChIP-chip and ChIP-Seq have been applied to analyze known transcription factors and their interacting regulatory DNA elements in the intestine. The intestine is an example of a dynamic tissue where stem cells in the crypt proliferate and undergo a differentiation process toward the villus. During this differentiation process, specific regulatory networks of transcription factors are activated to target specific genes, which determine the intestinal cell fate. The expanding genomewide mapping of transcription factor binding sites and construction of transcriptional regulatory networks provide new insight into how intestinal differentiation occurs. This review summarizes the current overview of the transcriptional regulatory networks driving epithelial differentiation in adult intestine. The novel technologies that have been implied to study these networks are presented and their prospects for implications in future research are also addressed.