Rineke Steenbergen

Disruption of the Phosphatidylserine Decarboxylase Gene in Mice Causes Embryonic Lethality and Mitochondrial Defects

Most of the phosphatidylethanolamine (PE) in mammalian cells is synthesized by two pathways, the CDP-ethanolamine pathway and the phosphatidylserine (PS) decarboxylation pathway, the final steps of which operate at spatially distinct sites, the endoplasmic reticulum and mitochondria, respectively. We investigated the importance of the mitochondrial pathway for PE synthesis in mice by generating mice lacking PS decarboxylase activity. Disruption of Pisd in mice resulted in lethality between days 8 and 10 of embryonic development. Electron microscopy of Pisd-/- embryos revealed large numbers of aberrantly shaped mitochondria. In addition, fluorescence confocal microscopy of Pisd-/- embryonic fibroblasts showed fragmented mitochondria. PS decarboxylase activity and mRNA levels in Pisd+/- tissues were approximately one-half of those in wild-type mice. However, heterozygous mice appeared normal, exhibited normal vitality, and the phospholipid composition of livers, testes, brains, and of mitochondria isolated from livers, was the same as in wild-type littermates. The amount and activity of a key enzyme of the CDP-ethanolamine pathway for PE synthesis, CTP:phosphoethanolamine cytidylyltransferase, were increased by 35-40 and 100%, respectively, in tissues of Pisd+/- mice, as judged by immunoblotting; PE synthesis from [3H]ethanolamine was correspondingly increased in hepatocytes. We conclude that the CDP-ethanolamine pathway in mice cannot substitute for a lack of PS decarboxylase during development. Moreover, elimination of PE production in mitochondria causes fragmented, misshapen mitochondria, an abnormality that likely contributes to the embryonic lethality.

Activity-based Protein Profiling Identifies a Host Enzyme, Carboxylesterase 1, Which Is Differentially Active during Hepatitis C Virus Replication

Hepatitis C virus (HCV) relies on many interactions with host cell proteins for propagation. Successful HCV infection also requires enzymatic activity of host cell enzymes for key post-translational modifications. To identify such enzymes, we have applied activity-based protein profiling to examine the activity of serine hydrolases during HCV replication. Profiling of hydrolases in Huh7 cells replicating HCV identified CES1 (carboxylesterase 1) as a differentially active enzyme. CES1 is an endogenous liver protein involved in processing of triglycerides and cholesterol. We observe that CES1 expression and activity were altered in the presence of HCV. The knockdown of CES1 with siRNA resulted in lower levels of HCV replication, and up-regulation of CES1 was observed to favor HCV propagation, implying an important role for this host cell protein. Experiments in HCV JFH1-infected cells suggest that CES1 facilitates HCV release because less intracellular HCV core protein was observed, whereas HCV titers remained high. CES1 activity was observed to increase the size and density of lipid droplets, which are necessary for the maturation of very low density lipoproteins, one of the likely vehicles for HCV release. In transgenic mice containing human-mouse chimeric livers, HCV infection also correlates with higher levels of endogenous CES1, providing further evidence that CES1 has an important role in HCV propagation.

Human serum leads to differentiation of human hepatoma cells, restoration of very‐low‐density lipoprotein secretion, and a 1000‐fold increase in HCV Japanese fulminant hepatitis type 1 titers

In this study, we differentiated the human hepatoma cell line Huh7.5 by supplementing tissue culture media with human serum (HS) and examined the production of hepatitis C virus (HCV) by these cells. We compared the standard tissue culture protocol, using media supplemented with 10% fetal bovine serum (FBS), to media supplemented with 2% HS. Cells cultured in HS undergo rapid growth arrest, have a hepatocyte‐like morphology, and increase the expression of hepatocyte differentiation markers. In addition, expression of cell adhesion proteins claudin‐1, occludin, and e‐cadherin are also increased. The lipid droplet content of these cells is highly increased, as are key lipid metabolism regulators liver X receptor alpha, peroxisome proliferator‐activated receptor (PPAR)‐α, and PPAR‐γ. Very‐low‐density lipoprotein secretion, which is absent in FBS‐grown cells, is restored in Huh7.5 cells that are cultured in HS. All these factors have been implicated in the life cycle of HCV. We show that viral production of Japanese fulminant hepatitis type 1 increases 1,000‐fold when cells are grown in HS, compared to standard FBS culture conditions. The virus produced under these conditions is associated with apolipoprotein B, has a lower density, higher specific infectivity, and has a longer half‐life than virus produced in media supplemented with FBS. Conclusion: We describe a convenient, cost‐effective method to produce hepatocyte‐like cells, which produce large amounts of virus that more closely resemble HCV present in serum of infected patients. (Hepatology 2013; 58:1907–1917)

Hepatitis C Virus-Induced Cytoplasmic Organelles Use the Nuclear Transport Machinery to Establish an Environment Conducive to Virus Replication

Hepatitis C virus (HCV) infection induces formation of a membranous web structure in the host cell cytoplasm where the viral genome replicates and virions assemble. The membranous web is thought to concentrate viral components and hide viral RNA from pattern recognition receptors. We have uncovered a role for nuclear pore complex proteins (Nups) and nuclear transport factors (NTFs) in the membranous web. We show that HCV infection leads to increased levels of cytoplasmic Nups that accumulate at sites enriched for HCV proteins. Moreover, we detected interactions between specific HCV proteins and both Nups and NTFs. We hypothesize that cytoplasmically positioned Nups facilitate formation of the membranous web and contribute to the compartmentalization of viral replication. Accordingly, we show that transport cargo proteins normally targeted to the nucleus are capable of entering regions of the membranous web, and that depletion of specific Nups or Kaps inhibits HCV replication and assembly.

Lipoprotein profiles in SCID/uPA mice transplanted with human hepatocytes become human-like and correlate with HCV infection success

Although multiple determinants for hepatitis C virus (HCV) infection are known, it remains partly unclear what determines the human specificity of HCV infection. Presumably, the presence of appropriate entry receptors is essential, and this may explain why HCV is unable to infect nonhuman hepatocytes. However, using mice with chimeric human livers, we show in this study that the presence of human hepatocytes, and therefore human entry receptors, is not sufficient for HCV infection. In successfully transplanted SCID/Alb-uPA mice, infection with HCV is reliable only when ∼70-80% of the liver consists of human hepatocytes. We show that chimeric mice, which are hard to infect with HCV, have significant groups of human hepatocytes that are readily infected with hepatitis B virus. Thus it is unlikely that the lack of infection with HCV can simply be attributed to low hepatocyte numbers. We investigated whether the humanization of lipoprotein profiles is positively associated with infection success. We show that the lipoprotein profiles of chimeric mice become more human-like at high levels of engraftment of human hepatocytes. This and expression of markers of human lipoprotein biosynthesis, human apolipoprotein B (ApoB) and cholesterol ester transfer protein (CETP), show a strong positive correlation with successful infection. Association of HCV in the blood of chimeric mice to ApoB-containing lipoproteins is comparable to association of HCV in patient serum and provides further support for a critical role for ApoB-containing lipoproteins in the infectious cycle of HCV. Our data suggest that the weakest link in the HCV infection chain does not appear to be the presence of human hepatocytes per se. We believe that HCV infection also depends on the presence of sufficient levels of human lipoproteins.

Human serum activates CIDEB-mediated lipid droplet enlargement in hepatoma cells.

Human hepatocytes constitutively express the lipid droplet (LD) associated protein cell death-inducing DFFA-like effector B (CIDEB). CIDEB mediates LD fusion, as well as very-low-density lipoprotein (VLDL) maturation. However, there are limited cell culture models readily available to study CIDEBs role in these biological processes, as hepatoma cell lines express negligible levels of CIDEB. Recent work has highlighted the ability of human serum to differentiate hepatoma cells. Herein, we demonstrate that culturing Huh7.5 cells in media supplemented with human serum activates CIDEB expression. This activation occurs through the induced expression of PGC-1α, a positive transcriptional regulator of CIDEB. Coherent anti-Stokes Raman scattering (CARS) microscopy revealed a correlation between CIDEB levels and LD size in human serum treated Huh7.5 cells. Human serum treatment also resulted in a rapid decrease in the levels of adipose differentiation-related protein (ADRP). Furthermore, individual overexpression of CIDEB was sufficient to down-regulate ADRP protein levels. siRNA knockdown of CIDEB revealed that the human serum mediated increase in LD size was CIDEB-dependent. Overall, our work highlights CIDEBs role in LD fusion, and presents a new model system to study the PGC-1α/CIDEB pathways role in LD dynamics and the VLDL pathway.

Investigating the antiviral role of cell death‐inducing DFF45‐like effector B in HCV replication

Cell‐death‐inducing DFF45‐like effector B (CIDEB) is an apoptotic host factor, which was recently found to also regulate hepatic lipid homeostasis. Herein we delineate the relevance of these dual roles of CIDEB in apoptosis and lipid metabolism in the context of hepatitis C virus (HCV) replication. We demonstrate that HCV upregulates CIDEB expression in human serum differentiated hepatoma cells. CIDEB overexpression inhibits HCV replication in HCV replicon expressing Huh7.5 cells, while small interfering RNA knockdown of CIDEB expression in human serum differentiated hepatoma cells promotes HCV replication and secretion of viral proteins. Furthermore, we characterize a CIDEB mutant, KRRA, which is deficient in lipid droplet clustering and fusion and demonstrate that CIDEB‐mediated inhibition of HCV is independent of the proteins lipid droplet fusogenic role. Our results suggest that higher levels of CIDEB expression, which favour an apoptotic role for the host factor, inhibit HCV. Collectively, our data demonstrate that CIDEB can act as an anti‐HCV host factor and contribute to altered triglyceride homeostasis.

Establishing normal metabolism and differentiation in hepatocellular carcinoma cells by culturing in adult human serum

Tissue culture medium routinely contains fetal bovine serum (FBS). Here we show that culturing human hepatoma cells in their native, adult serum (human serum, HS) results in the restoration of key morphological and metabolic features of normal liver cells. When moved to HS, these cells show differential transcription of 22–32% of the genes, stop proliferating, and assume a hepatocyte-like morphology. Metabolic analysis shows that the Warburg-like metabolic profile, typical for FBS-cultured cells, is replaced by a diverse metabolic profile consistent with in vivo hepatocytes, including the formation of large lipid and glycogen stores, increased glycogenesis, increased beta-oxidation and ketogenesis, and decreased glycolysis. Finally, organ-specific functions are restored, including xenobiotics degradation and secretion of bile, VLDL and albumin. Thus, organ-specific functions are not necessarily lost in cell cultures, but might be merely suppressed in FBS. The effect of serum is often overseen in cell culture and we provide a detailed study in the changes that occur and provide insight in some of the serum components that may play a role in the establishment of the differentiated phenotype.

Induction of differentiation and metabolic reprogramming in human hepatoma cells by adult human serum

Tissue culture medium routinely contains fetal bovine serum (FBS). Here we show that culturing human hepatoma cells in their native, adult serum (human serum, HS) results in the restoration of key morphological and metabolic features of normal liver cells. When moved to HS, these cells show differential transcription of 22-32% of the genes, stop proliferating, and assume a hepatocyte-like morphology. Metabolic analysis shows that the Warburg-like metabolic profile, typical for FBS-cultured cells, is replaced by a diverse metabolic profile consistent with in vivo hepatocytes. We demonstrate the formation of large lipid and glycogen stores, increased glycogenesis, increased β-oxidation, increased ketogenesis, and decreased glycolysis. Finally, organ-specific functions are restored, including xenobiotics degradation and secretion of bile, very low density lipoprotein, and albumin. Thus, organ-specific functions are not necessarily lost in cell cultures, but might be merely suppressed in FBS. Together, we showed that cells that are representative of normal physiology can be produced from cancer cells simply by replacing FBS by HS in culture media. The effect of serum is often overseen in cell culture and we provide a detailed study in the changes that occur, provide insight in some of the serum components that may play a role in the establishment of the different phenotypes, and discuss how these finding might be beneficial to a variety of research fields.