p38α Deficiency Reduces Hepatocyte Growth Through AKT/mTOR Down-regulation.
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 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 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. Endoplasmic reticulum stress could also contribute to reduced albumin synthesis but this idea was discarded as GADD 153 and eIF2α phosphorylation did not increase in liver-specific p38α-deficient mice upon cholestasis (Supporting Fig. S7).
p38α may inactivate GSK3β by direct phosphorylation of Ser389 or indirectly through phosphorylation of Thr9 by Akt, leading to β-catenin accumulation.20 Thus, p38 modulates canonical Wnt-β-catenin signaling, which is critical for normal cell proliferation and homeostasis.21
Inactivation of GSK3β produces embryonic lethality caused by severe liver degeneration associated with hypersensitivity to TNF-α and reduced NF-κB function.22 Inhibition of GSK3β may sensitize rat hepatocytes to apoptosis by reducing p65 phosphorylation and down-regulating NF-κB transactivation.23 In p38α-deficient livers, activation of GSK3β due to reduced phosphorylation (Fig. 3B) does not seem to be associated with changes in apoptosis or p65 phosphorylation upon BDL (Fig. S4).
p38α Deficiency Enhances the Long-Term Inflammatory Response in the Liver.
The p38α pathway is also involved in the up-regulation of inflammatory cytokines. p38 may positively regulate NF-κB activity by different mechanisms, including chromatin remodeling through Ser10 phosphorylation of histone H3 at NF-κB-dependent promoters or by impinging on IκB kinase (IKK) or the p65 subunit.20 However, in chronic cholestasis p38α deficiency did not significantly affect NF-κB activation (Supporting Fig. S4) or the expression of TNF-α and interleukin-6 (see Fig. 4). Nevertheless, RANTES and receptor 1 of TNF-α were up-regulated in the liver of p38α-deficient mice under basal conditions and remained high during the first 12 days after cholestasis (Fig. 4B). RANTES is one of the major adjacent cysteines motif (CC) chemokines that is produced by T-lymphocytes, monocytes, endothelial cells, and fibroblasts. It is worth noting that expression of antiinflammatory IL-10 was markedly up-regulated at 12 days after cholestasis induction only in p38α KO mice, which should provide protection restraining the inflammatory response, but this protection was lost in the long term (i.e., at 28 days) leading to up-regulation of Icam-1 and chemokine (C-C motif) ligand 2 (Ccl2) (Fig. 4B).
Although previous reports have associated p38α with the regulation of apoptosis and fibrogenesis, liver-specific p38α-deficient mice did not show a higher degree of apoptosis or fibrosis upon chronic cholestasis compared with WT mice (see Fig. 4 and Supporting Fig. S5). Hence, neither apoptosis nor fibrosis would contribute to the increased mortality of these animals.
p38α Deficiency Induces Mitosis Blockade and Cytokinesis Failure in Hepatocytes.
p38α controls the differentiation and proliferation of many cell types, including hepatocytes.4, 24, 25 p38α may negatively regulate cell cycle progression at the G1/S and the G2/M transitions triggering cell cycle arrest by down-regulation of cyclins, up-regulation of cyclin-dependent kinase inhibitors, and by inducing p53 phosphorylation and the up-regulation of p16.1, 24 p38α-deficient myogenic cells exhibited delayed cell-cycle exit and continued proliferation under conditions that normally induce cell-cycle withdrawal and differentiation.26 p38α controls myoblast proliferation by antagonizing the proliferation-promoting function of JNK, and this effect is at least in part mediated by up-regulation of the phosphatase MAPK phosphase-1 (MKP-1).26 Hence, p38α and JNK MAPKs may exert antagonistic effects on cell proliferation and survival.1 However, phospho-JNK did not increase upon cholestasis in the liver of p38α-deficient mice (Fig. S8) and therefore the JNK pathway would not contribute to the reduced cell proliferation in our chronic model.
PCNA is expressed in replicating cells during S phase, thus allowing detection of dividing cells. The number of PCNA-expressing cells was higher in skeletal muscle from mice deficient in p38α than in WTs.26 Continuous myoblast proliferation and reduced myofiber growth were attributed to the persistence of cyclin D1.26 Indeed, down-regulation of cyclin D1 by p38α has been reported in different cell types.26 Accordingly, inhibition of p38α in vivo was sufficient to stimulate hepatocyte cell cycle activity, whereas p38α activation resulted in hepatocyte growth arrest and decreased cyclin D1 in cultured fetal rat hepatocytes.4 Accordingly, cyclin D1 and cyclin B1 were up-regulated in liver of p38α-deficient mice upon chronic cholestasis (see Fig. 8). However, PCNA was surprisingly down-regulated at 12 days after cholestasis induction and the mitotic index was extremely high in long-term cholestasis in p38α-deficient mice (i.e., at 28 days) (see Fig. 7). Hence, unexpectedly p38α deficiency blockades progression of mitosis towards the S phase in hepatocytes during the initial course of chronic cholestasis. The increased death rate that occurs in liver-specific p38α KO mice could be due to the blockade of hepatocyte growth with impaired protein synthesis and lack of proliferative adaptive response in the liver.
Cardiac-specific p38α-KO mice exhibited an increase in neonatal cardiomyocyte mitoses and inhibition of p38α in adult cardiomyocytes promotes karyokinesis and cytokinesis.25 However, liver-specific p38α-KO mice exhibit cytokinesis failure evidenced by enhanced binucleation rate (see Fig. 8). Moreover, as chronic cholestasis evolves, the binucleation rate decreases in WT animals, whereas it remains high in p38α-deficient mice.
Incomplete cytokinesis may be associated with developmental or pathological cell division programs leading to polyploid progenies.27, 28 AKT activity regulates cytoskeleton organization and its down-regulation might be involved in cytokinesis failure.29 Indeed, during postnatal development binucleated tetraploid cells arise in the liver due to AKT-mediated failure in cytokinesis.29 Down-regulation of mTOR might also contribute to the p38α-dependent AKT-mediated cytokinesis failure since complex mTORC2 also controls the actin cytoskeleton.19
AKT and GSK3β cooperate in spindle formation.29 AKT phosphorylates GSK3β decreasing its activity. By limiting the activity of GSK3β at the cell cortex and at the centrosomes, AKT allows for the selective stabilization of microtubules. GSK3β is crucial for the regulation of microtubule organization and dynamics, particularly for mitotic spindle organization.29 p38α deficiency alters the balance between AKT and GSK3β leading to AKT down-regulation and GSK3β activation (Fig. 3), which seems to impair normal cytokinesis completion.
MK2 also plays a significant role downstream of p38α in remodeling the actin cytoskeleton.30 Particularly, MK2 triggers phosphorylation of HSP27 inducing its release from F-actin.30 HSP27 protects against apoptosis and actin fragmentation, promoting resistance against cell death.31 We found an increase in HSP27 levels, which may be an adaptive response against liver injury, with significant changes in phosphorylation.
Mnk1 and Polo-like kinase 1 (Plk1), two potential downstream targets of p38α signaling, may contribute to cytokinesis failure in p38α-deficient liver. Inhibition of Mnk1, a kinase target for MAPK pathways, causes cytokinesis failure inducing the formation of multinucleated cells.32 In addition, MK2 directly phosphorylates Plk1, and down-regulation of p38α or MK2 induces mitotic defects that can be rescued by Plk1.33
In conclusion, the present work shows that liver-specific p38α deficiency leads to reduced hepatocyte size, blockade of mitosis, cytokinesis failure, and eventually shorter life span upon chronic cholestasis induced by BDL. These results highlight the key role of p38α in cell proliferation, in the development of hepatomegaly, and in survival during chronic inflammation such as biliary cirrhosis.