Genes In Hepatic Cells Research Articles

Hepatitis B Virus-KMT2B Integration Drives Hepatic Oncogenic Processes in a Human Gene-edited Induced Pluripotent Stem Cells-derived Model

Background & AimsHepatitis B virus (HBV)-DNA integration into the host genome contributes to hepatocellular carcinoma (HCC) development. KMT2B is the second most frequent locus of HBV-DNA integration in HCC, however its role and function remain unclear. We aimed to clarify the impact of HBV-KMT2B integration in HCC development using a human genome-edited induced pluripotent stem cells (iPSCs) model. MethodsBased on the genetic information on HBV-KMT2B integration in HCC, we determined its complete DNA sequence and transcript variants. To exclude the effect of other oncogenic mutations, we reproduced HBV integration in healthy donor iPSCs with an intact genome and analyzed its effects using iPSC-derived hepatic progenitor cells (HPCs) and hepatocytes (iPS-Heps). ResultsThe reproduced HBV-KMT2B integration significantly upregulated the proliferation of hepatic cells. Comprehensive transcriptional and epigenetic analyses revealed enhanced expression of cell cycle-related genes in hepatic cells with HBV-KMT2B integration based on perturbation of histone 3 lysine 4 tri-methylation(H3K4me3), mimicking that in the original HCC sample. Long-read RNA-sequence detected the common KMT2B transcript variants in the HCC sample and HPCs. Overexpression of the truncated variant significantly enhanced proliferation of hepatic cells, whereas HBV-KMT2B fusion transcripts did not enhance proliferation. HBV-KMT2B-integrated HPCs exhibited replication stress and DNA damage, indicating that our model initiated the process of hepatocarcinogenesis due to abnormally promoted KMT2B function. ConclusionsOur disease model using genetically engineered iPSCs provides the first insight into both the KMT2B function in HCC development and the oncogenic processes by HBV-KMT2B integration. We clarified the novel oncogenic mechanism in HBV-related HCC due to aberrant KMT2B function.

Dietary licorice enhances in vivo cadmium detoxification and modulates gut microbial metabolism in mice.

Mass cadmium (Cd) poisoning is a serious health problem in many parts of the world. We propose that dietary intervention can be a practical solution to this problem. This study aimed to identify effective dietary products from traditional Chinese herbs that can detoxify Cd. Five candidate herbal foods with detoxifying potential were selected and subjected to mouse toxicological tests. The chemical composition and dose-response effects of licorice on mouse hepatocytes were determined. Licorice was selected for further tests to examine its effects on growth, tissue Cd accumulation, and gut and liver fitness of mice. The expression of hepatic metallothionein (Mt) genes was quantified in vitro in hepatocytes and in vivo in liver tissues of mice. The results showed that licorice dietary intervention was effective in reducing blood Cd by >50% within 1 month. Cd was also substantially reduced in the heart and lung tissues, but increased 2.1-fold in the liver. The liver of Cd poisoned mice improved with licorice intervention. Licorice treatment significantly induced Cd accumulation and expression of the Mt1 gene in hepatic cells both in vitro and in vivo. Licorice intake substantially altered gut microbial structure and enriched Parabacteroides distasonis. Omics results showed that licorice improved gut metabolism, particularly the metabolic pathways for glycyrrhizin, bile acids, and amino acids. Dietary licorice effectively reduced mouse blood Cd and had a profound impact on liver and gut fitness. We conclude that herbal licorice can be used as a dietary intervention for mass Cd poisoning.

Hepatoprotective Potential of Malabar Tamarind (Garcinia gummi-gutta) Fruit-Rind Extract in Acetaminophen-Induced Toxicity Through Modulation of CYP2E1 Gene Expression in Hepatic Cells of Wistar Rats

The present study was aimed to assess the hepatoprotective effect of ethanolic extract of Garcinia gummi-gutta (GGE) fruit rind on acetaminophen-induced toxicity in a rat model and to explore the possible mechanism of action by assessing hepatic histopathology, serum bio­chemical parameters and expression of CYP2E1 gene in hepatic cells. The group I and 11 animals served as normal control and hepatotoxic control group, respectively; while group III, IV and V were administrated with GGE fruit-rind extract @ 50, 100 & 200 mg kg-1 orally, respectively, for 10 days. The group VI (positive control) received drug silymarin @ 100 mg kg-1 day-1 orally for 10 days. Hepatotoxicity was induced by single oral administration of acetaminophen (2 g kg-1) on 8th day. The results revealed that the extract produced dose-dependent hepatoprotective effect against acetaminophen-induced hepatic damage by improving the serum bio­chemical profile towards the normal range. Histopathology of liver showed that pretreatment with fruit-rind extract attenuated the toxicant-induced hepatocellular necrosis and enhanced the regeneration of hepatic cells. The hepatic expression level of CYP2E1 gene was found down-regulated in a dose dependent manner in extract-treated group as compared to the normal control group. The study revealed remarkable hepatoprotective effect of GGE in acetaminophen-induced toxicity in rats through regeneration of hepatic cells and by decreasing the formation of reactive metabolite, NAPQ1 by modulation of CYP2E1 gene expression in liver.

Sialic acid-engineered mesoporous polydopamine dual loaded with ferritin gene and SPIO for achieving endogenous and exogenous synergistic T2-weighted magnetic resonance imaging of HCC

BackgroundHepatocellular carcinoma (HCC) is a common malignant tumor with poor prognosis. Magnetic resonance imaging (MRI) is one of the most effective imaging methods for the early diagnosis of HCC. However, the current MR contrast agents are still facing challenges in the early diagnosis of HCC due to their relatively low sensitivity and biosafety. Thus, the development of effective MR agents is highly needed for the early diagnosis of HCC.ResultsHerein, we fabricated an HCC-targeted nanocomplexes containing SPIO-loaded mesoporous polydopamine (MPDA@SPIO), sialic acid (SA)-modified polyethyleneimine (SA-PEI), and alpha-fetoprotein regulated ferritin gene (AFP-Fth) which was developed for the early diagnosis of HCC. It was found that the prepared nanocomplexes (MPDA@SPIO/SA-PEI/AFP-Fth) has an excellent biocompatibility towards the liver cells. In vivo and in vivo studies revealed that the transfection of AFP-Fth gene in hepatic cells significantly upregulated the expression level of ferritin, thereby resulting in an enhanced contrast on T2-weighted images via the formed endogenous MR contrast.ConclusionsThe results suggested that MPDA@SPIO/SA-PEI/AFP-Fth had a superior ability to enhance the MR contrast of T2-weighted images of tumor region than the other preparations, which was due to its HCC-targeted ability and the combined T2 contrast effect of endogenous ferritin and exogenous SPIO. Our study proved that MPDA@SPIO/SA-PEI/AFP-Fth nanocomplexes could be used as an effective MR contrast agent to detect HCC in the early stage.

Screening Dietary Herbs for Health Intervention on Mass Cadmium Poisoning

Objective: Mass cadmium (Cd) poisoning is a serious health problem in many parts of the world. We propose that dietary intervention can be a practical solution to this problem. This study aimed to identify effective dietary products from traditional Chinese herbs that can detoxify Cd. Design: Five candidate herbal foods with detoxifying potential were selected and subjected to mouse toxicological tests. The chemical composition and dose-response effects of licorice on mouse hepatocytes were determined. Licorice was selected for further tests to examine its effects on growth, tissue Cd accumulation, and gut and liver fitness of mice. The expression of hepatic metallothionein (Mt) genes was quantified in vitro in hepatocytes and in vivo in liver tissues of mice. Results: The results showed that licorice dietary intervention was effective in reducing blood Cd by > 50% within 1 month. Cd was also substantially reduced in the heart and lung tissues, but increased 2.1-fold in the liver. The liver of Cd poisoned mice improved with licorice intervention. Licorice treatment significantly induced Cd accumulation and expression of the Mt1 gene in hepatic cells both in vitro and in vivo. Licorice intake substantially altered gut microbial structure and enriched Parabacteroides distasonis. Omics results showed that licorice improved gut metabolism, particularly the metabolic pathways for glycyrrhizin, bile acids, and amino acids. Conclusion: Dietary licorice effectively reduced mouse blood Cd and had a profound impact on liver and gut fitness. We conclude that herbal licorice can be used as a dietary intervention for mass Cd poisoning. Funding: This work was supported by the Hebei Provincial Science Fund or Distinguished Young Scholars (No. D2018503005), the National Key Research and Development Program of China (No.2018YFD0800306), and the National Natural Science Foundation of China (Nos. 41877414 and 31700228). Declaration of Interest: None to declare. Ethical Approval: The study was approved by the Animal Care and Use Committee of the Hebei Science and Technical Bureau, China

Melatonin stimulates transcription of the rat phosphoenolpyruvate carboxykinase gene in hepatic cells.

Melatonin plays physiological roles in various critical processes, including circadian rhythms, oxidative stress defenses, anti-inflammation responses, and immunity; however, the current understanding of the role of melatonin in hepatic glucose metabolism is limited. In this study, we examined whether melatonin affects gene expression of the key gluconeogenic enzyme, phosphoenolpyruvate carboxykinase (PEPCK). We found that melatonin treatment increased PEPCK mRNA levels in rat highly differentiated hepatoma (H4IIE) cells and primary cultured hepatocytes. In addition, we found that melatonin induction was synergistically enhanced by dexamethasone, whereas it was dominantly inhibited by insulin. We also report that the effect of melatonin was blocked by inhibitors of mitogen-activated protein kinase/extracellular signal-regulated protein kinase (MAPK/ERK), RNA polymerase II, and protein synthesis. Furthermore, the phosphorylated (active) forms of ERK1 and ERK2 (ERK1/2) increased 15min after melatonin treatment. We performed luciferase reporter assays to show that melatonin specifically stimulated promoter activity of the PEPCK gene. Additional reporter analysis using 5'-deleted constructs revealed that the regulatory regions responsive to melatonin mapped to two nucleotide regions, one between -467 and -398 nucleotides and the other between -128 and +69 nucleotides, of the rat PEPCK gene. Thus, we conclude that melatonin induces PEPCK gene expression via the ERK1/2 pathway at the transcriptional level, and that induction requires denovo protein synthesis.

Transcriptional regulation of the human Liver X Receptor α gene by Hepatocyte Nuclear Factor 4α

Liver X Receptors (LXRs) are sterol-activated transcription factors that play major roles in cellular cholesterol homeostasis, HDL biogenesis and reverse cholesterol transport. The aim of the present study was to investigate the mechanisms that control the expression of the human LXRα gene in hepatic cells. A series of reporter plasmids containing consecutive 5′ deletions of the hLXRα promoter upstream of the luciferase gene were constructed and the activity of each construct was measured in HepG2 cells. This analysis showed that the activity of the human LXRα promoter was significantly reduced by deleting the −111 to −42 region suggesting the presence of positive regulatory elements in this short proximal fragment. Bioinformatics data including motif search and ChIP-Seq revealed the presence of a potential binding motif for Hepatocyte Nuclear Factor 4 α (HNF-4α) in this area. Overexpression of HNF-4α in HEK 293T cells increased the expression of all LXRα promoter constructs except −42/+384. In line, silencing the expression of endogenous HNF-4α in HepG2 cells was associated with reduced LXRα protein levels and reduced activity of the −111/+384 LXRα promoter but not of the −42/+384 promoter. Using ChiP assays in HepG2 cells combined with DNAP assays we mapped the novel HNF-4α specific binding motif (H4-SBM) in the −50 to −40 region of the human LXRα promoter. A triple mutation in this H4-SBM abolished HNF-4α binding and reduced the activity of the promoter to 65% relative to the wild type. Furthermore, the mutant promoter could not be transactivated by HNF-4α. In conclusion, our data indicate that HNF-4α may have a wider role in cell and plasma cholesterol homeostasis by controlling the expression of LXRα in hepatic cells.

Novel mechanism of transcriptional repression of the human ATP binding cassette transporter A1 gene in hepatic cells by the winged helix/forkhead box transcription factor A2

ATP binding cassette transporter A1 (ABCA1) plays a key role in the biogenesis of HDL by promoting the efflux of cellular cholesterol and phospholipids to lipid free apoA-I. Mutations in the ABCA1 gene cause Tangier disease which is characterized by near or complete absence of circulating plasma HDL. In the present study we show that the winged helix/forkhead box containing transcription factor A2 (FOXA2) shown previously to play a role in glucose and bile acid homeostasis in the liver and in energy utilization in adipose tissue is a negative modulator of ABCA1 gene expression in hepatic cells. We show that the ABCA1 promoter contains three FOXA2 binding elements in the proximal region. Two of the sites are localized in a region of the ABCA1 promoter enriched in binding elements for transcriptional repressor proteins whereas the third site is the core of the TATA element of the ABCA1 promoter. Inhibition of FOXA2 binding to the ABCA1 promoter by site-directed mutagenesis or FOXA2 gene expression by siRNA was associated with increased ABCA1 promoter activity and protein levels. Overexpression of FOXA2 inhibited both the constitutive ABCA1 gene expression as well as ABCA1 gene induction by oxysterols and retinoids via nuclear receptors LXRα/RXRα. In summary, the present study identifies transcription factor FOXA2 as a negative modulator of ABCA1 gene expression in hepatic cells and reveals a novel mechanism of transcriptional repression by FOXA2 which involves the TATA element of the ABCA1 gene.

MiR-206 controls LXRα expression and promotes LXR-mediated cholesterol efflux in macrophages

Liver X receptors (LXRα and LXRβ) are key transcription factors in cholesterol metabolism that regulate cholesterol biosynthesis/efflux and bile acid metabolism/excretion in the liver and numerous organs. In macrophages, LXR signaling modulates cholesterol handling and the inflammatory response, pathways involved in atherosclerosis. Since regulatory pathways of LXR transcription control are well understood, in the present study we aimed at identifying post-transcriptional regulators of LXR activity. MicroRNAs (miRs) are such post-transcriptional regulators of genes that in the canonical pathway mediate mRNA inactivation. In silico analysis identified miR-206 as a putative regulator of LXRα but not LXRβ. Indeed, as recently shown, we found that miR-206 represses LXRα activity and expression of LXRα and its target genes in hepatic cells. Interestingly, miR-206 regulates LXRα differently in macrophages. Stably overexpressing miR-206 in THP-1 human macrophages revealed an up-regulation and miR-206 knockdown led to a down-regulation of LXRα and its target genes. In support of these results, bone marrow-derived macrophages (BMDMs) from miR-206 KO mice also exhibited lower expression of LXRα target genes. The physiological relevance of these findings was proven by gain- and loss-of-function of miR-206; overexpression of miR-206 enhanced cholesterol efflux in human macrophages and knocking out miR-206 decreased cholesterol efflux from MPMs. Moreover, we show that miR-206 expression in macrophages is repressed by LXRα activation, while oxidized LDL and inflammatory stimuli profoundly induced miR-206 expression. We therefore propose a feed-back loop between miR-206 and LXRα that might be part of an LXR auto-regulatory mechanism to fine tune LXR activity.

RAF‐targeted therapy for hepatocellular carcinoma in the regenerating liver

Post-operative liver regeneration may contribute to tumor recurrence. There is a theoretical need for an adjuvant therapy that can suppress tumor growth without adversely affecting post-operative liver regeneration. To evaluate the effect of RAF inhibitor Sorafenib on cell viability and proliferation of hepatoma cells and hepatocytes in vitro and in an in vivo rat model. Cell viability, DNA synthesis, and RAF/MAPK kinase activity in the primary hepatocyte and hepatoma cell lines were investigated after Sorafenib exposure. Sequence analysis of the B-RAF gene in hepatic cells was determined. Tumor markers were compared within the rats after 70% hepatectomy with or without daily oral gavages of Sorafenib. Liver regeneration was assessed by liver function tests and proliferation markers. Primary hepatocytes showed higher cell viability, proliferation rate, and stronger RAF/MAPK kinase activity compared with hepatoma cell lines. The in vivo tumor volumes, size, and metastases were significantly decreased (P < 0.05) whereas no significant change in liver regeneration related to Sorafenib exposure was found (P > 0.05). B-RAF V600E mutation was not detected neither in the hepatic cells nor untransformed hepatocytes. The RAF targeted inhibitor can reduce tumor growth without retarding liver regeneration in this experiment.

Genomics Meets Glycomics—The First GWAS Study of Human N-Glycome Identifies HNF1α as a Master Regulator of Plasma Protein Fucosylation

Over half of all proteins are glycosylated, and alterations in glycosylation have been observed in numerous physiological and pathological processes. Attached glycans significantly affect protein function; but, contrary to polypeptides, they are not directly encoded by genes, and the complex processes that regulate their assembly are poorly understood. A novel approach combining genome-wide association and high-throughput glycomics analysis of 2,705 individuals in three population cohorts showed that common variants in the Hepatocyte Nuclear Factor 1α (HNF1α) and fucosyltransferase genes FUT6 and FUT8 influence N-glycan levels in human plasma. We show that HNF1α and its downstream target HNF4α regulate the expression of key fucosyltransferase and fucose biosynthesis genes. Moreover, we show that HNF1α is both necessary and sufficient to drive the expression of these genes in hepatic cells. These results reveal a new role for HNF1α as a master transcriptional regulator of multiple stages in the fucosylation process. This mechanism has implications for the regulation of immunity, embryonic development, and protein folding, as well as for our understanding of the molecular mechanisms underlying cancer, coronary heart disease, and metabolic and inflammatory disorders.

Interleukin-1 Receptor Antagonist Induction as an Additional Mechanism for Liver Receptor Homolog-1 to Negatively Regulate the Hepatic Acute Phase Response

The liver receptor homolog-1 (LRH-1) is an orphan nuclear receptor believed to play a key role in bile acid metabolism, cholesterol homeostasis, and intestinal cell crypt renewal. LRH-1 has recently been reported to negatively regulate the hepatic acute phase response by antagonizing, at least in part, the CCAAT/enhancer-binding protein signaling pathway. Here we have shown, using adenovirus-mediated LRH-1 overexpression and gene-silencing experiments, that the interleukin-1 receptor antagonist (IL-1RA) gene is a novel LRH-1 target gene in hepatic cells. Promoter mapping and chromatin immunoprecipitation experiments revealed that LRH-1 regulates IL-1RA gene expression under inflammatory conditions at the transcriptional level via the binding to an LRH-1 response element. Interestingly, IL-1RA induction by an intraperitoneal injection of lipopolysaccharide is significantly lower in LRH-1 heterozygous compared with wild-type mice, demonstrating the contribution of LRH-1 in IL-1RA gene regulation. Finally, RNA interference experiments indicate that LRH-1 blocks the hepatic acute phase response by, at least in part, inducing IL-1RA expression. Taken together, these results lead to the identification of IL-1RA as a novel LRH-1 target gene and demonstrate the existence of multiple mechanisms contributing to the overall anti-inflammatory properties of LRH-1 in hepatic cells.

Expression of E-Cadherin and Other Paracellular Junction Genes Is Decreased in Iron-Loaded Hepatocytes

Iron overload in the liver may occur in the clinical conditions hemochromatosis and transfusion-dependent thalassemia or by long-term consumption of large amounts of dietary iron. As iron concentrations increase in the liver, cirrhosis develops, and subsequently the normal architecture of the liver deteriorates. The underlying mechanisms whereby iron loading of hepatocytes leads to the pathology of the liver are not understood. Similarly, a direct relationship between the expression levels of paracellular junction genes and altered hepatocellular physiology has been reported; however, no relationship has been identified between iron loading and the expression of paracellular junction genes. Here, we report that the expression of numerous paracellular junction genes was decreased in iron-loaded hepatocytes, leading to increased cellular permeability, increased baculovirus-mediated gene transfer, and decreased gap junction communication. Iron loading of hepatocytes resulted in decreased E-cadherin promoter activity and subsequently decreased E-cadherin mRNA and protein expression. The data presented in this study describe a clear relationship between iron overload and decreased expression of paracellular junction genes in hepatic cells of rat and human origin.

Characterization of the human protein S gene promoter: a possible role of transcription factors Sp1 and HNF3 in liver.

Protein S is a vitamin-K-dependent plasma glycoprotein that serves as a cofactor for activated protein C in the protein C anticoagulation pathway, which regulates blood coagulation by inactivating factors Va and VIIIa. Mechanisms regulating the expression of the protein S gene remain unknown to date. The aim of this study was to characterize the cis-acting DNA elements of the human protein S gene in liver. We found that liver cell lines (HepG2 and PLC) transcribed the human protein S gene to mRNA, whereas non-liver cell lines (HEK293 and HeLa cells) either transcribed the gene weakly or not at all. Isolation and analysis of tissue-specific gene expression in HepG2 and HeLa cells of the 5'-flanking region from -6183 to +294 of the protein S gene indicated that the consensus binding motifs to HNF3 and Sp1 or MAZ transcription factors in the flanking region are essential for protein S gene expression. Exogenous expression of the Sp1 gene augmented the protein S-reporter gene expression in HepG2 or PLC cells but had no effect in HeLa cells. Taken together, we would conclude that transcription factors of HNF3, MAZ, and Sp1 are required for high-level expression of the protein S gene in hepatic cells, but in non-hepatic cells such as HeLa cells, an unknown factor(s) binds to the Sp1 region and disturbs the action of Sp1 and MAZ.

Regulation of the human p21/WAF1/Cip1 promoter in hepatic cells by functional interactions between Sp1 and Smad family members.

The cell cycle inhibitor p21/WAF1/Cip1 is expressed in many cell types and is regulated by p53-dependent and p53-independent mechanisms. p21 is an important regulator of hepatocyte cell cycle, differentiation, and liver development, but little is known about the regulation of its synthesis in hepatocytes. We report herein that the p21 gene is constitutively expressed in human hepatoma HepG2 cells. Deletion analysis of the p21 promoter showed that it contains a distal (positions -2,300/-210) and a proximal (positions -124 to -61) region that act synergistically to achieve high levels of constitutive expression. The proximal region that consists of multiple Sp1 binding sites is essential for constitutive p21 promoter activity in hepatocytes. This region also mediates the transcriptional activation of the p21 promoter by members of the Smad family of proteins, which play important role in the transduction of extracellular signals such as transforming growth factor beta, activin, etc. Constitutive expression of p21 was severely reduced by a C-terminally truncated form of Smad4 that was shown previously to block signaling through Smads. Smad3/4 and to a much lesser extent Smad2/4 caused high levels of transcriptional activation of the p21 promoter. Transactivation was compromised by N- or C-terminally truncated forms of Smad3. By using Gal4-Sp1 fusion proteins, we show that Smad proteins can activate gene transcription via functional interactions with the ubiquitous factor Sp1. These data demonstrate that Smad proteins and Sp1 participate in the constitutive or inducible expression of the p21 gene in hepatic cells.

Protein tyrosine phosphatase 2 (SHP-2) moderates signaling by gp130 but is not required for the induction of acute-phase plasma protein genes in hepatic cells.

Signals propagated via the gp130 subunit of the interleukin-6 (IL-6)-type cytokine receptors mediate, among various cellular responses, proliferation of hematopoietic cells and induction of acute-phase plasma protein (APP) genes in hepatic cells. Hematopoietic growth control by gp130 is critically dependent on activation of both STAT3 and protein tyrosine phosphatase 2 (SHP-2). To investigate whether induction of APP genes has a similar requirement for SHP-2, we constructed two chimeric receptors, G-gp130 and G-gp130(Y2F), consisting of the transmembrane and cytoplasmic domains of gp130 harboring either a wild-type or a mutated SHP-2 binding site, respectively, fused to the extracellular domain of the granulocyte colony-stimulating factor (G-CSF) receptor. Rat hepatoma H-35 cells stably expressing the chimeric receptors were generated by retroviral transduction. Both chimeric receptors transmitted a G-CSF-induced signal characteristic of that triggered by IL-6 through the endogenous gp130 receptor; i.e., both activated the appropriate JAK, induced DNA binding activity by STAT1 and STAT3, and up-regulated expression of the target APP genes, those for alpha-fibrinogen and haptoglobin. Notwithstanding these similarities in the patterns of signaling responses elicited, mutation of the SHP-2 interaction site in G-gp130(Y2F) abrogated ligand-activated receptor recruitment of SHP-2 as expected. Moreover, the tyrosine phosphorylation state of the chimeric receptor, the associated JAK activity, and the induced DNA binding activity of STAT1 and STAT3 were maintained at elevated levels and for an extended period of time in G-gp130(Y2F)-expressing cells following G-CSF treatment compared to that in cells displaying the G-gp130 receptor. H-35 cells ectopically expressing G-gp130(Y2F) were also found to display an enhanced sensitivity to G-CSF and a higher level of induction of APP genes. Overexpression of the enzymatically inactive SHP-2 enhanced the signaling by the wild-type but not by the Y2F mutant G-gp130 receptor. These results indicate that gp130 signaling for APP gene induction in hepatic cells differs qualitatively from that controlling the proliferative response in hematopoietic cells in not being strictly dependent on SHP-2. The data further suggest that SHP-2 functions normally to attenuate gp130-mediated signaling in hepatic (and, perhaps, other) cells by moderating JAK action.

Transmembrane Domain of gp130 Contributes to Intracellular Signal Transduction in Hepatic Cells

Interleukin-6 (IL-6) induces the expression of acute phase plasma protein genes in hepatic cells through the action of gp130, the signal-transducing subunit of the IL-6 receptor. To identify whether the transmembrane domain of gp130 is required for signaling function, cytoplasmic forms of gp130 were constructed that consisted of the tetramerizing N-terminal domain of Bcr linked to the transmembrane and cytoplasmic domains of gp130 (Bcr/gp130) or just to the cytoplasmic domain of gp130 (Bcr/gp130DeltaTM). The expression and function of both constructs were determined in transiently transfected COS-1 and HepG2 cells. Bcr/gp130 is capable of interacting with JAK1, JAK2, and TYK2; is constitutively active; and induces gene expression through IL-6-responsive elements. In contrast, Bcr/gp130DeltaTM, while expressed at a higher level than Bcr/gp130 and still able to interact with JAK1, is ineffective in recruiting the endogenous signal transduction pathways for inducing gene expression. However, Bcr/gp130DeltaTM initiates partial signaling in the presence of overexpressed JAK1 and TYK2, but not JAK2. The data suggest that the transmembrane domain of gp130 is necessary for signal transduction and determines the interaction with members of the Janus kinase family.

Influence of Subunit Combinations on Signaling by Receptors for Oncostatin M, Leukemia Inhibitory Factor, and Interleukin-6

Oncostatin M (OSM), leukemia inhibitory factor (LIF), and interleukin-6 (IL-6) induce expression of a similar set of acute phase plasma protein genes in hepatic cells. The redundant action of these cytokines has been ascribed to the involvement of the common signal-transducing receptor subunit, gp130, in combination with cytokine-specific, ligand-binding subunits. To define the specificity of the signal transduction by the LIF/OSM receptor (a heterodimer of gp130 and LIF receptor (LIFR)) and the OSM-specific receptor (a heterodimer of gp130 and OSM receptor (OSMR)), we reconstituted the receptor function by transfection into receptor-negative Hep3B hepatoma cells. Both receptors activate DNA binding activity of STAT1, -3, and -5B and induce gene transcription through IL-6-responsive elements. The signaling-competent cytoplasmic domain regions of OSMR and LIFR were defined by the analysis of progressive carboxyl-terminal deletion constructs. The 36 residue carboxyl-terminal region containing the distal box 3 sequence motif of OSMR is required for signal transduction by the OSM-specific receptor. In contrast, signaling by LIFR did not display the same requirement for receptor domains and was not strictly dependent on the box 3 elements. The signaling by endogenous LIF and OSM receptors differed from that by IL-6R by the prominent activation of STAT5 as shown in the mouse hepatoma cell line, Hepa-1. The data suggest that the signaling specificity of the receptors for the three cytokines is determined by the composition of the cytoplasmic domains associated in the signal-competent receptor complex and that the signaling is not identical among these cytokine receptors.

Characterization of Hepatic-specific Regulatory Elements in the Promoter Region of the Human Cholesterol 7α-Hydroxylase Gene

Cholesterol 7alpha-hydroxylase is the rate-limiting enzyme in the degradation of cholesterol to bile salts and plays a central role in regulating cholesterol homeostasis. The mechanisms involved in the transcriptional control of the human gene are largely unknown. HepG2 cells represent an appropriate model system for the study of the regulation of the gene. To identify liver-specific DNA sequences in the promoter of the human CYP7 gene, we first examined the DNase I hypersensitivity in the 5'-region of the gene. An area of hypersensitivity was observed in the region from -50 to -200 of the human gene in nuclei from transcriptionally active HepG2 cells, but was absent in transcriptionally inactive HeLa cell nuclei or in free DNA. Various 5'-promoter deletion constructs were made and transfected into HepG2 cells. About 300 base pairs of upstream sequence are required for high level promoter activity of the human CYP7 gene in HepG2 cells. DNase I footprinting of the hypersensitive region revealed nine protected sequences. Gel retardation experiments demonstrated binding of HNF-3 to the segment from -80 to -70 and of hepatocyte nuclear factor HNF-4 (and ARP-1) to the segment from -148 to -127 of the human CYP7 promoter. Deletion of either of these sites depressed promoter activity in HepG2 cells. A third region from -313 to -285 is bound by members of the HNF-3 family and acts as an enhancer. Additionally, the segment from -197 to -173 binds a negative regulatory protein that is present in Chinese hamster ovary cell extracts and in HepG2 cell extracts. These experiments define the key control elements responsible for basal transcription of the human CYP7 gene in HepG2 cells.

Interleukin-1β Induces Production of Granulocyte Colony-Stimulating Factor in Human Hepatoma Cells

Interleukin-1 (IL-1) is a proinflammatory cytokine that participates in the activation of the acute-phase plasma protein genes in hepatic cells during infection and injury. In human hepatoma HepG2 and Hep3B cells, IL-1 beta induced production of the granulocyte colony-stimulating factor (G-CSF) in a dose-dependent manner. Activation of G-CSF gene expression was an early and transient response. In HepG2 cells, G-CSF mRNA was strongly upregulated 2 hours after IL-1 beta treatment and returned to the pretreatment level by 6 hours. The secreted G-CSF was biologically active, as shown by the induction of gene transcription through the G-CSF receptor. Maximal G-CSF activity released to culture medium occurred after 8 hours. Previous studies have shown that liver expression of G-CSF was augmented in mice challenged by inflammatory stimuli. Our data suggest that IL-1 beta mediates, at least in part, this cytokine activation program in parenchymal cells and that liver-derived G-CSF may contribute to the regulation of hematopoiesis during the acute-phase response.