Hnf4a and Spontaneous IBD Figure 3. Increased cytokine and chemokine secretion associated with the inflammatory state of Hnf4a colon mice. (A) Summarized results (means 6 SEM; at least 3 individuals aged of 12 months) of the semi-quantitative cytokine/chemokine array shown in Table S1. (B) ELISA performed on total protein lysates (means 6 SEM; n = 6 per group). (C) Labelling of granulocytic cells by MPO immunohistochemistry was negative in controls as opposed to (D) mutants. Scale bars, 100 mm. (E) Western blot of accumulated phospho-IkBa relative to total IkBa and relative quantification (mean 6 SEM), *P#.05. doi:10.1371/journal.pone.0007609.g003 expression of these classes of genes except for the down modulation of claudin-15 transcript and a probable indirect induction of claudin-4 and -8 gene transcripts (Table S3). Gene transcript quantification for each of these claudins confirmed an induction of claudin-4 and claudin-8 and a reduction of claudin-15 in Hnf4a colon mice (Figure 5A). These changes were observed as early as postnatal day 7, a period that preceded detection of crypt distortion and inflammation. Western blot analysis confirmed that these variations were of consequence on the respective protein level in the colon of adult mice (Figure 5B, C). Immunolocalization of both claudin-4 and claudin-8 showed a stronger signal on the colon epithelial surface of Hnf4a colon mice as compared to controls (Figure 5D). Although claudin-15 protein was localized throughout the colonic crypt in control mice, its distribution changed drastically in the Hnf4a colon mice with the upper half of the crypt being devoid of claudin-15 (Figure 5D and E). Because claudin-15 was reported to be crucial in maintaining ion transport but not mucosal permeability in the gut [23,24], mucosal barrier assays were next performed. Young adult mutant mice did not show significant alteration in colon mucosal permeability to 51Cr-EDTA as compared to controls (Figure 5F). This observation coincided with the fact that a limited subset of tight and adherens junction related genes were modulated in the Hnf4a colon mice (Table S3). However, baseline Isc was significantly decreased in the colon of young adult Hnf4a mice as compared to controls demonstrating that a decrease in active epithelial ion transport in absence of Hnf4a was taking place before the onset of the disease (Figure 5G). In addition to claudin-15 being crucial for ion transport function, more than 39 gene transcripts directly associated with ion transport function were predicted to be significantly reduced in diseased Hnf4a colon mice (Table S4). Claudin-15 Is a Direct Gene Target for Hnf4a and Increases Paracellular Conductance in Polarized T84 Colonic Epithelial Cells Since Claudin-15 (Cldn15) was one of the earliest modified transcripts in Hnf4a colon mice, we verified its status as a direct gene target for Hnf4a. Analysis of the first 1 kb of the mouse Cldn15 gene promoter with the TRANSFAC database predicted 4 putative Hnf4a binding sites (Figure 6A). The ability of Hnf4a to interact with these sites was monitored by EMSA. Nuclear extracts from 293T cells expressing exogenous Hnf4a led to a specific shift complex for two of the predicted sites (BS-1 and BS-4) in a comparable manner to a previously validated Hnf4a site within the Apoc3 gene promoter [10] (Figure 6A). This shift complex was specific for Hnf4a interaction as demonstrated with the formation of a supershift complex when Hnf4a antibodies were included in the binding reaction but not when substituted with control antibodies (Figure 6A). ChIP assays were next performed on PLoS ONE | www.plosone.org 5 October 2009 | Volume 4 | Issue 10 | e7609