BIR repeat-containing ubiquitin conjugating enzyme (BRUCE) regulation of β-catenin signaling in the progression of drug-induced hepatic fibrosis and carcinogenesis

Figure 6 BRUCE-dependent regulation of β-catenin links to protein kinase activity. A: Whole cell lysates of HepG2 cells transfected with either an siCtrl or siBRUCE were blotted for BRUCE, phospho- and total-β-catenin, as well as a tubulin control; B: Lysates described in (A) were blotted for phospho-protein kinase A (PKA) substrates to measure PKA activity, as well as a glyceraldehyde-3-phosphate dehydrogenase control; C: Western blot analysis of mouse liver tissue lysates from control and liver-specific BRUCE knockout (LKO) exposed to diethylnitrosamine (DEN) for phospho-PKA substrates showing an increase in PKA activity in LKO livers at the time of tumor onset (8 mo); D: Immunofluorescence staining showing colocalization of BRUCE (red) and PKA (green) in endosomes (arrows) in normal human THLE2 hepatocyte line with cell nucleus counterstained with DAPI. The cellular areas outlined in dashed squares are enlarged and shown below; scale bar 20 μm; E: A working model showing a new BRUCE-PKA-β-catenin signaling axis involved in the regulation of fibrosis and HCC. BRUCE regulates β-catenin activation by inhibiting PKA-dependent phosphorylation-activation of β-catenin for hepatic proliferation and carcinogenesis. Mechanistically, BRUCE interacts with PKA in the hepatocyte cytoplasm to restrain PKA activity. When this interaction is disrupted by KO of BRUCE in the mouse liver, or by KD of BRUCE expression in liver cancer cell line, the repression of PKA is derepressed and PKA-dependent phosphorylation-activation of β-catenin at Ser-675 occurs which results in hepatic proliferation. Meanwhile hepatocytes undergo apoptosis induced by DEN-DNA damage and these apoptotic hepatocytes release damage associated molecular patterns to activate hepatic stellate cells. The BRUCE-PKA-β-catenin signaling axis, together with DEN induced DNA damage, hepatic cell death, and oxidative stress, result in an early onset of fibrosis and accelerated HCC. DEN: Diethylnitrosamine; LKO: Liver-specific knockout; BRUCE: BIR repeat-containing ubiquitin conjugating enzyme; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; PKA: Protein kinase A; DAPI: 4',6-diamidino-2-phenylindole; HCC: Hepatocellular carcinoma; DAMPs: Damage associated molecular patterns; CTRL: Control.