Serine 574 phosphorylation alters transcriptional programming of FOXO3 by selectively enhancing apoptotic gene expression.

Forkhead box O3 (FOXO3) is a multispecific transcription factor that is responsible for multiple and conflicting transcriptional programs such as cell survival and apoptosis. The protein is heavily post-translationally modified and there is considerable evidence that post-transcriptional modifications (PTMs) regulate protein stability and nuclear-cytosolic translocation. Much less is known about how FOXO3 PTMs determine the specificity of its transcriptional program. In this study we demonstrate that exposure of hepatocytes to ethanol or exposure of macrophages to lipopolysaccharide (LPS) induces the c-Jun N-terminal kinase (JNK)-dependent phosphorylation of FOXO3 at serine-574. Chromatin immunoprecipitation (ChIP), mRNA and protein measurements demonstrate that p-574-FOXO3 selectively binds to promoters of pro-apoptotic genes but not to other well-described FOXO3 targets. Both unphosphorylated and p-574-FOXO3 bound to the B-cell lymphoma 2 (Bcl-2) promoter, but the unphosphorylated form was a transcriptional activator, whereas p-574-FOXO3 was a transcriptional repressor. The combination of increased TRAIL (TNF-related apoptosis-inducing ligand) and decreased Bcl-2 was both necessary and sufficient to induce apoptosis. LPS treatment of a human monocyte cell line (THP-1) induced FOXO3 S-574 phosphorylation and apoptosis. LPS-induced apoptosis was prevented by knockdown of FOXO3. It was restored by overexpressing wild-type FOXO3 but not by overexpressing a nonphosphorylatable S-574A FOXO3. Expression of an S-574D phosphomimetic form of FOXO3 induced apoptosis even in the absence of LPS. A similar result was obtained with mouse peritoneal macrophages where LPS treatment increased TRAIL, decreased Bcl-2 and induced apoptosis in wild-type but not FOXO3(-/-) cells. This work thus demonstrates that S-574 phosphorylation generates a specifically apoptotic form of FOXO3 with decreased transcriptional activity for other well-described FOXO3 functions.

Liver-Regenerative Transplantation: Regrow and Reset.

Liver transplantation, either a partial liver from a living or deceased donor or a whole liver from a deceased donor, is the only curative therapy for severe end-stage liver disease. Only one-third of those on the liver transplant waiting list will be transplanted, and the demand for livers is projected to increase 23% in the next 20 years. Consequently, organ availability is an absolute constraint on the number of liver transplants that can be performed. Regenerative therapies aim to enhance liver tissue repair and regeneration by any means available (cell repopulation, tissue engineering, biomaterials, proteins, small molecules, and genes). Recent experimental work suggests that liver repopulation and engineered liver tissue are best suited to the task if an unlimited availability of functional induced pluripotent stem (iPS)-derived liver cells can be achieved. The derivation of iPS cells by reprogramming cell fate has opened up new lines of investigation, for instance, the generation of iPS-derived xenogeneic organs or the possibility of simply inducing the liver to reprogram its own hepatocyte function after injury. We reviewed current knowledge about liver repopulation, generation of engineered livers and reprogramming of liver function. We also discussed the numerous barriers that have to be overcome for clinical implementation.

Apoptosis induced in normal human hepatocytes by tumor necrosis factor-related apoptosis-inducing ligand.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has been reported to induce apoptosis in various tumor cells but not in nontransformed, normal cells. Preclinical studies in mice and nonhuman primates have shown that administration of TRAIL can induce apoptosis in human tumors, but that no cytotoxicity to normal organs or tissues is found. The susceptibility of tumor cells to TRAIL and an apparent lack of activity in normal cells has lead to a proposal to use TRAIL in cancer therapy. Here, we assessed the sensitivity of hepatocytes from rat, mouse, rhesus monkey and human livers to TRAIL-induced apoptosis. TRAIL induced apoptosis in normal human hepatocytes in culture but not in hepatocytes isolated from the other species. Human hepatocytes showed characteristic features of apoptosis, including cytoplasmic shrinkage, the activation of caspases and DNA fragmentation. Apoptosis and cell death in human hepatocytes was massive and rapid, occurring in more than 60% of the cells exposed to TRAIL within 10 hours. These results indicate that there are species differences in sensitivity to TRAIL, and that substantial liver toxicity might result if TRAIL were used in human cancer therapy.

Cross-talk between epidermal growth factor receptor and c-Met signal pathways in transformed cells.

In rat liver epithelial cells constitutively expressing transforming growth factor alpha (TGFalpha), c-Met is constitutively phosphorylated in the absence of its ligand, hepatocyte growth factor. We proposed that TGFalpha and the autocrine activation of its receptor, epidermal growth factor receptor (EGFR), leads to phosphorylation and activation of c-Met. We found that there is constitutive c-Met phosphorylation in human hepatoma cell lines and the human epidermoid carcinoma cell line, A431 which express TGFalpha, but not in normal human hepatocytes. Constitutive c-Met phosphorylation in A431, HepG2, AKN-1, and HuH6 cells was inhibited by neutralizing antibodies against TGFalpha and/or EGFR. Exposure to exogenous TGFalpha or EGF increased the phosphorylation of c-Met in the human epidermoid carcinoma cell line, A431. The increase of c-Met phosphorylation by TGFalpha in A431 cells was inhibited by neutralizing antibodies against TGFalpha and/or EGFR and by the EGFR-specific inhibitor tyrphostin AG1478. These results indicate that constitutive c-Met phosphorylation, and the increase of c-Met phosphorylation by TGFalpha or EGF, in tumor cell lines is the result of the activation via EGFR. We found that c-Met in tumor cells co-immunoprecipitates with EGFR regardless of the existence of their ligands in tumor cells, but not in normal human hepatocytes. We conclude that c-Met associates with EGFR in tumor cells, and this association facilitates the phosphorylation of c-Met in the absence of hepatocyte growth factor. This cross-talk between c-Met and EGFR may have significant implications for altered growth control in tumorigenesis.

Isolation and characterization of a human hepatic epithelial-like cell line (AKN-1) from a normal liver.

The isolation and characterization of human liver cell lines are rather difficult due to limited material and poor growth in cell culture. In this report, we present the isolation, culture and characterization of a new epithelial-like liver cell line (AKN-1) with a heterogeneous cell population and many characteristics of the biliary epithelium. The AKN-1 cell line stained positively with antibodies to epithelial cytokeratin polypetides CK 8, 18, and 19. In addition, the cell line expressed the anti-human epithelial-related antigen (MOC-31), the human epithelial antigen (HEA), and the gamma-glutamyl transpeptidase, the hematopoietic growth factor, stem cell factor, and also its receptor, c-kit. The cell line failed to express albumin and factor 8 by immunohistochemistry. It did show, however, a twofold increase in 7-ethoxyresorufin-O-deethylase activity. Cytogenetic characterization revealed rare breakpoints in chromosome 2, which to our knowledge, have not yet been reported in liver cells.

CD14 expression and production by human hepatocytes.

BACKGROUND/AIMS: CD14 has been identified as a receptor for LPS and is present both in a membrane-bound and a soluble form. Membrane CD14 (mCD14) is predominantly expressed on monocytes, macrophages and granulocytes. The source of soluble CD14 (sCD14) is as yet unclear. Previous investigation using monocytes has shown that sCD14 can be derived either from the shedding of mCD14 or from direct secretion by monocytes. Whether the monocyte is the sole or even the major source of sCD14 is as yet uncertain. Clearance of LPS from the bloodstream is thought to be primarily mediated by the liver. Production of CD14 by hepatocytes would potentially provide a powerful mechanism by which the liver could clear LPS, and therefore we examined the ability of human hepatocytes to produce CD14. METHODS: Human hepatocytes were isolated using collagenase perfusion. RESULTS: Human hepatocytes were found to have CD14 mRNA by Northern blot analysis. Western blot analysis and immunohistochemical staining confirmed CD14 protein in primary hepatocyte cultures. Further studies showed that a liver epithelial-like cell line AKN-1 is capable of producing CD14. Comparisons of the size of hepatocyte-derived CD14 protein with the sCD14 protein from the human monocytic leukemia cell line HL60 suggested that a slightly larger form of CD14 is expressed by human liver cells. CONCLUSION: This is the first study to demonstrate production of CD14 by human hepatocytes.

Troglitazone increases cytochrome P-450 3A protein and activity in primary cultures of human hepatocytes.

Troglitazone (TRO) is an insulin sensitizer used in the treatment of type II diabetes. TRO is known to increase the activity of cytochrome P-450 (CYP) 3A in vivo. We have investigated the effect of TRO on CYP3A protein content and the activity of CYP3A (as measured by the formation of 6beta-hydroxytestosterone formation) in primary cultures of human hepatocytes in comparison with rifampicin (RIF). Hepatocytes were isolated from four human livers by perfusion with collagenase, plated on collagen-coated plates, and maintained in William's E medium. After 48 h in culture, cells were exposed to RIF (10 microM) or TRO (0-50 microM) twice, each over a period of 24 h, and the activity of CYP3A was measured. TRO increased the activity of CYP3A in a concentration-dependent manner, reaching a maximal response at 5 microM. Pretreatment of the hepatocytes with 10 microM TRO or 10 microM RIF resulted in a 4- to 15-fold increase in the activity of CYP3A. Maximum increase in CYP3A protein was observed at 5 microM TRO. There was a significant correlation (R(2) = 0.89) between the content of immunoreactive CYP3A protein in the hepatocytes and the rate of formation of 6beta-hydroxytestosterone. These results indicate that TRO is a potent inducer of CYP3A and is similar to RIF in inducing CYP3A in human hepatocytes. At concentrations of 25 microM and above, TRO was toxic to the cells, as determined by a decrease in the activity of CYP3A, a reduction in the amount of immunoreactive protein, and changes in the morphology of the cells.

The use of human hepatocyte cultures to study the induction of cytochrome P-450.

We have previously reported that paclitaxel (Taxol) is a potent inducer of cytochrome P-450 (CYP) 3A protein and CYP3A mRNA in human hepatocyte cultures. Here we report that Taxol increased CYP3A-dependent testosterone 6beta-hydroxylation in intact hepatocytes. This effect was concentration-dependent, with maximal increase in enzyme activity being observed at 10 microM Taxol. Treatment of hepatocyte cultures with concentrations of Taxol higher than 10 microM caused a dose-dependent decrease in testosterone 6beta-hydroxylase activity, amount of CYP3A protein, and total protein synthesis. The maximal CYP3A activity detected after treatment with Taxol or rifampicin was similar in six separate human hepatocyte cultures, suggesting that the cultures have achieved a limit of maximally inducible CYP3A. The fold increase in enzyme activity, however, was different and was inversely related to the level of expression in untreated hepatocytes, with the greatest increases being observed in the hepatocytes that expressed the lowest basal level of CYP3A. Pretreatment of hepatocytes with triacetyloleandomycin resulted in a 90% inhibition of testosterone 6beta-hydroxylase activity. Our results demonstrate the use of human hepatocyte cultures to investigate the induction of cytochrome P-450 by xenobiotics in intact cells and stress the importance of large dose-response studies as well as the need to assess toxicity in these investigations. The response to inducers of CYP3A activity were very consistent among different hepatocyte donors. Absolute values of testosterone 6beta-hydroxylase activity did not vary more than 2- and 5-fold in induced and untreated hepatocytes, respectively.

Epidermal growth factor- and hepatocyte growth factor-receptor activity in serum-free cultures of human hepatocytes.

BACKGROUND/AIMS: Serum-free primary cultures of hepatocytes are a useful tool to study factors triggering hepatocyte proliferation and regeneration. We have developed a chemically defined serum-free system that allows human hepatocyte proliferation in the presence of epidermal growth factor and hepatocyte growth factor. METHODS: DNA synthesis and accumulation were determined by [3H]thymidine incorporation and fluorometry, respectively. Western blot analyses and co-immunoprecipitations were used to investigate the association of proteins involved in epidermal growth factor and hepatocyte growth factor activation and signaling: epidermal growth factor receptor, hepatocyte growth factor receptor (MET), urokinase-type plasminogen activator and its receptor, and a member of the signal transducer and activator of transcription family, STAT-3. RESULTS: Primary human hepatocytes proliferated under serum-free conditions in a chemically defined medium for up to 12 days. Epidermal growth factor-receptor and MET were present and functional, decreasing over time. MET, urokinase-type plasminogen activator and urokinase-type plasminogen activator receptor co-precipitated to varying degrees during the culture period. STAT-3 co-precipitated with epidermal growth factor-receptor and MET to varying degrees. CONCLUSIONS: Proliferation of human hepatocytes can improve by modification of a chemically defined medium originally used for rat hepatocyte cultures. In these long-term cultures of human hepatocytes, hepatocyte growth factor and epidermal growth factor can stimulate growth and differentiation by interacting with their receptors and initiating downstream signaling. This involves complex formation of the receptors with other plasma membrane components for MET (urokinase-type plasminogen activator in context of its receptor) and activation of STAT-3 for both receptors.

Primary human hepatocyte culture for the study of HCV.

Research since 1983 has demonstrated that human hepatocytes can be isolated, cultured, and used for biological investigations, including studies of gene transcription and drug metabolism (1,2). In addition, the ability to cyropreserve hepatocytes has facilitated clinical research of hepatitic cell transplantation (3). We have used primary human heptocytes as host tissue for viral infection with hepatitis C. The availability of HCV-infected livers has also allowed for the culturing and analysis of HCV-positive cells. Our laboratory (4) and others (5) have confirmed the ability of these cells to display molecular markers of HCV replication. This chapter will review the basic steps of hepatocyte isolation and culturing and analysis for HCV by RT-PCR. We have also attempted to indicate alternative techniques that may be better suited to an individual investigator's needs.

Effects of hepatocellular mitogens on cytokine-induced nitric oxide synthesis in human hepatocytes.

The synthesis of induced nitric oxide (NO) is regulated by several cytokines, including growth factors produced following hepatic injury and inflammation. However, little information is available on the role of growth factors in regulating the inducible NO synthase in human hepatocytes. The capacity of hepatocellular mitogens (HGF, EGF, and TGF-alpha) to regulate the inducible NO synthase (iNOS) was studied in human hepatocytes incubated with inflammatory cytokines and lipopolysaccharide (LPS). Furthermore, the effects of hepatic mitogens on NO-induced changes in DNA and protein synthesis was studied. It was found that NO-mediated decrease of protein and DNA synthesis were partially reversed by the mitogens. This was associated with a down-regulation in cytokine-mediated hepatocyte NO formation, iNOS mRNA expression, and NOS enzyme activity. Cytokine-induced NO formation or SNAP, an NO donor, added with cytokines increased hepatocyte chromatin condensation but no DNA fragmentation was observed. The increase in chromatin condensation was partially reversed by hepatic mitogens and corresponded with the inhibition of NO production. Thus, the hepatic mitogens, HGF, EGF, and TGF-alpha, all suppress iNOS expression and it is the suppression of iNOS that appears to be responsible for the mitogen-reduced preservation of DNA and protein synthesis and prevention of chromatin condensation.

Further characterization and comparison of inducible nitric oxide synthase in mouse, rat, and human hepatocytes.

Marked differences in induced nitric oxide (NO) synthesis occur between species. We have previously shown that both human and rat hepatocytes express an inducible NO synthase in response to cytokines and lipopolysaccharide. In this study, we compare the expression and regulation of cytokine-induced NO synthase in hepatocytes isolated from three species, human, rat, and mouse. On stimulation with tumor necrosis factor alpha (TNF alpha), interleukin-1 beta (IL-1 beta), interferon gamma (IFN gamma), and lipopolysaccharide (LPS), it was found that hepatocytes from all three species produce high levels of NO with levels of production exhibiting the following hierarchy: rat hepatocytes > mouse hepatocytes > human hepatocytes. Whereas rat and mouse hepatocytes express inducible NO synthase messenger RNA (mRNA) in response to TNF alpha, IL-1 beta, or IFN gamma as a single stimulus, human hepatocytes respond to LPS alone. Inhibition of NO generation through transforming growth factor (TGF-beta 1) was seen in mouse (77% +/- 5.9) and rat hepatocytes (17% +/- 2.6) whereas only about 10% was seen in human hepatocytes. Epidermal growth factor (EGF) was shown to inhibit NO synthesis in human and mouse hepatocytes but not rat. A marked NO-dependent inhibition of total protein synthesis was seen in rat and human hepatocytes, whereas mouse hepatocytes showed almost no inhibition in protein synthesis when stimulated. NO-dependent cyclic guanosine monophosphate (cGMP) release was found in all three species.(ABSTRACT TRUNCATED AT 250 WORDS)

A new technique for isolating and culturing human hepatocytes from whole or split livers not used for transplantation.

Large numbers of human hepatocytes were obtained from split and whole livers by using an adaptive form of the collagenase perfusion technique employed in rodent and human biopsies. In order to guarantee a homogenous distribution of the perfusate within the whole specimen, major hepatic veins were cannulated with large bore catheters. This technique allowed for the isolation of human hepatocytes on a large scale (up to 18.5 x 10(9) in one case) from normal and diseased liver specimens. The yield of isolated normal viable hepatocytes is inversely proportional to the donor age. In addition, it was noted that a short time between declared death and organ harvest (cross clamp time) results in higher viability of hepatocytes. In contrast, the time of cold organ preservation did not correlate with the viability or the yield of isolated hepatocytes. We conclude that the technique presented here allows isolation of large numbers of human hepatocytes from specimens unsuitable for transplantation but very valuable for biomedical research.