001).

Interestingly, greater hepatocyte proliferation in rAd.A20 versus rAd.βgal livers occurred despite significantly higher basal hepatic IL-6 levels in the latter (Fig. S5; n = 4-5; P < 0.001). Higher IL-6 in rAd.βgal-treated livers is caused by the inflammatory response to rAd. that is contained in rAd.A20-treated livers by the anti-inflammatory function of this protein. We evaluated SOCS3 expression in livers of A20 HT and WT mice before and 24 hours following EH. A20 KO mice die within 3-6 weeks of age, and hence cannot be used for these experiments.21 HT livers showed a tendency towards higher SOCS3 mRNA and protein (IHC) (Fig PD0325901 molecular weight 6A; n = 5-6) and lower miR203 levels than WT, 24 hours after EH (Fig. 6B; n = 5-6). Increased SOCS3 levels in HT livers associated with impaired hepatocyte proliferation and delayed LR, as evidenced by lower number of Ki67/HNF4α-positive hepatocytes in HT versus WT livers,

36 hours after EH (Fig. 6C; n = 2 (WT) and 5 (HT); P < 0.001). This also correlated with lower P-STAT3 levels in HT versus WT livers 4 hours following EH (Fig. 6D; n = 2), and with inadequate up-regulation of CCND1 (P < 0.05) and CCNA (P < 0.05) mRNA, 24 hours after EH (Fig. 6E,F; n = 5-6). IL-6/STAT3 signaling also triggers an acute inflammatory response in hepatocytes that, DNA Damage inhibitor if uncontrolled, causes liver damage and counterproductively stuns cell cycle progression.9 Accordingly, we measured mRNA levels of STAT3-dependent liver-derived acute phase response proteins serum amyloid A 1 (SAA1) and fibrinogen (FGG) in A20-overexpressing and knockdown livers, at baseline and following EH. Overexpression of A20 significantly decreased EH-induced up-regulation of FGG (Fig. 7A,B; n = 6; P < 0.01), and reduced baseline and EH-induced Cyclic nucleotide phosphodiesterase up-regulation of SAA1. Conversely, A20 HT livers expressed higher levels of SAA1 (Fig 7C; n = 5-6; P < 0.01) and FGG (Fig. 7D; n = 6) following EH. These findings suggest that A20 does not aggravate and may even moderately decrease IL-6-induced pro-inflammatory signals. A20, a newly identified player in LR, is induced in mice and humans as part of the hepatocyte's

regenerative response following hepatectomy.15, 20 Overexpression of A20 dramatically improves LR and survival in mouse models of extended (78%) and lethal (87%) hepatectomy in part through decreasing the expression of CDKI, p21.15 In contrast, A20 knockdown significantly impairs and delays LR (Studer et al., in prep.). We initiated this work to determine which A20 domain supports this function in hepatocytes. Our results showed that both Nter and 7Zn mutants independently promote hepatocyte proliferation, but neither recapitulates A20′s effect on p21. Akin to A20′s NF-κB inhibitory function, both domains were required to decrease p21. Inhibition of NF-κB by A20 has been ascribed to its sequential N-terminus-mediated deubiquitination of signaling mediators such as RIP and NEMO, followed by C-terminus-mediated degradative polyubiquitination of the same substrates.