The Role of CDX2 in Intestinal Homeostasis and Inflammation

Biochimica et Biophysica Acta 1812 (2011) 283–289

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Review The role of CDX2 in intestinal homeostasis and inﬂammation

Mehmet Coskun a,b,⁎, Jesper Thorvald Troelsen b, Ole Haagen Nielsen a a Department of Gastroenterology, Medical Section 54 O3, Herlev Hospital, University of Copenhagen, Herlev Ringvej 75, DK-2730 Herlev, Denmark b Institute of Cellular and Molecular Medicine, the Panum Institute, University of Copenhagen, Denmark article info abstract

Article history: Many transcription factors are known to control transcription at several promoters, while others are only Received 23 September 2010 active at a few places. However, due to their importance in controlling cellular functions, aberrant Received in revised form 19 November 2010 transcription factor function and inappropriate gene regulation have been shown to play a causal role in a Accepted 22 November 2010 large number of diseases and developmental disorders. Inflammatory bowel disease (IBD) is characterized by Available online 30 November 2010 a chronically inflamed mucosa caused by dysregulation of the intestinal immune homeostasis. The aetiology of IBD is thought to be a combination of genetic and environmental factors, including luminal bacteria. The Keywords: CDX2 Caudal-related homeobox transcription factor 2 (CDX2) is critical in early intestinal differentiation and has IBD been implicated as a master regulator of the intestinal homeostasis and permeability in adults. When Inflammation expressed, CDX2 modulates a diverse set of processes including cell proliferation, differentiation, cell Intestine adhesion, migration, and tumorigenesis. In addition to these critical cellular processes, there is increasing MAPK evidence for linking CDX2 to intestinal inflammation. The aim of the present paper was to review the current κ NF- B knowledge of CDX2 in regulation of the intestinal homeostasis and further to reveal its potential role in inflammation. © 2010 Elsevier B.V. All rights reserved.

1. Introduction [1] and to modulate cellular processes such as cell differentiation, proliferation, cell adhesion, migration, and tumorigenesis. Therefore, The intestinal epithelium is the most vigorously self-renewing by modulating these processes and regulating genes, CDX2 is essential tissue of adult mammals. The continuous renewal of intestinal in intestinal homeostasis and for the maintenance of an intestinal cell epithelium provides several unique challenges. phenotype. Thus, loss of accurate control of CDX2 expression has been Thus rates of intestinal cell production must be precisely balanced demonstrated to cause serious disruption of the mucosal architecture, by cell loss or destruction; otherwise, the epithelial barrier function is leading to intestinal diseases and developmental disorders. In compromised. Cell proliferation and differentiation are tightly addition to these specific roles, increasing knowledge indicates that controlled in the normal intestinal epithelium. Various genes and CDX2 may be pivotal in intestinal inflammation. In fact, a linkage transcription factors may take part in this process, in which some are between the key pathways involved in inflammation and regulators of up-regulated and others are down-regulated. One of these well- homeostasis is often seen [2], supporting the hypothesis that there is a studied factors is the homeodomain protein CDX2. CDX2 is an connection between inflammation and CDX2 expression. intestine-specific transcription factor known to act as a major protein In this review, we summarize the importance of CDX2 in gut regulator, governing expression of numerous intestine-specific genes homeostasis and additionally discuss the relationship between molecular pathways involved in intestinal inflammation and CDX2 expression. Abbreviations: AP, activator protein; CD, Crohn's disease; CDX2, Caudal-related homeobox transcription factor 2; CLDN 2, claudin-2; COX, cyclooxygenase; CREB, cAMP response element binding protein; DSS, dextran sodium sulphate; E, embryonic day; 2. Review criteria ERK, extracellular signal-regulated kinase; IBD, inflammatory bowel disease; IL, interleukin; JNK, c-Jun N-terminal kinase; MAPK, mitogen-activated protein kinase; MUC2, mucin 2; NF, nuclear factor; PEPT1, H+-coupled peptide transporter 1; PI3K, The search “Cdx2 cancer, Cdx2 ERK, Cdx2 homeostasis, Cdx2 phosphatidylinositol 3-kinase; PTEN, phosphatase and tensin homolog; TNF-α, tumor inflammation, Cdx NF-κB, Cdx2 p38, inflammatory bowel disease, and necrosis factor α; UC, ulcerative colitis MAP kinase inflammation” was performed in the PubMed database up ⁎ Corresponding author. Department of Gastroenterology, Medical Section 54 O3, to November 2010. English-language original papers, reviews, short Herlev Hospital, University of Copenhagen, Herlev Ringvej 75, DK-2730 Herlev, Denmark. Tel.: +45 4488 3421; fax: +45 4488 4009. communications, and articles were evaluated. Subsequently, articles E-mail address: [email protected] (M. Coskun). were selected based on their scientific and clinical relevance.

3. CDX genes: their role in intestinal development factor 4 (Tcf4), and β-catenin. In HeLa cells, these transcription factors and homeostasis activated endogenous expression of Cdx2 synergistically in co- transfection experiments with a Cdx2-luciferase construct. Further- The homeobox gene, Caudal, was originally identified in Drosophila more, CDX2 has been found to be able to positively autoregulate its [3], but subsequently other Caudal homologue transcription factors own expression [1,32]. have been identified in a wide array of organisms having pivotal roles The transcriptional activity of the CDX2 protein is also regulated at in intestinal epithelial development and maintenance [4–7]. Three the post-translational level. CDX2 expression persists throughout life Caudal homologue genes (CDX1, CDX2, and CDX4) have been identified in the intestinal epithelium and various phosphorylated forms coexist, in mammals, which are expressed during embryonic development, exerting different effects on cell behavior [33]. It has been established and they contribute to axial patterning [8–11]. In adult mammals, the that CDX2 is phosphorylated within the amino-terminal region by the CDX1 and CDX2 homeoproteins have been found to be intestine- mitogen-activated protein kinases (MAPKs) and that this modulates specific transcription factors regulating homeostasis of the continu- CDX2 transcriptional activity [15,34]. The phosphorylation of CDX2 ously renewing intestinal epithelium. However, the role of CDX4 in has also been shown to influence the stability of the protein. Gross adults is yet unknown. During adulthood, the CDX1 and CDX2 genes et al. [33] demonstrated that cyclin-dependent kinase 2 (CDK2) seem to be differently expressed in the intestinal epithelial cells and phosphorylates CDX2 at serine 281 and controls its degradation via along the crypt–villus axis [12]. The expression of CDX1 is restricted to polyubiquitination. the proliferating cells of the crypt compartment [13], while CDX2 is In summary, CDX2 is regulated by a combination of transcription found in all epithelial cells located in the crypt–villus epithelium of the factors of importance for intestinal differentiation and functional small intestine and colon [6,14], but CDX2 is active in differentiating properties of the intestinal epithelial cell. enterocytes [15]. By transactivating the promoters of several intestine- specific genes, both CDX1 and CDX2 may be involved in the regulation 4. CDX2 and colon cancer of proliferation and differentiation of intestinal epithelial cells [16–18]. In fact, CDX2 is directly involved in the activation of some of the genes Different lines of evidence suggest that CDX2 may suppress characteristic for enterocytic functions such as Sucrase-isomaltase, colorectal tumorigenesis [25,35,36]. Indeed, the expression of CDX2 Lactase-phlorizin hydrolase, Calbindin-D9K,andHephaestin genes is often reduced in a subset of primary colon cancers [37–39], and cell [6,19–21]. differentiation is poor in tumors that loose CDX2 [40]. Moreover, In the mouse embryo, the first stage of Cdx1 expression is from E heterozygous Cdx2+/− mice are viable and fertile; however, they (embryonic day) 7.5 to 12, with early expression in the ectoderm and develop multiple polyp-like lesions of the colon [25], and Cdx2+/− mesoderm of the primitive streak and later in the developing neu- mice treated with a carcinogenic compound, azoxymethane, seems to roectoderm, somites, and developing limb buds [22]. Cdx2 expression possess an increased risk for tumor development [36]. Some reports begins as early as E3.5 and is confined to the trophectoderm and have, however, questioned whether CDX2 has a tumor suppressor role, persists in the extra-embryonic ectoderm. From E8.5, Cdx2 is as some studies did not show any reduction in the CDX2 expression expressed in the posterior gut endoderm, neural tube, and tail bud level in tumors, as compared with normal mucosa [41,42], and [23]. By E12.5, the expression of Cdx2 is restricted to the endoderm of additionally heterozygous Cdx2+/− mice seemed to be more resistant the gut [8,24]. The expression of both Cdx1 and Cdx2 increases to colonic cancers induced by chronic inflammation [43]. Therefore, significantly during the transformation of endoderm into a columnar revealing the underlying mechanisms of a diminished CDX2 expres- epithelium (E14–E17) [12]. sion during tumorigenesis might clarify the pathogenesis of colon To directly address the function of CDX1 and CDX2 proteins during cancers. early development, transgenic models have been investigated. Mice embryos with inactivated Cdx2 alleles (Cdx2−/−)leadstoearlylethality 5. CDX2 regulates intestinal permeability due to an implantation failure [25],whileCdx1-null mice are viable and show anterior homeotic transformation of the axial skeleton [26]. Cdx2 The intestinal epithelial barrier is established by a single cellular heterozygotes are viable and fertile; however, the colon and small layer, and it plays a critical role in the transport of nutrients. Injury of intestine shows Cdx2-deficient lesions with gastric-like epithelium [27]. the epithelial layer requires a complex interplay between prolifera- This has further been supported by conditional homozygotes Cdx2 tion and migration of cells to restore the intestinal morphology. Apart knockout mice created by Gao et al. [28]. These mice had an abnormal from its importance in the development of the gut, CDX2 contributes colon, as the colonocytes differentiate into a gastric rather than into an to the balance between differentiation, proliferation, and cell renewal intestinal phenotype and thus exhibits loss of intestinal morphology. of the mature intestine. Even though the precise role of CDX2 on the Additionally, ectopic expression of Cdx2 in the stomach of transgenic migratory process of cells has not yet been established, there are some mice triggers intestinal-like heterodifferentiation of the gastric mucosa, indications for its influence on cell migration. Rao et al. [44] showed supporting the notion that Cdx2 is critical in both intestinal cell that overexpression of CDX2 in intestinal epithelial cells increased cell differentiation and in maintaining the intestinal phenotype [29,30]. migration in wound healing, while a more recent work of Gross et al. Taken together, these transgenic data demonstrate that CDX2 has [45] indicated that a decreasing CDX2 expression enhanced the mi- a crucial homeotic function during intestinal differentiation. gration of intestinal cells. Moreover, recently it has been demon- Owing to the essential role of CDX2 in intestinal development and strated that CDX2 regulates the transcriptional regulation of the cell phenotype, the transcriptional gene regulation of CDX2 has been engulfment and cell motility 3 (ELMO3) gene [46], which is assumed to the focus of numerous studies. In this context, Benahmed et al. [31] function as an essential upstream regulator of the monomeric GTP- used various transgenic genomic fragments of the mouse Cdx2 locus binding protein, Rac, during cell migration [47]. and demonstrated that the fragments containing the first −5kb Cell–cell interactions and the expression of adhesion molecules are upstream of the transcriptional start site is necessary in order to important regulators of intestinal proliferation, apoptosis, and migra- activate Cdx2 expression during gastrulation but became inactive at tion. The intestinal epithelial cells are held together by intercellular mid-gestation. However, the authors reported that genomic fragments junctions that include both adherence and tight junctions. The seal that extending to −9 kb is required to maintain the expression of Cdx2 in regulates paracellular permeability is established by the tight junction, the midgut region of the endoderm into adulthood. They demonstrat- which is composed of transmembrane adhesions molecules, e.g., ed a 250-bp segment around −8.5 kb that revealed interactions with claudins and occludin, and cytosolic proteins [48]. An intact intestinal hepatic nuclear factor 4α (HNF4α), GATA-binding protein 6, T-cell epithelial tight junction barrier is crucial in providing a barrier function M. Coskun et al. / Biochimica et Biophysica Acta 1812 (2011) 283–289 285 against translocation of bacteria and toxic antigens but also for the The link between CDX2 and intestinal inflammation has also been immune system homeostasis. Accordingly, three adhesion genes, LI- investigated in other species than humans. In Drosophila, inhibition of cadherin, E-cadherin,andClaudin-2 (CLDN2), have been reported as Caudal resulted in overexpression of antimicrobial peptide genes, CDX2-targets [49–51]. These proteins are critical in the establishment which led to an increased bacterial growth with elevated apoptosis and maintenance of cell adhesion and the intestinal cell polarity. In [78]. Moreover, Calon et al. [43] have shown a linkage of CDX2 to addition to tight junction proteins, the continuity and stability of the inflammation with experimental colitis models and demonstrated mucus layer is of importance for the intestinal homeostasis. that dextran sodium sulphate (DSS) in the drinking water of Cdx2+/− Mucins produced by goblet cells comprise the main structural mice led to an increased intestinal permeability. These animals components of the mucus layer and forms the primary barrier against showed a high susceptibility to development of DSS-induced acute pathogenic infections [52]. The predominant secretory mucin pro- colitis [43], suggesting that Cdx2 is involved in protection against duced by goblet cells is mucin 2 (MUC2) [53–55], and interestingly, DSS-induced colitis. MUC2 has also been reported to be a CDX2-target gene [56,57]. Taken together with the observations that (1) CDX2 is a downstream Studies on human tissues have shown that a dysfunction of the target of inflammatory cytokines, (2) expression of CDX2 is reduced in intestinal innate immunity and an altered expression and structural inflamed tissues, (3) heterozygotes Cdx2 mice are sensitive to DSS- changes of the intestinal tight junction proteins and mucins are induced acute colitis, and (4) CDX2 is essential in the regulatory closely associated with the development of cancer and inflammatory mechanism of genes involved in mucosal permeability, these facts bowel disease (IBD) [58–61]. Given the strategic position of CDX2 at indicate that CDX2 is involved in intestinal inflammation. Therefore, an the crossroads of regulating numerous intestinal genes on the one altered CDX2 expression and activity may change several functions and hand and preserving intestinal homeostasis and permeability on the properties of the epithelium to trigger infections and inflammation. other hand, it is obvious that a proper regulation of CDX2 expression and activity is of importance for maintaining a normal intestinal 6.1. Cytokine induced NF-κB pathway regulates CDX2 expression epithelia barrier. Inflammatory cytokines are key mediators in the pathogenesis of 6. Linking CDX2 to intestinal inflammation IBD, and a study has reported a TNF-α-mediated regulation of the CDX2 gene expression during inflammation [67]. This study indicates IBD is characterized as a chronic idiopathic inflammation of the that the TNF-α regulated CDX2 expression in vitro is actually intestine and is an umbrella term for different diseases of which mediated by nuclear factor (NF)-κB activation. ulcerative colitis (UC) and Crohn's disease (CD) are the two most The NF-κB family of transcription factors consists of five mamma- common entities. The inflammation in UC is localized exclusively to lian members (p50, p52, p65, cRel, and RelB) [79,80], which can form the colon, whereas CD is characterized by deep (transmural) homodimers or heterodimers. NF-κB is a key regulator in IBD [81,82]. segmental inflammatory lesions, which might occur anywhere in The expression and activation of NF-κB are strongly enhanced in the the gastrointestinal tract, the prevailing site is, however, the terminal inflamed gut of patients with IBD [83,84] and promote the expression ileum. The most common symptoms seen in UC are bloody stools and of various pro-inflammatory cytokines including IL-1, IL-2, IL-6, IL-8, diarrhea, while in CD, it is diarrhea, weight loss, abdominal cramps, IL-12, and TNF-α [85,86] (Fig. 1). In addition to an enhanced ex- and pain. pression and activity of NF-κB in IBD patients, a constitutive activation The aetiology of IBD is thought to be a combination of genetic and of the NF-κB pathway is involved in some malignancies including environmental factors. Thus, chronic intestinal inflammation might leukemia, lymphoma, colon cancer, and ovarian cancer [87,88]. NF-κB occur among genetic predisposed individuals affecting the mucosal additionally promotes the expression of a wide variety of genes that immune system [62–64], combined with luminal bacteria or infec- are important for the activation of immune responses, including genes tions (e.g., inflammatory signals) [65,66]. Beside pro- and anti- encoding chemokines, adhesion molecules, enzymes, and genes that inflammatory mediators and the molecular pathways regulating these facilitate proliferation, tumor promotion, and metastatic development factors, studies have investigated the role of CDX2 in the inflamma- [89]. Furthermore, NF-κB is important for regulation of genes coding tory process. One study has shown that CDX2 expression is repressed for regulators of apoptosis, such as cIAP, BCL-X, FLIP, and XIAP [90,91]. by the pro-inflammatory cytokine tumor necrosis factor α (TNF-α) Interestingly, Kim et al. [67] have identified two putative NF-κB [67] (Fig. 1), which are found in increased concentrations in the binding sites in the CDX2 promoter, suggesting a direct transcriptional inflamed intestines of active UC and CD [68,69]. Surprisingly, Sipos et regulation controlled by the balance between p50 and p65 subunits of al. [70] have recently reported a low expression level of CDX2 in NF-κB. They have shown that the CDX2 promoter is able to bind both inflamed UC, but with no significant correlation to inflammation. As p50/p50 and p65/p50 dimers, where the activity of the CDX2 previously described, CDX2 is a positive regulator of CLDN2 [51] and promoter is increased by the p50/p50 homodimer, and the binding MUC2 genes [56,57] with an essential role of the intestinal epithelial of p65/p50 heterodimer decreased CDX2 expression [67] (Fig. 1). barrier function. Interestingly, both CLDN2 [61,71] and MUC2 [72,73] Furthermore, they demonstrated that the DNA-binding activities of expressions are affected by the inflammatory mediators interleukin the p50/p50 or p65/p50 dimers are mediated by the phosphatase and (IL)-13 and TNF-α. Additionally, secretion of MUC2 is also reduced in tensin homolog (PTEN) and the phosphatidylinositol 3-kinase (PI3K) the active stage of UC resulting in a thinner layer of mucus as pathways [67] (Fig. 1). PTEN is a tumor suppressor protein that compared to healthy individuals [74]. Furthermore, Kaneko et al. [75] antagonizes the activity of PI3K by dephosphorylating the D3 have reported that CDX2 and MUC2 expressions are decreased in the phosphate group of a lipid second messenger, phopshatidylinositol ulcer-associated cell lineage, which occurs at sites of chronic 3,4,5-triphosphate, that is produced by PI3K [92]. Additionally, studies ulceration in CD, with a histological differentiation resembling gastric have reported that CDX2 can reduce NF-κB regulated gene expres- mucosa. sion [93–95]. In these studies, CDX2 interacts with the p65/p50 CDX2 has additionally been reported to positively regulate the H+- heterodimer of NF-κB and prevent it from forming transcriptional coupled peptide transporter 1 (PEPT1) [76]. PEPT1 is an integral active DNA complexes and thereby reduces cyclooxygenase (COX)-2 membrane-spanning protein transporting di- and tripeptides and is and cyclin D1 expressions, respectively. expressed in the inflamed colon of IBD patients [77]. Reducing CDX2 COX-2 is an inducible pro-inflammatory enzyme [96] playing a key could affect the regulation of PEPT1 and the intake of bacterial role in inflammatory cascades and cancer where arachidonic acid is peptides responsible for the production of immune molecules, involved [97,98], and it is positively regulated by NF-κB activation thereby increasing the risk for infections. [99]. Taken together, the interaction between NF-κB and CDX2 could 286 M. Coskun et al. / Biochimica et Biophysica Acta 1812 (2011) 283–289

Fig. 1. Regulation of CDX2 expression by inflammatory mediators. Extracellular stimuli from cytokines or inflammatory mediators activate either the MAPK signaling or the NF-κB signaling pathway to induce CDX2 expression. The mammalian MAPK family includes ERK, p38, and JNK. Activated MAPKs phosphorylate various substrate proteins including transcription factors resulting in the regulation of various cellular activities including cell survival, inflammation, and proliferation. Both p38 and ERK can phosphorylate CDX2, but p38 activates whereas ERK inactivates the transcriptional activity of CDX2. NF-κB has a pivotal role in inflammation. PTEN activity via NF-κB upregulates CDX2 expression and is antagonized by PI3K signaling. When expressed, CDX2 modulates a diverse set of processes including cell differentiation, cell adhesion, and tumorigenesis.

be the mechanism of linking CDX2 to inflammation by regulating the cAMP response element binding protein (CREB) [107]. Through DNA-binding activity of p50/p50 and p65/p50 in gene expression phosphorylation and activation of AP-1 and CREB, ERK1/2 mediates (Fig. 1). In fact, as previously described, inhibition of Caudal in its transcriptional function. ERK1/2 has been found to induce Drosophila resulted in overexpression of antimicrobial peptide genes expression of the pro-inflammatory cytokine IL-1 in IBD [108] and is leading to an increased bacterial growth with elevated apoptosis [78]. overexpressed in inflamed tissues with increased phosphorylation and Furthermore, it has been reported that the expression of antimicrobial elevated activation [109]. peptides is controlled by the balance between Caudal and NF-κB. Thus, In addition to ERK1/2, increased activation of p38 and JNK MAPKs it is likely that stimuli from pro-inflammatory cytokines, e.g., TNF-α, has also been found in IBD patients [109,110]. p38 and JNK MAPKs activate the NF-κB pathway and through PTEN or PI3K activity, NF-κB have many downstream targets in common, including IL-1, IL-6, IL-8, might regulate the CDX2 expression in IBD (Fig. 1). TNF-α, and members of the AP-1 family of transcription factors [111,112] (Fig. 1). However, p38 MAPK also activates transcription 6.2. Stimuli-dependent MAPK signaling regulates CDX2 activity factors like CREB and NF-κB [99]. Interestingly, ERK1/2 has been shown to phosphorylate CDX2 at Serine 60 to reduce its transcrip- As illustrated in Fig. 1, in addition to the above described NF-κB tional activity [15], and recently Krueger et al. [113] have confirmed pathway, extracellular stimuli from, e.g., cytokines or inflammatory these findings in cancer cells, while phosphorylation of CDX2 by p38 mediators also activate the MAPK pathway. The MAPK super-family is MAPK accompanies cell differentiation and enhances its transcrip- composed of three major sets of kinases in mammals: the extracellular tional activity [34]. However, the p38 MAPK mediated phosphoryla- signal-regulated kinases (ERKs), the c-Jun N-terminal kinase (JNK), tion of CDX2 is dependent on type of stimulus and signaling. In fact, and p38 MAPKs [100,101]. Activation of specific MAPKs involves the previously described regulation of COX-2 by NF-κB activation is phosphorylation and activation of upstream kinases. MAPKs are p38 MAPK-dependent, as the p65 subunit of NF-κB is repressed when members of intracellular kinases that are involved in a wide range of p38 activity was inhibited [99]. cellular events associated with the inflammatory response, as well as The last subfamily of the above-described MAPKs is the JNK MAPK. cell proliferation and survival [101,102]. Consistent with their critical JNK was originally identified by its ability to specifically phosphorylate roles in various key cellular activities, the MAPK signaling pathways the transcription factor c-Jun on its N-terminal transactivation have been implicated in the pathogenesis of several human diseases domain. There are yet no evidence for a direct regulatory link between [103–105]. ERK1/2 are the most studied ERK MAPKs and works JNK and CDX2 activity. 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