Figure 6. The claudin-15 gene is a direct target of Hnf4a. (A) EMSA of 4 Hnf4a binding sites found in the 1 kb mouse claudin-15 promoter performed with 293T cells transfected with pBabe Hnf4a1 vector and 293T nuclear extracts as negative controls. Shifts (white arrow head) are compared to previously published APOC3 and mutated APOC3 (APOC3m) for control binding site. NS is non- specific binding (grey arrow head). Supershifts (black arrow head) were done with Hnf4a antibody (sc-6556). (B) ChIP assays of the 4 putative sites performed on epithelial cells from adult mouse colon. (C) qRT-PCR of claudin-15 on IEC6 cells infected with pBabe Hnf4a as compared to control vector pBabe (n = 4). (D) Western blot of 3 stable T84 cell populations expressing mouse claudin-15. (E) Conductance measured for three consecutive days after the reach of confluence on three populations of T84 cells expressing mouse claudin-15 or control plenti eGFP vector. Represented as mean values6SEM, *** P#.001. doi:10.1371/journal.pone.0007609.g006 Hnf4a and Spontaneous IBD Discussion HNF4a transcriptional regulator is critical during the ontogeny and normal development of certain organs as well as in the pathogenesis of human disorders. Its roles have been ascertained for homeostasis of liver and pancreatic function and have been functionally linked to diabetes [25]. HNF4a is crucial to the polarization of cultured intestinal epithelial cells and to the initiation of their morphological and genetic differentiation in vitro [5]. Hnf4a is also essential to colonic epithelial cell differentiation during early mouse embryonic development [10]. However, small intestine deletion of Hnf4a following embryonic development results in minor defects on epithelial differentiation [11]. Here, our findings identify HNF4a as an important regulator of gut mucosal ion transport rather than mucosal permeability. In addition, a long term and chronic inflammatory consequence arising from the colonic epithelial loss of this transcriptional regulator was observed. The delayed manifestation of the clinical symptoms related to the colonic disease indicates that loss of Hnf4a involves complex mechanistically interactions that ultimately lead to the development of the disease. The pathogenesis of IBD is complex and involves susceptibility genes, the epithelial barrier function, innate and adaptive immunity and environmental factors including bacteria [26]. The only premature defect we were able to identify in the colon of Hnf4a mice was a decline in ion transport. We propose that rapid down-modulation of Hnf4a- dependent transcriptional targets such as Cldn15 can cause defects in epithelial ion transport ultimately leading to histopathological changes resembling those seen in patients with IBD. Hnf4a was recently reported to be required in colon epithelial cells to protect against dextran sodium sulphate (DSS), a commonly used model of acute intestinal injury that does not result in chronic inflammatory disease [12]. The Muc3 gene, a transcriptional target of Hnf4a [10], was reported to be down- modulated in young adult Hnf4a colon mice and was proposed to account for the Hnf4a protective effect. This specific transmembrane mucin is localized both in absorptive and goblet cells [27] and can reduce mucosal ulceration and apoptosis in experimental acute colitis [28]. Thus reduction in Muc3 could be partly involved in the long term spontaneous disease of Hnf4a colon mice. However and in contrast to Ahn et al. [12], we observed that goblet cell maturation was not significantly altered in young adult Hnf4a colon mutant mice and that the Muc2 gene transcript was only decreased in later stages, in association with the depletion of goblet cells in hyperplasic crypts. The results suggest the latter is not a primary event in the initiation of the disease but rather a consequence of progression of colonic inflammation. Colonic goblet cell maturation was reported to be altered in embryonic Hnf4a colon mutant mice with the use of another mouse conditional knockout system [10]. The precise window of time in which intestinal Hnf4a was altered differs in our study, as compared to what had been previously reported [10]. The Foxa3- Cre transgene utilized in the latter study efficiently removed Hnf4a at embryonic day 7.0, a period that precedes the onset of intestinal epithelial monolayer formation and cytodifferentiation. The Villin- Cre transgene has been reported to be active at a period following the onset of intestinal cytodifferentiation [11]. We conclude that the presence of Hnf4a no longer interferes with cell epithelial differentiation or goblet cell maturation after determination has occurred during early intestinal ontogeny. Increased mucosal permeability was also reported to occur in adult Hnf4a colon mice when administrated with DSS for several days [12]. Although the exact mechanism by which DSS induces acute colitis are still not well described, there are suggestions that PLoS ONE | www.plosone.org 8 October 2009 | Volume 4 | Issue 10 | e7609