Hnf4a and Spontaneous IBD Figure 4. The apoptotic pathway is overly active and the proliferative dynamic is altered in regenerative epithelium. (A) Increased abundance of cleaved and active forms of caspase-3 in 12 month-old mutants (Open bar) relative to controls (Black bar). (B) Quantification of band intensities of cleaved and active caspase-3 combined (n = 4) normalized to b-actin (Black bars, controls; open bars, mutants). Represented as means 6 SEM, * P#.05. (C) E2f2 and AurkA modulations seen in microarray data were verified at various time points in mutants (Open bars) relative to controls (Black bars). Expression levels are shown as mean values (6 SEM) relative to controls for each time point (3–14 mice per group) and were normalized with the Tbp housekeeping gene. (D) Measurements (n = 4 per group) of crypt length. (E) Ki67 immunochemistry of 12 month-old control mice compared to a hyperplasic area seen in mutants of same age. * P#.05, ** P#.01 and *** P#.001. doi:10.1371/journal.pone.0007609.g004 >isolated mouse colonocytes. Immunoprecipitations of the chromatin with Hnf4a antibodies followed by PCR amplifications of different Cldn15 regions were performed. Hnf4a consistently pulled down the BS-1 and BS-4 containing genomic regions of Cldn15 in contrast to the BS-2 region, negative for Hnf4a interaction by EMSA and not immunoprecipitated under these conditions (Figure 6B). Forced expression of Hnf4a in cultured intestinal epithelial cells resulted in a significant increase of Cldn15 gene transcript (Figure 6C). Exogenous expression of Cldn15 in the T84 colon cell line was next achieved to monitor the function of Cldn15 on ion conductance in the context of colonic epithelial cells (Figure 6D). Controls and Cldn15 T84 cell monolayers were seeded onto semipermeable filters and conductance was monitored after each cell populations had reached confluence. A significant increase in ion conductance was maintained for several days in T84/Cldn15 as compared to T84 control cell populations (Figure 6E). These results indicated that Hnf4a was able to activate Cldn15 expression probably via direct transcriptional mechanisms and confirmed that Cldn15 was potent to increase ion conductance in the context of a colonic epithelial cell monolayer. Down-Modulation of HNF4A Correlates with Decreased Expression of CLDN15 in Experimental Colitis and IBD HNF4a was reported to be decreased during experimental colitis and IBD [12]. The expression status of CLDN15 in the disease was assessed by qRT-PCR in comparison to HNF4A. DSS- induced colitis led to a significant Cldn15 reduction in the colon similarly to Hnf4a levels (Figure 7A). The expression of both HNF4A (Figure 7B) and CLDN15 (Figure 7C) gene transcripts were significantly reduced in intestinal biopsies from IBD patients as compared to controls. These observations further supported a close relationship between Hnf4a and Cldn15 expression during intestinal inflammatory diseases. PLoS ONE | www.plosone.org 6 October 2009 | Volume 4 | Issue 10 | e7609