Hnf4a and Spontaneous IBD Figure 7. HNF4A expression is reduced in IBD patient samples in association with Claudin-15. (A) qRT-PCR of Hnf4a and claudin-15 expression following 10 days of 2.5% DSS mild treatment in the distal colon of CD1 mice relative to water treated mice. HNF4A expression was retained at 22% (B) and claudin-15 was retained at 37% (C) in ulcerative colitis (UC) patient samples (n = 21) as compared to healthy patient samples (CTRL) (n = 6). Represented as mean values 6 SEM, * P#.05, ** P#.01, *** P#.001. doi:10.1371/journal.pone.0007609.g007 epithelial cells might become sensitive to this chemical to result in a loss of epithelial integrity. We did not observe any change in paracellular permeability as measured by 51Cr-EDTA flux in adult Hnf4a colon mice suggesting that spontaneous inflammatory disorder in this mouse model was not due to initial defects in tight junction dysfunction. In support of this, it was recently demonstrated that intestinal barrier loss in transgenic mice can accelerate the onset and severity of experimental colitis but is insufficient to lead in spontaneous inflammatory disease [29]. Our findings support the important conclusion that the solely loss of intestinal Hnf4a can initiate, by itself, epithelial defects that are resulting in spontaneous inflammatory disease. A detailed gene profiling of Hnf4a colon mice identified important classes of molecules with known functions in cell death, ion transport and cell cycle regulation. Our data indicate that three claudin family members (claudin-4, -8 and -15), crucial components of epithelial tight junctions, are deregulated long before the histopathological manifestations of the disease. Hnf4a colon mice displayed a significant increase in the expression of claudin-4 and claudin-8 without any suggestion of relocalization in the mucosa, as studied by immunofluorescence. As expected, these modulations did not have an impact on mucosal permeability. Cldn15 was recently demonstrated to function as a paracellular ion transport without interfering in tight junction barrier function [23]. This function was confirmed in the mouse intestine by the generation of a claudin-15 knockout model that led to intestinal epithelial cell alterations, including enhanced proliferation and decreased electrical conductance without noticeable impact on mucosal permeability [24]. These findings are consistent with reduced claudin-15 expression in the upper half of the colonic mucosa in relation to the reduction of ion transport but not permeability that we observed in the Hnf4a colon mice. There is growing evidence that specific defects in ion transport can lead to altered water movements and contribute to symptom generation and diarhea in IBD and spontaneous colitis in mice [30,31]. Such defects can result in the alteration of acidic pH of the intestinal surface and favour bacterial adhesion to further facilitate microbial-epithelial interactions and translocation con- tributing to the inflammatory change. Functional variants of SLC22A4 and SLC22A5 ion transporter genes that alter their transcription and transporter functions are associated with the Crohn’s disease IBD5 locus [32]. Our observations are suggestive of a cascade effect of several ion transporters being altered in the Hnf4a colon mice and to become important contributors to the long term onset of the disease in this context. The detection of increased cleaved effector forms of caspase-3 as well as sporadic crypt hyperplasic regions in the colonic mucosa of Hnf4a mutant mice are consistent with the alterations predicted by the specific gene networks identified from the gene profiling analysis. Evidence is accumulating indicating that a defect in apoptosis is involved in the pathogenesis of IBD [33,34]. In addition, E2F family members are recognized as important modulators of both cell division and apoptosis within developing tumors [35]. The long term observation of rare polyp initiation in some of the Hnf4a mutant mice could suggest that cumulated defects associated with colonic crypt distortion could eventually lead to tumor initiation under specific circumstances. Hnf4a down-regulates cell proliferation through the transcriptional activation of the cyclin-dependent-kinase inhibitor p21/CIP1/ WAF1 [36]. However, the proliferation index of colonic epithelial cells in young adult mice was comparable between control and Hnf4a mutant mice (data not shown) and was consistent to what has been reported in the colonic embryonic situation [10]. Thus, the specific hyperplasic regions that develop in the absence of Hnf4a are probably the consequence of early molecular defects that influence the establishment of this phenotype. In accordance with this reasoning, IBD patients manifest an increased risk of developing colon cancer because persistent inflammation will lead to increased cellular turnover with selection pressures may result in the emergence of cells that are at high risk for malignant transformation [37]. We conclude that Hnf4a integrity is crucial to maintaining the fundamental properties of the colonic mucosa in protecting the host against luminal components that are likely to be disruptive, in the long term, to mucosal, immune homeostasis, potentially leading to episodes of colonic inflammation as observed in human IBD. HNF4A transcriptional regulator along with ion transport related target genes that include CLDN15 represent promising candidates to dissect at the molecular level the delayed manifestation of IBD and might help to better define the predicting risk of IBD susceptibility among human populations. Supporting Information Figure S1 Classification of modified genes in Hnf4a colon mice by biological function PLoS ONE | www.plosone.org 9 October 2009 | Volume 4 | Issue 10 | e7609