The transcription factor c-Jun protects against sustained hepatic endoplasmic reticulum stress thereby promoting hepatocyte survival.

UNLABELLED: Endoplasmic reticulum (ER) stress due to accumulation of hepatoviral or misfolded proteins is increasingly recognized as an important step in the pathogenesis of inflammatory, toxic, and metabolic liver diseases. ER stress results in the activation of several intracellular signaling pathways including Jun N-terminal kinase (JNK). The AP-1 (activating protein 1) transcription factor c-Jun is a prototypic JNK target and important regulator of hepatocyte survival, proliferation, and liver tumorigenesis. Because the functions of c-Jun during the ER stress response are poorly understood, we addressed this issue in primary hepatocytes and livers of hepatocyte-specific c-Jun knockout mice. ER stress was induced pharmacologically in vitro and in vivo and resulted in a rapid and robust induction of c-Jun protein expression. Interestingly, ER-stressed hepatocytes lacking c-Jun displayed massive cytoplasmic vacuolization due to ER distension. This phenotype correlated with exacerbated and sustained activation of canonical ER stress signaling pathways. Moreover, sustained ER stress in hepatocytes lacking c-Jun resulted in increased cell damage and apoptosis. ER stress is also a strong inducer of macroautophagy, a cell-protective mechanism of self-degradation of cytoplasmic components and organelles. Interestingly, autophagosome numbers in response to ER stress were reduced in hepatocytes lacking c-Jun. To further validate these findings, macroautophagy was inhibited chemically in ER-stressed wildtype hepatocytes, which resulted in cytoplasmic vacuolization and increased cell damage closely resembling the phenotypes observed in c-Jun-deficient cells. CONCLUSION: Our findings indicate that c-Jun protects hepatocytes against excessive activation of the ER stress response and subsequent cell death and provide evidence that c-Jun functionally links ER stress responses and macroautophagy.

Caspase-3 feeds back on caspase-8, Bid and XIAP in type I Fas signaling in primary mouse hepatocytes.

The TNF-R1 like receptor Fas is highly expressed on the plasma membrane of hepatocytes and plays an essential role in liver homeostasis. We recently showed that in collagen-cultured primary mouse hepatocytes, Fas stimulation triggers apoptosis via the so-called type I extrinsic signaling pathway. Central to this pathway is the direct caspase-8-mediated cleavage and activation of caspase-3 as compared to the type II pathway which first requires caspase-8-mediated Bid cleavage to trigger mitochondrial cytochrome c release for caspase-3 activation. Mathematical modeling can be used to understand complex signaling systems such as crosstalks and feedback or feedforward loops. A previously published model predicted a positive feedback loop between active caspases-3 and -8 in both type I and type II FasL signaling in lymphocytes and Hela cells, respectively. Here we experimentally tested this hypothesis in our hepatocytic type I Fas signaling pathway by using wild-type and XIAP-deficient primary hepatocytes and two recently characterized, selective caspase-3/-7 inhibitors (AB06 and AB13). Caspase-3/-7 activity assays and quantitative western blotting confirmed that fully processed, active p17 caspase-3 feeds back on caspase-8 by cleaving its partially processed p43 form into the fully processed p18 species. Our data do not discriminate if p18 positively or negatively influences FasL-induced apoptosis or is responsible for non-apoptotic aspects of FasL signaling. However, we found that caspase-3 also feeds back on Bid and degrades its own inhibitor XIAP, both events that may enhance caspase-3 activity and apoptosis. Thus, potent, selective caspase-3 inhibitors are useful tools to understand complex signaling circuitries in apoptosis.

Hepatocyte Ploidy Is a Diversity Factor for Liver Homeostasis.

Polyploidy, the existence of cells containing more than one pair of chromosomes, is a well-known feature of mammalian hepatocytes. Polyploid hepatocytes are found either as cells with a single polyploid nucleus or as multinucleated cells with diploid or even polyploid nuclei. In this study, we evaluate the degree of polyploidy in the murine liver by accounting both DNA content and number of nuclei per cell. We demonstrate that mouse hepatocytes with diploid nuclei have distinct metabolic characteristics compared to cells with polyploid nuclei. In addition to strong differential gene expression, comprising metabolic as well as signaling compounds, we found a strongly decreased insulin binding of nuclear polyploid cells. Our observations were associated with nuclear ploidy but not with total ploidy within a cell. We therefore suggest ploidy of the nuclei as an new diversity factor of hepatocytes and hypothesize that hepatocytes with polyploid nuclei may have distinct biological functions than mono-nuclear ones. This diversity is independent from the well-known heterogeneity related to the cells' position along the porto-central liver-axis.