8A). This could be related to the cell cycle alterations observed upon p38α down-regulation. The mitotic delay could be reflected in an increase in polyploid nuclei (nonmitotic nuclei division) or binucleated cells (nonmitotic cytoplasm division). By identifying nuclei with 4,6-diamidino-2-phenylindole (DAPI) and cellular membranes with anti-β-catenin immunostaining, we found that p38α KO mice exhibited a higher rate of binucleated hepatocytes than WT mice both before and after Selleck AZD1152 HQPA BDL, whereas no differences were observed between BDL and sham groups in p38α KO animals (Fig. 8). In contrast, the percentage of binucleated

cells in WT sham livers was markedly reduced when they start proliferating after induction of cholestasis.

The two major groups of proteins that are regulated by p38 MAPK-mediated phosphorylation are protein kinases, such as MK2, and transcription factors, such as p53.1 p38α may negatively regulate AKT activity independently of PI3K, by regulating its interaction with PP2A or through the activation of MK2 (Fig. 2A). Indeed, MK2 mediates HSP27-dependent selleck activation of AKT by way of phosphorylation on Ser473.13 Accordingly, phosphorylation of MK2 on Thr334 and of AKT on Ser473 were markedly reduced in liver of p38α-deficient mice upon chronic cholestasis (Figs. 2B, 3A). Activation of AKT triggers a key antiapoptotic Alanine-glyoxylate transaminase signaling pathway in the liver. However, in our model of chronic cholestasis the lack of AKT activation did not lead to increased apoptosis (Supporting Fig. S5).

PDK1 and AKT are required for normal cell growth and liver regeneration after partial hepatectomy.17 Taking into account the absence of significant PDK1-mediated AKT phosphorylation on Thr308 upon chronic cholestasis (Fig. 3A), it seems that p38α/MK2-dependent AKT activation is essential for liver regeneration and hepatomegaly in this chronic disorder. In vitro in rat hepatoma cells, AKT activation increased cell size through mTOR-dependent and mTOR-independent pathways and the latter also involved inhibition of protein degradation.18 Accordingly, p38α deficiency may reduce hepatocyte growth during chronic cholestasis through down-regulation of both AKT and mTOR (Fig. 3A). Similar to yeast, in mammals two distinct protein kinase mTOR complexes have been characterized. mTORC1 is rapamycin-sensitive and controls protein synthesis, whereas mTORC2 is rapamycin-insensitive and controls the actin cytoskeleton.19 Hence, down-regulation of mTOR may contribute to reduce albumin levels in liver of p38α-deficient mice. The Akt/mTOR pathway may lead to activation of the p70 S6 kinase/S6 pathway. Our findings suggest that blockade of this pathway seems to be involved in the lack of hepatocyte proliferation and growth that occurs upon p38α deficiency during chronic cholestasis.