However, the association in KO livers was dramatically SCH772984 reduced in KO livers, suggesting the presence of a NF-κB/β-catenin complex in hepatocytes and nonparenchymal cells. Next, to investigate whether the p65/β-catenin complex undergoes changes and thus modulates NF-κB activation, we harvested WT livers at baseline and at 1, 2, and 3 hours after treatment with LPS only. Disruption of β-catenin and p65 association was observed as early as 1 hour after LPS (Fig. 6B) along with concomitant p65 nuclear translocation

(Fig. 6C). Although p65 phosphorylation began to increase simultaneously, it peaked at 2 hours after LPS treatment, as shown by the appearance of ser536-phospho-p65 in the nuclei (Fig. 6C). IHC confirmed the presence of active p65 in approximately 50% of hepatocytes (Fig. 6C), consistent with previous reports.24, 25 We hypothesized that lack of β-catenin in hepatocytes may be lowering the threshold of p65 activation after apoptotic stimuli. To test this hypothesis, we treated both KO and WT with LPS to compare kinetics of p65 nuclear translocation and activation. While some animal-to-animal variation was evident, KO livers showed a greater increase

in nuclear p65 at 1 hour after LPS treatment compared with WT livers (Fig. 6E). Additionally, at 1 hour after LPS, KO but not WT livers showed active ser536-phospho-p65 Selleckchem GSK2126458 via both IHC and WB (Fig. 6D,E). These results were also confirmed by calorimetric measurement of NF-κB transcriptional activity

after 1 hour of LPS, in which KO shows a significant increase over WT (Fig. 6F). Thus, loss of β-catenin lowers the threshold to prime the KO livers for early and robust p65 nuclear translocation and activation in response to TNF-α. To directly address how p65-β-catenin interactions may influence NF-κB activity, we first transfected HepG2 cells, which harbor a monoallelic exon-3-deleted constitutively active β-catenin,26 with control or β-catenin small interfering RNA (siRNA) concomitantly with either TOPflash (a luciferase reporter that measures β-catenin/Tcf-dependent transcriptional activation) or p65 luciferase reporter plasmid. Although RNA inhibition caused a reduction in full-length β-catenin at 48 hours, as shown by WB and TOPflash others reporter assay, there was no significant change in p65 activity (Fig. 7A). While this was unexpected, further analysis of p65/β-catenin association in HepG2 cells by p65 immunoprecipitation revealed an association between p65 and the predominant truncated as well as the full-length form of β-catenin (Fig. 7B), suggesting that despite knockdown of the WT form, the presence of the truncated form was sufficient to bind and disallow p65 activation. However, when Hep3B cells that contain full-length, nonmutated β-catenin were transfected with siRNA and reporter plasmids, β-catenin was effectively suppressed, leading to a significant decrease in TOPflash reporter activity and an increase in p65 reporter activity (Fig. 7C).