angiotensinogen and Liver-Neoplasms

There are currently no accurate predictive markers of metachronous liver metastasis (MLM) from colorectal cancer.. Magnetic bead-based fractionation coupled with mass spectrometry analysis was used to compare serum samples from 64 patients with MLM and 64 without recurrence or metastasis for at least 3 y after radical colorectal surgery (NM). A total of 40 MLM and 40 NM serum samples were randomly selected to build a decision tree, and the remainder were tested as blinded samples. Selected peptides were identified.. The patients in the two groups were matched for gender, age, tumor location, TNM staging, and histologic differentiation grade. Preoperative serum carcinoembryonic antigen retained no independent power to predict MLM. The decision tree model with eight proteomic features (m/z 3315, 6637, 1207, 1466, 4167, 4210, 2660, and 4186) correctly classified 33 of 40 NM sera (82.5%) and 32 of 40 MLM sera (80%) in the training set and 19 of 24 NM sera (79.2%) and 17 of 24 MLM sera (70.8%) in the test set. The peptides were identified as fragments of alpha-fetoprotein, complement C4-A, fibrinogen alpha, eukaryotic peptide chain release factor GTP-binding subunit ERF3B, and angiotensinogen.. In patients matched for gender, age, tumor location, TNM staging, and histologic differentiation grade, preoperative carcinoembryonic antigen retained no independent power to predict MLM. The decision tree model of eight proteomic features demonstrated promising value for predicting MLM in patients who underwent radical resection of colorectal cancer.

Topics: Aged; alpha-Fetoproteins; Angiotensinogen; Biomarkers, Tumor; Colorectal Neoplasms; Complement C4a; Decision Support Techniques; Female; Fibrinogen; Humans; Liver Neoplasms; Male; Middle Aged; Models, Statistical; Neoplasm Recurrence, Local; Neoplasm Staging; Neoplasms, Second Primary; Peptide Mapping; Peptide Termination Factors; Predictive Value of Tests; Proteomics

We examined the effects of hyperglycemic conditions on liver metastasis of colorectal cancer (CRC). Angiotensin (A)-II increased growth, invasion, and anti-apoptotic survival in HT29 and CT26 cells. In contrast, angiotensinogen (ATG) increased these features in HT29 cells but not in CT26 cells. HT29 cells expressed A-II type 1 receptor, chymase, and rennin, whereas CT26 cells did not express renin. Renin expression and ATG-induced cell growth, invasion, and survival induced and increased as glucose concentration increased in HT29 cells and also CT26 cells. An inhibitor of renin or chymase abrogated A-II production in HT29 cells. Reduction of hepatic ATG production by cholesterol-conjugated antisense S-oligodeoxynucleotide suppressed liver metastasis of HT29 cells. An examination of 121 CRC patients showed that diabetes in CRC cases was associated with higher blood HbA1c, higher renin and A-II concentrations in the primary tumors, and higher incidence of liver metastasis than in nondiabetic cases. These results suggest that diabetes-associated angiotensin activation enhances liver metastasis of CRC and may therefore provide a possible target for antimetastatic therapy in CRC.

Topics: Angiotensin I; Angiotensin II; Angiotensin II Type 1 Receptor Blockers; Angiotensinogen; Animals; Apoptosis; Cell Line, Tumor; Cell Proliferation; Chymases; Colonic Neoplasms; Diabetes Complications; Diabetes Mellitus; Glucose; Glycated Hemoglobin; HT29 Cells; Humans; Hyperglycemia; Liver Neoplasms; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplasm Invasiveness; Oligoribonucleotides, Antisense; Receptor, Angiotensin, Type 1; Renin; RNA Interference; RNA, Small Interfering

Angiotensinogen (AGT) is the precursor of one of the most important vasoactive hormone angiotensin II and this gene locus is associated with human essential hypertension. AGT is an acute phase protein and its gene expression is regulated by IL-6. Previous studies have identified three potential STAT-3 binding sites (APREs) located between -160 and -280 of the hAGT gene promoter but only APRE-1 (located between -271 and -279) was shown to be a bonafide enhancer for IL-6-induced promoter activity. We show here that APRE-2, located between -236 and -247, is indeed an HNF-1alpha-binding site and plays an important role in basal and IL-6 induced promoter activity of this gene. Our chromatin immunoprecipitation (ChIP) assay shows that HNF-1alpha binds to this region of the hAGT gene promoter and its recruitment is increased in the presence of IL-6 in Hep3B cells. We also show that the promoter activity of a deletion construct containing only 223 bp of the hAGT gene promoter (that contains only APRE-3) is increased after IL-6 treatment. Our ChIP assay shows that IL-6 treatment recruits STAT-3 to APRE-3 and suggests that this is also an IL6 responsive element. We have previously shown that GR binds to the proximal promoter of the hAGT gene. Since GR physically interacts with STAT-3, we propose that transcription factors GR, STAT-3, and HNF-1alpha that bind to the nucleotide sequence located between -160 and -280 of the hAGT gene promoter are responsible for IL-6 induced promoter activity of this gene.

Topics: Angiotensinogen; Base Sequence; Carcinoma, Hepatocellular; Cell Line, Tumor; Chromatin; Chromatin Immunoprecipitation; Electrophoretic Mobility Shift Assay; Hepatocyte Nuclear Factor 1-alpha; Humans; Interleukin-6; Liver Neoplasms; Molecular Sequence Data; Mutation; Promoter Regions, Genetic; Receptors, Glucocorticoid; RNA Interference; RNA, Small Interfering; STAT3 Transcription Factor; Transcription, Genetic; Transfection

Hypertension is a serious health problem particularly for African-Americans. Previous studies have suggested that angiotensinogen (AGT) gene locus is involved in human essential hypertension. We have recently shown that an A/G polymorphism at -217 in the promoter of the AGT gene is associated with essential hypertension especially in African-Americans. We report here that A/G polymorphism at -217 affects the glucocorticoid-induced promoter activity of the human AGT gene. We show that recombinant glucocorticoid receptor (GR) binds strongly to the AGT gene promoter when nucleoside A is present at -217, and dexamethasone treatment increases the interleukin 6 induced promoter activity of reporter constructs containing nucleoside A at -217. Similarly cotransfection of GR and C/EBP beta or C/EBP delta increases the promoter activity of reporter construct containing nucleoside A at -217. Since AGT is an acute phase protein, we propose that increased expression of -217A allele of the AGT gene by glucocorticoids and C/EBP family of transcription factors may be involved in essential hypertension.

Topics: Angiotensinogen; Carcinoma, Hepatocellular; CCAAT-Enhancer-Binding Protein-beta; CCAAT-Enhancer-Binding Protein-delta; CCAAT-Enhancer-Binding Proteins; Cells, Cultured; Dexamethasone; DNA Primers; Gene Expression Regulation; Glucocorticoids; Humans; Hypertension; Interleukin-6; Kidney; Liver Neoplasms; Mutation; Polymorphism, Single Nucleotide; Promoter Regions, Genetic; Receptors, Glucocorticoid; Recombinant Proteins; Regulatory Sequences, Nucleic Acid; Transcription Factors; Transcription, Genetic; Transfection

The signal transducers and activators of transcription (STATs) are cytoplasmic transcription factors mediating acute-phase response (APR) of the human angiotensinogen (hAGT) gene in hepatocytes. The mechanisms of how STAT3 activates target genes are unknown. Here we analyzed the biochemistry of STAT3 activation by interleukin (IL)-6 in hepatocellular carcinoma HepG2 and Balb/C mice.. Immunoprecipitation-Western assays and Matrix Assisted Laser Desorption-Time of Flight mass spectrometry determined sites of STAT3 acetylation by the 300-kilodalton target of E1A (p300) co-activator. The subcellular localization of acetylation-deficient STAT3 molecules were studied by microscopic imaging, effects on DNA binding measured by gel shift and chromatin immunoprecipitation (ChIP) assays, and gene transactivation by Northern blot and reporter assays.. Two Lys residues at amino acids 49 and 87 in the STAT3 NH2 terminus are acetylated by p300. Lys-to-Arg point mutations (STAT3 K49R/K87R) had no effect on inducible DNA binding, but blocked p300-mediated acetyl(Ac)-STAT3 formation and abrogated IL-6-induced hAGT activation. Although STAT3 K49R/K87R rapidly translocated into the nucleus, it did not bind p300 and had delayed cytoplasmic redistribution. ChIP assays show IL-6-inducible acetylated STAT3 and p300 binding to the native hAGT promoter. Activation of the APR in mice induces nuclear Tyr phosphorylated and acetylated STAT3 in hepatic nuclei. We also observed that STAT3 interacts with histone deacetylases (HDACs), specifically HDAC 1, that down-regulate IL-6-induced hAGT transactivation.. IL-6-induced target gene activation requires p300-mediated STAT3 acetylation, and HDACs are involved in the termination of STAT3 action. These studies indicate the acetylation-deacetylation reaction as a novel signaling mechanism controlling the IL-6-STAT3 pathway in the hepatic APR.

Topics: Acetylation; Active Transport, Cell Nucleus; Acute-Phase Reaction; Angiotensinogen; Animals; Carcinoma, Hepatocellular; Cell Line, Tumor; Cytoplasm; E1A-Associated p300 Protein; Humans; Interleukin-6; Lipopolysaccharides; Liver Neoplasms; Lysine; Mice; Mice, Inbred BALB C; Mutagenesis; Phosphorylation; Promoter Regions, Genetic; Signal Transduction; STAT3 Transcription Factor; Transcriptional Activation; Tyrosine

Several genetic polymorphisms have been identified in the proximal promoter of angiotensinogen ( AGT). Gene titration experiments in transgenic animals have demonstrated that small increases in the basal expression of AGT can lead to elevated blood pressure. The direct proof that promoter variants of AGT can lead to elevated blood pressure will ultimately require the development of specific animal models. Before such work can be contemplated, however, a formal understanding of the mechanisms controlling transcriptional activation of AGT needs to be developed. Analysis of DNA-protein interactions in vitro and transactivation experiments in cultured cells reveal the critical role of an Sp1 binding site immediately upstream of the TATA box of AGT in both mouse and human. Both sites are required for transcription initiation in the mouse. By contrast, a minimal human AGT promoter can initiate transcription in the absence of either this Sp1 site or the TATA box, albeit at a lower level. Further analysis and consideration of these interspecific differences will be essential for the development of meaningful animal models to probe the mechanism by which AGT may predispose to human essential hypertension.

Topics: Angiotensinogen; Animals; Base Sequence; Carcinoma, Hepatocellular; Deoxyribonuclease I; DNA Footprinting; DNA Primers; Electrophoretic Mobility Shift Assay; Humans; Liver Neoplasms; Luciferases; Mice; Molecular Sequence Data; Plasmids; Promoter Regions, Genetic; Rats; Sequence Homology, Nucleic Acid; Sp1 Transcription Factor; Transcription, Genetic; Transfection; Tumor Cells, Cultured

The circulating level of angiotensinogen (AGT) is dynamically regulated as an important determinant of blood pressure and electrolyte homeostasis. Because the mechanisms controlling the regulated expression of human angiotensinogen (hAGT) are unknown, we investigated the inducible regulation of the hAGT gene in well differentiated HepG2 cells. Interleukin-6 (IL-6) stimulation produced a 3.2-fold increase in hAGT mRNA peaking at 96 h after stimulation. Deletional mutagenesis of the hAGT promoter in transient transfection assays identified an IL-6 response domain between nucleotides -350 and -122 containing three reiterated motifs, termed human acute phase response elements (hAPREs). Although mutation of each site individually caused a fall in IL-6-inducible luciferase activity, mutation of all three sites was required to block the IL-6 effect. Electrophoretic mobility shift assay (EMSA), supershift, and microaffinity DNA binding assays indicate IL-6-inducible high-affinity binding of signal transducers and activators of transcription 1 and -3 (STAT1 and -3) to hAPRE1 and -3 but only low-affinity binding to hAPRE2. Expression of a dominant-negative form of STAT3, but not STAT1, produced a concentration-dependent reduction in IL-6-induced hAGT transcription and endogenous mRNA expression. These data indicate that STAT3 plays a major role in hAGT gene induction through three functionally distinct hAPREs in its promoter and suggest a mechanism for its up-regulation during the acute-phase response.

Topics: Acute-Phase Proteins; Angiotensinogen; Base Sequence; Binding, Competitive; Carcinoma, Hepatocellular; DNA-Binding Proteins; Enhancer Elements, Genetic; Gene Expression Regulation; Genes, Dominant; Genes, Reporter; Humans; Interleukin-6; Kinetics; Liver Neoplasms; Luciferases; Molecular Sequence Data; Promoter Regions, Genetic; Regulatory Sequences, Nucleic Acid; Response Elements; RNA, Messenger; Sequence Deletion; Signal Transduction; STAT1 Transcription Factor; STAT3 Transcription Factor; Trans-Activators; Transcriptional Activation; Tumor Cells, Cultured

Angiotensinogen is the glycoprotein precursor of one of the most potent vasoactive hormones angiotensin-II which plays an important role in the regulation of blood pressure. We show here that the promoter activity of reporter constructs containing human angiotensinogen promoter is increased by cAMP treatment on transient transfection in HepG2 cells. We have identified a composite cAMP responsive element, located around 840 bases upstream from the transcriptional initiation site, in the promoter of human angiotensinogen gene. This element is recognized by members of CREB/ATF as well as C/EBP family of transcription factors. Another C/EBP binding site that is not recognized by CREB is located 10 bases upstream from this site. We show that co-transfection of CREB increases the promoter activity of reporter constructs containing human angiotensinogen gene promoter attached to the CAT gene. We also show that co-transfection of DBP (which is a member of C/EBP family of transcription factors) increases promoter activity of these reporter constructs.

Topics: Activating Transcription Factor 1; Angiotensinogen; Base Sequence; Binding Sites; Carcinoma, Hepatocellular; CCAAT-Enhancer-Binding Proteins; Chloramphenicol O-Acetyltransferase; Cyclic AMP; Cyclic AMP Response Element-Binding Protein; DNA-Binding Proteins; Gene Expression Regulation; Genes, Reporter; Humans; Liver Neoplasms; Promoter Regions, Genetic; Transcription Factors; Transfection; Tumor Cells, Cultured

-Angiotensinogen is the glycoprotein precursor of 1 of the most potent vasoactive hormones, angiotensin II. Human angiotensinogen gene contains a C/A polymorphism at -20 located between the TATA box and transcriptional initiation site. We show here that when nucleoside A is present at -20, this sequence binds to the estrogen receptor. We also show that transcriptional activity of reporter constructs containing human angiotensinogen gene promoter with nucleoside A at -20 is increased on cotransfection of an expression vector containing human estrogen receptor-alpha coding sequence in human hepatoma cells (HepG2) followed by estrogen treatment. On the other hand, adenoviral major late transcription factor binds preferentially to this region of the promoter when nucleoside C is present at -20. We also show that reporter constructs containing human angiotensinogen gene promoter with nucleoside C at -20 have increased basal promoter activity on transient transfection in HepG2 cells as compared with reporter constructs with nucleoside A at -20. Our data suggest that C/A polymorphism at -20 may modulate the expression of human angiotensinogen gene in a sex-specific manner.

Topics: Adenoviridae; Angiotensinogen; Base Sequence; Carcinoma, Hepatocellular; Estradiol; Gene Expression; Gene Expression Regulation; Genes, Reporter; Genetic Vectors; Humans; Liver Neoplasms; Molecular Sequence Data; Plasmids; Polymorphism, Genetic; Promoter Regions, Genetic; Receptors, Estrogen; Transcription Factors; Transfection; Tumor Cells, Cultured

Human angiotensinogen gene contains a C/A polymorphism at 20 bases upstream from the transcriptional initiation site. This sequence binds to the estrogen receptor when nucleoside A is present at this site and reporter constructs containing human angiotensinogen gene promoter with nucleoside A at -20 are transactivated on co-transfection of estrogen receptor in HepG2 cells followed by estrogen treatment. We show here that orphan receptor, Arp-1, which belongs to the COUP family of transcription factors also binds to this sequence. Co-transfection of Arp-1 reduces estrogen induced promoter activity of reporter constructs containing human angiotensinogen gene promoter. On the other hand co-transfection of Arp-1 does not have a significant effect on estrogen induced promoter activity of reporter constructs containing rat angiotensinogen gene promoter. Our data suggests that human and rat angiotensinogen genes are regulated in a different manner by estrogens.

Topics: Angiotensinogen; Animals; Base Sequence; Carcinoma, Hepatocellular; Chloramphenicol O-Acetyltransferase; COUP Transcription Factor II; COUP Transcription Factors; DNA-Binding Proteins; Estrogens; Genes, Reporter; Humans; Liver Neoplasms; Polymorphism, Genetic; Promoter Regions, Genetic; Rats; Receptors, Estrogen; Receptors, Steroid; Recombinant Fusion Proteins; Recombinant Proteins; Regulatory Sequences, Nucleic Acid; Sequence Alignment; Sequence Homology, Nucleic Acid; TATA Box; Transcription Factors; Transcription, Genetic; Transfection; Tumor Cells, Cultured

Angiotensinogen (AGT) has been linked to hypertension. Because there are no direct inhibitors of AGT, we have developed antisense (AS) inhibition of AGT mRNA delivered in an adeno-associated virus (AAV)-based plasmid vector. This plasmid, driven by the cytomegalovirus promoter, contains a green fluorescent protein reporter gene and AS cDNA for rat AGT. Transfection of the plasmid into rat hepatoma cells brought a strong expression of the transgenes and a significant reduction in the level of AGT. In the in vivo study, naked plasmid DNA was intravenously injected into adult spontaneously hypertensive rats at different doses (0.6, 1.5, and 3 mg/kg). Expression of AGT AS mRNA was present in liver and heart, and it lasted longer in the liver. All three doses produced a significant decrease in blood pressure (BP). BP decreased for 2, 4, and 6 days, respectively. The lowest dose decreased BP by 12 +/- 3.0 mmHg, whereas the higher doses decreased BP by up to 22.5 +/- 5.2 mmHg compared with the control rats injected with saline (P < 0.01). The injection of the plasmid with liposomes produced a more profound and longer reduction (8 days) in BP. Consistent changes in plasma AGT level were observed. Sense plasmid had no effect. No liver toxicity was observed after injection of AS plasmid with or without liposomes. Our results suggest that the systemic delivery of AS against AGT mRNA by AAV-based plasmid vector, especially with liposomes, may have potential for gene therapy of hypertension and that further studies with the plasmid packaged into a recombinant AAV vector for a longer-lasting AS effect are warranted.

Topics: Angiotensinogen; Animals; Blood Pressure; Carcinoma, Hepatocellular; Cytomegalovirus; Dependovirus; Genes, Reporter; Genetic Vectors; Green Fluorescent Proteins; Hypertension; Injections, Intravenous; Liver; Liver Neoplasms; Luminescent Proteins; Male; Myocardium; Oligodeoxyribonucleotides, Antisense; Promoter Regions, Genetic; Rats; Rats, Inbred SHR; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Systole; Time Factors; Transcription, Genetic; Transfection; Tumor Cells, Cultured

Angiotensinogen is the glycoprotein precursor of one of the most potent vasoactive hormones, angiotensin-II. It has been shown recently that an ATF like element (ALE) located between bases -102 and -87 of the human angiotensinogen gene plays an important role in liver specific expression of this gene and binds to CREB/ATF family of transcription factors and a novel factor (ALF). We show here that this sequence binds to the liver enriched transcription factor DBP and cotransfection of expression vector CMV-DBP increases the expression of reporter constructs containing this sequence. In addition, we show that transcription factor C/EBP-delta binds to this sequence and an expression vector containing C/EBP-delta coding region increases the expression of reporter constructs containing this sequence. Since DBP is involved in circadian rhythm, our studies suggest that this sequence may be involved in circadian expression of the human angiotensinogen gene.

Topics: Activating Transcription Factors; Angiotensinogen; Blood Proteins; Carcinoma, Hepatocellular; DNA-Binding Proteins; Gene Expression Regulation; Genes, Reporter; Humans; Liver; Liver Neoplasms; Organ Specificity; Promoter Regions, Genetic; Recombinant Fusion Proteins; Transcription Factors; Tumor Cells, Cultured

Angiotensinogen is the precursor protein of angiotensin II that is involved in regulating blood pressure and electrolyte homeostasis, and it is mainly synthesized in the liver. In the present study, we analyzed the human angiotensinogen proximal promoter region by means of Chloramphenicol acetyltransferase assays, and suggested that the region from -106 to +44 is sufficient for hepatoma cell line (HepG2)-specific expression. Electrophoretic mobility shift assays using ALE (ATF-like element, -102 to -87) fragment identified CREB/ATF family nuclear factors and novel ones, ALF (ALE-binding factor). The deletion and in vivo competition of ALE decreased the human angiotensinogen promoter activity. Furthermore, the heterologous promoter analysis demonstrated that ALE acts as a HepG2-dependent activating element. These results indicate that ALE plays an important role in hepatic expression of human angiotensinogen gene.

Topics: Activating Transcription Factors; Angiotensinogen; Blood Proteins; Carcinoma, Hepatocellular; Chloramphenicol O-Acetyltransferase; Humans; Kinetics; Liver; Liver Neoplasms; Nuclear Proteins; Promoter Regions, Genetic; Recombinant Fusion Proteins; Transcription Factors; Transfection; Tumor Cells, Cultured

The renin-angiotensin system controls blood pressure through the enzymatic production of the vasopressor angiotensin II (AII) from the angiotensinogen (AGT) precursor. Intravascular AII production stimulates de novo synthesis of its precursor in a positive feedback loop through increased gene expression. In this study, we investigate the effects of AII on AGT gene expression. At nanomolar concentrations, All activates transcription of the native AGT gene; this region is mapped to the AGT gene multihormone-inducible enhancer (-615 to -470). Within the multihormone-inducible enhancer, site-directed mutations of the acute-phase response element (APRE) that interfere with nuclear factor-kappa B (NF-kappa B) transcription factor binding also abolish All responsiveness. The APRE functions as a rapidly inducible All-inducible enhancer with peak reporter activity detected after a 4-h stimulation; this effect occurs only when the type 1 AII receptor is expressed. All induces sequence-specific NF-KB binding to the APRE in HepG2 nuclear extracts. Moreover, AII infusions of primary rat hepatocyte cultures produces a rapid 4-fold increase in sequence-specific NF-kappa B binding to the APRE. Antibodies against the transcriptional activator subunit, Rel A, quantitatively supershift the nucleoprotein complex, whereas antibodies to other NF-kappa B members do not, demonstrating that Rel A APRE-binding activity is AII-inducible. Transient overexpression of Rel A(1-551) activates the AGT multihormone-inducible enhancer. AII-inducible domains of Rel A were mapped by cotransfecting a chimeric GAL4-Rel A fusion protein with a reporter gene containing GAL4-binding sites. GAL4-Rel A(1-551) was an AII-inducible transactivator. Deletion of the NH(2)-terminal 254 amino acids of Rel A produces a constitutive transactivator, indicating that Rel A is activated by AII in a manner dependent on its NH(2) terminus. These studies define one mechanism for the renin-angiotensin system positive feedback loop in hepatocyctes.

Topics: Angiotensin II; Angiotensinogen; Animals; Blood Pressure; Carcinoma, Hepatocellular; Enhancer Elements, Genetic; Feedback; Gene Expression Regulation; Humans; Liver; Liver Neoplasms; Macromolecular Substances; Models, Biological; NF-kappa B; Proto-Oncogene Proteins; Rats; Rats, Sprague-Dawley; Recombinant Fusion Proteins; Renin-Angiotensin System; Signal Transduction; Transcription Factor RelA; Transcription Factor RelB; Transcription Factors; Transcriptional Activation; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha

In the present study effects of estrogens (natural estradiol and synthetic ethinyl estradiol) on liver derived proteins (angiotensinogen, IGF-I) were investigated in vivo in ovariectomized rats and in vitro in a rat hepatoma cell line (Fe33). The aim of this study was to establish both an animal and an in vitro model for quantification of the hepatic activity of given estrogenic compounds, and to study underlying mechanisms as regards the question of direct or indirect mode of estrogen action. In ovariectomized rats subcutaneous (s.c.)-treatment for 11 days with either estradiol (E2) or ethinyl estradiol (EE) (dose range 0.1-3 micrograms/animal/day) induced a comparable dose-dependent increase in uterine weight indicating a similar estrogenic potency of the two estrogens. Equipotency was also found as regards the effects on IGF-I plasma levels which dose-dependently decreased by about 50% at the highest dose tested (3 micrograms/animal/day). The decrease in IGF-I serum levels was accompanied by a significant 40% decrease in liver IGF-I mRNA. In contrast angiotensinogen plasma levels were affected only by EE (60% increase for the 3 micrograms/animal/day dose) but not by E2. When rats, in addition to ovariectomy, were also hypophysectomized (substituted with human growth hormone and dexamethasone) angiotensinogen again increased by 80% upon administration of 3 micrograms/animal/day EE, whereas IGF-I remained unaffected by EE. In a rat hepatoma cell line (Fe33) which is stably transfected with an estrogen receptor expression plasmid, 10 nmol/l EE for 24 h caused a 2.4-fold increase in angiotensinogen mRNA level. We conclude from our studies that estrogen effects on angiotensinogen serum levels in the rat are direct effects via the hepatic estrogen receptor, whereas estrogen effects on IGF-I serum levels are indirect effects, the primary target of estrogen action being probably the pituitary. The changes in angiotensinogen serum levels in the rat model are comparable to the situation in humans indicating the rat model and the Fe33 model to be useful tools to study the hepatic activity of estrogenic compounds.

Topics: Angiotensinogen; Animals; Base Sequence; Carcinoma, Hepatocellular; Dose-Response Relationship, Drug; Estradiol; Ethinyl Estradiol; Female; Hypophysectomy; Insulin-Like Growth Factor I; Kinetics; Liver; Liver Neoplasms; Molecular Sequence Data; Organ Size; Ovariectomy; Rats; Rats, Wistar; RNA, Messenger; Tumor Cells, Cultured; Uterus

MyoD is one of the regulatory genes that causes activation of the expression of muscle specific genes in fibroblasts through interaction with a cis-acting DNA element CANNTG (E-box). We show here that forced expression of MyoD in mouse fibroblast C3H10T1/2 cells induces the expression of the liver specific rat angiotensinogen gene promoter. In the absence of MyoD, the angiotensinogen promoter is not expressed in C3H10T1/2 fibroblasts. Cotransfection of MyoD has no significant effect on the angiotensinogen promoter activity in the liver-derived HepG2 cells, suggesting that the regulatory programs of HepG2 and C3H10T1/2 cells are different, at least in terms of the requirement for MyoD-mediated transactivation of the target angiotensinogen promoter.

Topics: Angiotensinogen; Animals; Base Sequence; Carcinoma, Hepatocellular; Fibroblasts; Helix-Loop-Helix Motifs; Humans; Liver; Liver Neoplasms; Mice; Mice, Inbred C3H; Molecular Sequence Data; Muscle Proteins; MyoD Protein; Promoter Regions, Genetic; Protein Binding; Rats; Recombinant Fusion Proteins; Regulatory Sequences, Nucleic Acid; Repetitive Sequences, Nucleic Acid; Transcriptional Activation; Tumor Cells, Cultured

Topics: Acute-Phase Reaction; Angiotensinogen; Animals; Carcinoma, Hepatocellular; DNA Probes; Gene Expression Regulation; Liver; Liver Neoplasms; Mice; Mice, Inbred C57BL; Mice, Transgenic; Nucleic Acid Hybridization; RNA, Messenger; Sequence Homology, Nucleic Acid; Species Specificity; Transcription, Genetic; Tumor Cells, Cultured

We have generated two lines of transgenic mice with integrated copies of a 14-kilobase pair (kb) human DNA fragment containing the angiotensinogen gene, which includes 1.3 kb of 5'- and 3'-flanking regions. In both transgenic lines, a considerable quantity of the correctly initiated and processed angiotensinogen mRNA was detected in the liver and it was detectable in heart. Unexpectedly, mRNA for the transgene was accumulated in the kidney, where is normally the minor source of angiotensinogen, to levels comparable to that in the liver. In addition, an in vitro transfection analysis suggested that the 1.3-kb 5'-flanking sequences are essential for expression of the angiotensinogen gene in hepatic and renal cells and that neither DNA segment within the 14-kb construct contributes significantly to repression of the gene expression in renal cells.

Topics: Angiotensinogen; Animals; Base Sequence; Blotting, Northern; Carcinoma, Hepatocellular; Cell Line; Chloramphenicol O-Acetyltransferase; Gene Expression; Humans; Liver Neoplasms; Mice; Mice, Transgenic; Molecular Sequence Data; Oligodeoxyribonucleotides; Organ Specificity; Rats; Recombinant Proteins; Restriction Mapping; RNA Probes; RNA, Messenger; Transfection

Binding characteristics and effects of 3,5,-3'-triiodo-L-thyronine (T3) on angiotensinogen production in HepG2 were studied in serum-free medium. Binding was performed on intact cells and on partially purified isolated nuclei using [125I]T3. Scatchard plots revealed one class of high affinity binding sites with a Kd of approximately 80 pmol/liter. Calculation of maximum binding showed that HepG2 possess approximately 1000 binding sites per cell. Unlabeled T3 and T4 competed for binding sites on intact HepG2 with 50% inhibition of [125I] T3 binding at approximately 3.0 and 38.0 pmol/liter, respectively. The HepG2 showed a dose-dependent increase in angiotensinogen production in serum-free medium which was maximal at 10(-5) mol/liter (two-fold increase/10(6) cells/24 h) and had an EC50 of approximately 5.0 x 10(-8) mol/liter. T3 also produced after 24 h a dose-dependent increase in DNA highly correlated with T3 applied (r = 0.88, P less than 0.01). In conclusion, this study shows that HepG2 possess specific high affinity binding sites for T3 and that T3 stimulates angiotensinogen production and DNA synthesis in these cells.

Topics: Angiotensinogen; Binding, Competitive; Carcinoma, Hepatocellular; Cell Line; Cell Nucleus; Humans; Kinetics; Liver Neoplasms; Receptors, Thyroid Hormone; Thyroxine; Triiodothyronine

The hepatic transcription of the angiotensinogen gene is regulated by both glucocorticoids and cytokines generated as products of the acute phase reaction. We have identified a multimodular enhancer in the 5'-flanking region of the rat angiotensinogen gene that mediates these responses and consists of an acute phase response element (APRE) flanked on both sides by adjacent glucocorticoid response element consensus motifs (GREs). Induction of transcription by the cytokine interleukin-1 (IL-1) is glucocorticoid dependent and mediated through the APRE. The APRE binds in a mutually exclusive manner a cytokine/phorbol ester-inducible protein (BPi), indistinguishable from nuclear factor kB, and a family of constitutive liver proteins (BPcs) related to the heat-stable transcription factor C/EBP. Using mutated 5'-flanking sequences of the angiotensinogen gene fused to a firefly luciferase reporter gene transfected into hepatoblastoma (HepG2) cells, we have mapped enhanson sequences required for the transcriptional response to glucocorticoids. Two functionally distinct GREs are identified by deletion and site-directed mutagenesis, both of which mediate glucocorticoid-stimulated transcription in vivo. Glucocorticoid-induced transcription mediated by the angiotensinogen gene enhancer is, furthermore, dependent on the occupancy of the APRE by either the BPi or a member of the BPc family because a mutant APRE that binds neither BPi nor BPc exhibits an attenuated glucocorticoid responsiveness. Mutant APREs that permit exclusive binding of either BPi or BPc synergistically transmit the glucocorticoid response mediated by one or the other of the adjacent GREs. Thus, the induction of angiotensinogen gene transcription involves interaction between the glucocorticoid receptor and either one of the APRE-binding proteins: either the cytokine-inducible NFkB or the constitutive family of C/EBP-like proteins, bound to adjacent enhansons in a mutually synergistic enhancer complex.

Topics: Acute-Phase Proteins; Angiotensinogen; Animals; Base Sequence; Carcinoma, Hepatocellular; Chromosome Deletion; Enhancer Elements, Genetic; Glucocorticoids; Humans; Interleukin-1; Liver Neoplasms; Luciferases; Molecular Sequence Data; Mutagenesis, Site-Directed; Promoter Regions, Genetic; Rats; Recombinant Fusion Proteins; Transcription, Genetic; Transfection; Tumor Cells, Cultured

The gene encoding angiotensinogen, the glycoprotein precursor of the potent vasopressor peptide angiotensin II, is transcriptionally activated in hepatocytes during the acute-phase response through interactions of mutually cooperative glucocorticoid receptors and proteins that bind to an acute-phase response element (APRE) 5'-AGTTGGGATTTCCCAACC-3'. The APRE binds a family of constitutive proteins (BPcs) and a cytokine inducible protein (BPi) that is indistinguishable from nuclear factor kappa B (NF kappa B). The interactions of purified proteins with the APRE were studied by in vitro binding and in vivo transcriptional trans-activation assays. BPc is a family of heat-stable DNA binding proteins, the different sized members of which are capable of forming heterodimers. BPcs are recognized by anti-C/EBP antiserum and produce a footprint similar to bacterially expressed C/EBP on the APRE. BPi has a 4- to 5-fold greater affinity for the APRE than the BPcs, and contacts guanosine residues distinct from those contacted by the BPcs, demonstrating that these two classes of proteins contain functionally distinct DNA binding domains. Assays of APRE-luciferase reporter plasmids transfected into HepG2 cells show that a mutated APRE that binds only BPi functions as an IL-1 alpha inducible enhancer, whereas a mutated APRE that binds only BPc does not. The APRE mutant that binds the C/EBP-like BPcs to the exclusion of BPi functions as an uninducible basal enhancer both in the native context of the angiotensinogen gene and when multimerized and placed upstream of a minimal angiotensinogen promoter. The wild-type APRE that binds both BPi and BPc is less inducible by IL-1 alpha than the mutated APRE that binds only BPi. Gel shift competition assays demonstrate in vitro that the mechanism of transcriptional regulation by the APRE involves a competition between BPc and the inducible BPi for binding to the APRE. IL-1 alpha stimulation of hepatocytes leads to nuclear translocation of the NF kappa B-like BPi which competes with the constitutive C/EBP-like BPcs for overlapping binding sites on the APRE and thereby replaces weak transcriptional activators with a stronger one.

Topics: Angiotensinogen; Animals; Base Sequence; Binding Sites; Carcinoma, Hepatocellular; CCAAT-Enhancer-Binding Proteins; Cell Line; Cell Nucleus; DNA-Binding Proteins; Gene Expression Regulation; Genes; Humans; Interleukin-1; Liver; Liver Neoplasms; Models, Genetic; Molecular Sequence Data; NF-kappa B; Nuclear Proteins; Plasmids; Rats; Restriction Mapping; Templates, Genetic; Transcription Factors; Transcription, Genetic

The cellular mechanism by which the angiotensin II (AII) agonist, Sar1-AII, inhibits production and release of angiotensinogen in human hepatoma HepG2 cells was examined. Pretreatment of HepG2 cells with pertussis toxin attenuated the ability of Sar1-AII to block angiotensinogen production. This effect could be correlated with the in situ ADP-ribosylation of a protein(s) of apparent molecular weight 39,000-41,000 on SDS-PAGE, and attenuation of the ability of Sar1-AII to inhibit cAMP accumulation. The role of cAMP in angiotensinogen production was examined. A transient increase in cAMP accumulation above basal could be evoked by forskolin (8-fold) or by glucagon (5-fold) using insulin-deficient media. Although neither forskolin nor glucagon had a significant effect on angiotensinogen production agents producing a sustained increase in intracellular cAMP (8-bromo-cAMP, dibutyryl-cAMP, cholera toxin) were able to increase angiotensinogen production. Although these data indicate that intracellular cAMP is a regulatory factor in angiotensinogen production other evidence suggests that modulation of intracellular cAMP is not entirely responsible for the effects of Sar1-AII.

Topics: Adenosine Diphosphate Ribose; Angiotensin II; Angiotensinogen; Carcinoma, Hepatocellular; Colforsin; Cyclic AMP; Glucagon; GTP-Binding Proteins; Humans; Liver Neoplasms; Neoplasm Proteins; Pertussis Toxin; Protein Processing, Post-Translational; Second Messenger Systems; Tumor Cells, Cultured; Virulence Factors, Bordetella

Angiotensinogen is the glycoprotein precursor of angiotensin II, an octapeptide hormone important for the regulation of blood pressure and volume homeostasis. The gene encoding angiotensinogen is expressed in liver and several other tissues, providing a model gene for understanding the role of cis-acting DNA control elements and trans-acting factors in tissue-type specific gene expression. To identify DNA control elements in the rat angiotensinogen gene we prepared an array of fusion genes consisting of either 5' or 3'-deleted sequences of the 5'-flanking region of the gene linked to a firefly luciferase reporter gene and analyzed the relative cellular specificity of expression of these genes after their introduction into hepato-carcinoma cells (Hep G2) that do express and placental cells (JEG-3) that do not express the endogenous angiotensinogen gene. Six transcriptionally active elements were found within 688 base pairs of 5'-flanking DNA. The interactions of DNA binding proteins with these elements was demonstrated by their specific protection to digestion with DNase I in the presence of liver cell extracts. The orientation and spatial requirements for transcription of two of the elements were analyzed further by the construction and expression of synthetic oligonucleotide cassettes incorporating the sequences of these elements when linked to a homologous (angiotensinogen) or a heterologous Simian virus 40 promoter and enhancer. One element located between 60 and 108 base pairs from the start of gene transcription functioned either as a silencer or an enhancer of transcription (SOAP box element), depending upon the distance from the angiotensinogen and viral gene promoters. Moreover, the SOAP box element demonstrated enhancer activity in JEG-3 cells when linked to the Simian virus 40 early promoter. An oligonucleotide mutation of the SOAP box element interfered with protein binding in a gel mobility shift assay and this mutant was transcriptionally inactive in both homologous and heterologous promoters. These observations indicate that expression of the angiotensinogen gene in liver cells is coordinately regulated by multiple cis-acting elements that interact with DNA binding proteins.

Topics: Angiotensinogen; Animals; Base Sequence; Carcinoma, Hepatocellular; Cell Line; DNA; Gene Expression Regulation; Humans; Liver; Liver Neoplasms; Molecular Sequence Data; Rats; Rats, Inbred Strains

We have prepared and purified a rabbit polyclonal antibody (PcAb) and two mouse monoclonal antibodies (McAbs) against human angiotensinogen. The PcAb (Kd: 4.0 X 10(-12) M) inhibits 50% of the hydrolytic activity of renin on angiotensinogen, at a final dilution of 1:800. The two monoclonal antibodies (Kd: 5.0 X 10(-11) and 9.0 X 10(-13) M) do not inhibit the enzymatic reaction. None of the antibodies showed displacement of 125l-labeled angiotensinogen by angiotensin I, angiotensin II or human tetradecapeptide. The polyclonal antibodies recognize marmoset and baboon angiotensinogen with an affinity 10(3)lower than that of the human angiotensinogen, whereas the McAbs do not recognize primate angiotensinogen. Since the two monoclonal antibodies recognize different epitopes of the human angiotensinogen molecule than the polyclonal antibody, it is therefore possible to use them in various sandwich assays as ELISA. Thus, we have developed a liquid phase radioimmunoassay and an ELISA which allowed to measure human plasma angiotensinogen, under several pathophysiological conditions, and that produced by human hepatocyte cells in culture (HepG2).

Topics: Angiotensinogen; Animals; Antibodies, Monoclonal; Carcinoma, Hepatocellular; Enzyme-Linked Immunosorbent Assay; Humans; Liver; Liver Neoplasms; Mice; Rabbits; Radioimmunoassay; Renin; Tumor Cells, Cultured

Angiotensinogen is the precursor of biologically active peptide angiotensin II and its synthesis is increased in the liver during acute inflammation. We have used radiolabeled human angiotensinogen cDNA to study the effect of hepatocyte stimulating factor (HSF), a protein synthesized in differentiating monocytes which increases the synthesis of various hepatic proteins during inflammation, on angiotensinogen mRNA levels in human hepatoma cells (HepG2). Our results indicate that angiotensinogen mRNA is present in human hepatoma (HepG2) cells and its levels are decreased when treated with hepatocyte stimulating factor. Although dexamethasone elevated angiotensinogen mRNA levels, HSF reduced this increase. These results suggest that a factor other than HSF may be involved in elevating the angiotensinogen mRNA levels in the liver during inflammation.

Topics: Angiotensinogen; Carcinoma, Hepatocellular; Cell Line; Dexamethasone; Gene Expression Regulation; Genes; Humans; Interleukin-6; Liver Neoplasms; Monocytes; Nucleic Acid Hybridization; Proteins; RNA, Messenger; RNA, Neoplasm; Transcription, Genetic

The effects of the estrogen analog mestranol and of the antiestrogen tamoxifen on cell growth and the rate of angiotensinogen production were investigated in HepG2 cells, an hepato-carcinoma cell line of human origin. After 36 h of cell contact with high concentration of mestranol, a (10(-5) M) dose increased by 2-fold the rate of proliferation of HepG2 while reducing angiotensinogen production to below control level. Mestranol at 10(-6) M preferentially stimulated angiotensinogen production 5-fold, whereas cell growth rate was slightly increased. Comparable results were obtained for thymidine uptake in the course of the cell cycle, with a maximum increase for 10(-5) M mestranol, and an increase of angiotensinogen production for 10(-6) M mestranol. At 10(-6) M, tamoxifen acted as a pure antagonist by strongly inhibiting the stimulatory effect of mestranol and reducing angiotensinogen production to below the control level within 60 h. Tamoxifen did not affect the growth rate of HepG2 cells, either when administered alone or together with an equimolar concentration of mestranol.

Topics: Angiotensinogen; Carcinoma, Hepatocellular; Cell Cycle; Cell Line; Fluorescent Antibody Technique; Humans; Liver; Liver Neoplasms; Mestranol; Tamoxifen