br Material and Methods br Results br Discussion In

Material and Methods
Results
Discussion In the injured liver, the initial hepatocyte cell death stimulates subsequent inflammatory responses, leading to further liver injury and fibrosis. HSC activation and fibrogenesis are mediated by a complex cross-talk between damaged parenchymal and non-parenchymal cells, such as Kupffer cells, which activate HSCs through TGF-β production (Bataller and Brenner, 2005). In the CCl4- or BDL-induced liver injury model, the translocation of β-catenin and S100A4 expression were observed only in the non-parenchymal cells. In addition, PRI-724 did not affect the liver injuries examined histologically and biochemically. These results suggest that β-catenin is not involved in hepatocyte cell death. Moreover, the co-administration of PRI-724 had no effect on the number of F4/80-positive dpp-4 inhibitor in the liver of CCl4-treated mice (data not shown), and C-82 treatment suppressed the activation of quiescent HSCs on the plastic dishes, suggesting that the inhibitory effect of PRI-724 on fibrogenesis is due to its direct effect on HSCs. It has been reported that the levels of the β-catenin protein are elevated in fibroblasts during proliferation, that stabilized β-catenin-expressing mice show aggressive fibromatoses and hyperplastic cutaneous wounds (Cheon et al., 2002), and that Wnt signaling is required for TGF-β-mediated fibrosis to occur (Akhmetshina et al., 2012). In progenitor cells, the selective blocking of the CBP/β-catenin interaction by ICG-001 initiates a differentiation program (Lenz and Kahn, 2014). Thus, it is likely that C-82 shifts the balance from CBP/β-catenin to p300/β-catenin-mediated transcription, resulting in the induction of differentiation rather than proliferation, through which activation was inhibited in the quiescent HSCs. The death of activated HSCs has been reported to contribute to fibrosis resolution (Iredale et al., 1998; Wright et al., 2001; Issa et al., 2001), and the inhibition of Wnt signaling increases apoptosis in cultured HSCs (Cheng et al., 2008). In the activated HSCs, C-82-induced cell death is observed seven days after the beginning of the treatment. On the other hand, C-82 did not induce cell death in the quiescent HSCs for at least 15days (data not shown), and the removal of C-82 started its activation. These results suggest that CBP/β-catenin inhibition induces cell death only in activated HSCs, but not in quiescent cells. During the activation, CBP/β-catenin-mediated transcription, such as that of survivin is increased and these transcripts may be required for the survival of activated HSCs. The contribution of macrophages to fibrosis resolution has been reported (Duffield et al., 2005; Friedman, 2005). MMPs resolve the ECM resulting in fibrosis resolution (Iimuro and Brenner, 2008), and macrophages have been reported to produce MMPs (Huang et al., 2012; Song et al., 2000; Shapiro et al., 1993). The cell count of macrophages in the liver during fibrosis resolution was comparable in PRI-724-treated and untreated mice, suggesting that the reagent affects the property, but not the number, of cells. In prostate cancer PC3 cells, p300 knockdown by siRNA leads to decreased MMP-9 activity, accompanied by the decreased mRNA expression of MMP-9 and MMP-2 (Santer et al., 2011), suggesting that p300/β-catenin is involved in MMP expression. Thus, PRI-724 may shift the balance to p300/β-catenin transcription resulting in MMP expression. The expression of MMP-8, generally known as neutrophil collagenase, was also up-regulated by PRI-724. The neutrophil-dependent resolution of the repair of the cholestatic rat liver has been reported (Harty et al., 2008). However, Gr-1-positive cells, which indicate the presence of neutrophils, rapidly disappeared during fibrosis resolution. Besides neutrophils, MMP-8 expression is found in other inflammatory cells such as monocytes and macrophages (Prikk et al., 2002), plasma cells (Wahlgren et al., 2001), and T-cells (Kim et al., 2001), suggesting that neutrophils may play a minor role in the acceleration of fibrosis resolution by PRI-724.