Methods for simultaneous and quantitative isolation of mitochondrial DNA, nuclear DNA and RNA from mammalian cells.

The aim of this study was to assess two protocols for their capacities to simultaneously isolate RNA, mtDNA and ncDNA from mammalian cells. We compared the Invitrogen TRIzol-based method and Qiagen DNeasy columns, using the HepG2 cell line and human primary glioblastoma stem cells. Both methods allowed the isolation of all three types of nucleic acids and provided similar yields in mtDNA. However, the yield in ncDNA was more than tenfold higher on columns, as observed for both cell types. Conversely, the TRIzol method proved more reproducible and was the method of choice for isolating RNA from glioblastoma cells, as demonstrated for the housekeeping genes RPLP0 and RPS9.

Epidermal Growth Factor Represses Constitutive Androstane Receptor Expression in Primary Human Hepatocytes and Favors Regulation by Pregnane X Receptor.

Growth factors have key roles in liver physiology and pathology, particularly by promoting cell proliferation and growth. Recently, it has been shown that in mouse hepatocytes, epidermal growth factor receptor (EGFR) plays a crucial role in the activation of the xenosensor constitutive androstane receptor (CAR) by the antiepileptic drug phenobarbital. Due to the species selectivity of CAR signaling, here we investigated epidermal growth factor (EGF) role in CAR signaling in primary human hepatocytes. Primary human hepatocytes were incubated with CITCO, a human CAR agonist, or with phenobarbital, an indirect CAR activator, in the presence or absence of EGF. CAR-dependent gene expression modulation and PXR involvement in these responses were assessed upon siRNA-based silencing of the genes that encode CAR and PXR. EGF significantly reduced CAR expression and prevented gene induction by CITCO and, to a lower extent, by phenobarbital. In the absence of EGF, phenobarbital and CITCO modulated the expression of 144 and 111 genes, respectively, in primary human hepatocytes. Among these genes, only 15 were regulated by CITCO and one by phenobarbital in a CAR-dependent manner. Conversely, in the presence of EGF, CITCO and phenobarbital modulated gene expression only in a CAR-independent and PXR-dependent manner. Overall, our findings suggest that in primary human hepatocytes, EGF suppresses specifically CAR signaling mainly through transcriptional regulation and drives the xenobiotic response toward a pregnane X receptor (PXR)-mediated mechanism.

Mesenchymal stem cells seeded on a human amniotic membrane improve liver regeneration and mouse survival after extended hepatectomy.

Liver failure remains the leading cause of post-operative mortality after hepatectomy. This study investigated the effect of treatment with allogenic mesenchymal stem cells (MSCs) on survival and liver regeneration 48 hr and 7 days after 80% hepatectomy in C57Bl/6 mice. To optimize their biodistribution, MSCs were grown on acellular human amniotic membranes (HAM) and applied as a patch on the remnant liver. This approach was compared with MSC infusion and HAM patch alone. Hepatectomized mice without any treatment were used as control group. Survival rate was calculated and biological and histopathological parameters were analysed to monitor liver function and regeneration. MSCs grown on HAM retained their ability to proliferate, to differentiate into osteoblasts and adipocytes and to respond to pro-inflammatory stimuli. Extended hepatectomy (80%) led to liver failure that resulted in death within 72 hr in 76% of mice. MSC infusion showed an early but transitory positive effect on survival. MSC/HAM patches stimulated regeneration and significantly improved survival rate (54% vs. 24% in the control group at 7 days). They also decreased the severity of hepatectomy-induced steatosis, suggesting a modulation of lipid metabolism in hepatocytes. MSCs were still present on HAM at Days 2 and 7 posthepatectomy. In conclusion, engineered tissue constructs that combine MSCs and HAM improve survival and liver regeneration after 80% hepatectomy in mice. These encouraging results pave the way to potential clinical application.

Septin 9 induces lipid droplets growth by a phosphatidylinositol-5-phosphate and microtubule-dependent mechanism hijacked by HCV.

The accumulation of lipid droplets (LD) is frequently observed in hepatitis C virus (HCV) infection and represents an important risk factor for the development of liver steatosis and cirrhosis. The mechanisms of LD biogenesis and growth remain open questions. Here, transcriptome analysis reveals a significant upregulation of septin 9 in HCV-induced cirrhosis compared with the normal liver. HCV infection increases septin 9 expression and induces its assembly into filaments. Septin 9 regulates LD growth and perinuclear accumulation in a manner dependent on dynamic microtubules. The effects of septin 9 on LDs are also dependent on binding to PtdIns5P, which, in turn, controls the formation of septin 9 filaments and its interaction with microtubules. This previously undescribed cooperation between PtdIns5P and septin 9 regulates oleate-induced accumulation of LDs. Overall, our data offer a novel route for LD growth through the involvement of a septin 9/PtdIns5P signalling pathway.

Analysis of Glycogen Synthase Kinase Inhibitors That Regulate Cytochrome P450 Expression in Primary Human Hepatocytes by Activation of ß-Catenin, Aryl Hydrocarbon Receptor and Pregnane X Receptor Signaling.

Cytochrome P450 (CYP) expression and activity are not homogeneous in the liver lobules. Indeed, CYPs are mainly expressed and induced in centrilobular hepatocytes. The wingless-type MMTV integration site family (WNT)/ß-catenin pathway was identified as a major regulator of this zonal organization. We have recently demonstrated that in primary human hepatocytes (PHHs), the expression of CYP2E1, CYP1A2, and aryl hydrocarbon receptor (AhR), but not of CYP3A4, is regulated by the WNT/ß-catenin pathway in response to WNT3a, its canonical activator. Here, we investigated whether glycogen synthase kinase 3ß (GSK3ß) inhibitors, which mimic the action of WNT molecules, could be used in PHHs to activate the ß-catenin pathway to study CYP expression. We assessed the activity of 6BIO (6-bromoindirubin-3'-oxime), CHIR99021 (6-((2-((4-(2,4-dichlorophenyl)-5-(4methyl-1H-imidazol-2-yl)pyrimidin-2-yl)amino)ethyl)amino) nicotinonitrile), and GSK3iXV (Pyridocarbazolo-cyclopentadienyl Ruthenium complex GSK3 inhibitor XV) that belong to structurally different families of GSK3ß inhibitors. Using small interfering RNAs, reporter gene assays, and molecular docking predictions, we demonstrated that GSK3ß inhibitors can activate the WNT/ß-catenin pathway in PHHs to regulate CYP2E1 expression. We also found that 6BIO and GSK3iXV are AhR full agonists that participate, through AhR signaling, to CYP1A2 induction. Conversely, CHIR99021 is an AhR partial agonist, and a pregnane X receptor ligand and partial agonist, thus regulating CYP1A2 and CYP3A4 gene expression in a ß-catenin-independent manner. In conclusion, GSK3ß inhibitors can activate the WNT/ß-catenin pathway in PHHs. Nevertheless, their role in CYP regulation should be analyzed with caution as these molecules can interact with xenosensors.

The WNT/ß-catenin pathway is a transcriptional regulator of CYP2E1, CYP1A2, and aryl hydrocarbon receptor gene expression in primary human hepatocytes.

The wingless-type MMTV integration site family (WNT)/ß-catenin/adenomatous polyposis coli (CTNNB1/APC) pathway has been identified as a regulator of drug-metabolizing enzymes in the rodent liver. Conversely, little is known about the role of this pathway in drug metabolism regulation in human liver. Primary human hepatocytes (PHHs), which are the most physiologically relevant culture system to study drug metabolism in vitro, were used to investigate this issue. This study assessed the link between cytochrome P450 expression and WNT/ß-catenin pathway activity in PHHs by modulating its activity with recombinant mouse Wnt3a (the canonical activator), inhibitors of glycogen synthase kinase 3ß, and small-interfering RNA to invalidate CTNNB1 or its repressor APC, used separately or in combination. We found that the WNT/ß-catenin pathway can be activated in PHHs, as assessed by universal ß-catenin target gene expression, leucine-rich repeat containing G protein-coupled receptor 5. Moreover, WNT/ß-catenin pathway activation induces the expression of CYP2E1, CYP1A2, and aryl hydrocarbon receptor, but not of CYP3A4, hepatocyte nuclear factor-4α, or pregnane X receptor (PXR) in PHHs. Specifically, we show for the first time that CYP2E1 is transcriptionally regulated by the WNT/ß-catenin pathway. Moreover, CYP2E1 induction was accompanied by an increase in its metabolic activity, as indicated by the increased production of 6-OH-chlorzoxazone and by glutathione depletion after incubation with high doses of acetaminophen. In conclusion, the WNT/ß-catenin pathway is functional in PHHs, and its induction in PHHs represents a powerful tool to evaluate the hepatotoxicity of drugs that are metabolized by CYP2E1.

Use of human hepatocytes to investigate HCV infection.

Investigations on the biology of hepatitis C virus (HCV) have been hampered by the lack of small animal models. Efforts have therefore been directed to designing practical and robust cellular models of human origin able to support HCV replication and production in a reproducible and physiologically pertinent manner. Different systems have been constructed based on hepatoma or other cell lines, sub-genomic and genomic replicons, productive replicons, and immortalized hepatocytes. Although these models are practical for high-throughput screenings, they present several drawbacks related to the nature of the virions and the fact that the cells are not differentiated. Adult primary human hepatocytes infected with natural serum-derived HCV virions represent the model that most closely mimics the physiological situation. This chapter describes our experience with this culture model.