UMLS:C0019204 - FACTA Search

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Query: UMLS:C0019204 (hepatocellular carcinoma)
71,386 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Transcription of the phosphoenolpyruvate carboxykinase gene is stimulated by glucocorticoids, retinoic acid, and cAMP and is dominantly inhibited by insulin and phorbol esters. The glucocorticoid response is mediated by a complex regulatory unit that consists of two glucocorticoid receptor (GR) binding sites (GR1 and GR2) and two adjacent accessory factor elements (AF1 and AF2). Deletion of either the AF1 or the AF2 element results in a 50-75% reduction of the glucocorticoid response. In addition to their accessory role in glucocorticoid action, the AF1 and AF2 elements mediate retinoic acid and insulin/phorbol ester effects, respectively. Site-directed mutagenesis was performed on AF1 and AF2 to precisely locate the sequences responsible for accessory activity in each element. The glucocorticoid accessory activity of the AF1 element maps to the same 12-base pair sequence (TGACCTTTGGCC) involved in the response of the PEPCK gene to retinoic acid. The glucocorticoid accessory activity of the AF2 region maps to the same 10-base pair sequence (TGGTGTTTTG) responsible for mediating the insulin and phorbol ester responses through this element. The AF1 and AF2 elements bind different sets of nuclear proteins, and this binding is not qualitatively or quantitatively affected by treatment of the rat H4IIE hepatoma cells with retinoic acid (AF1) or insulin (AF2). AF2 functions in a heterologous context (a consensus glucocorticoid response element and the thymidine kinase promoter), whereas AF1 functions in this context only if the retinoic acid receptor is overexpressed in the cells. These results show that the AF1 and AF2 elements affect the glucocorticoid response through different protein DNA interactions, and that a small sequence in each serves multiple functions. Together with GR1 and GR2, they form a complex hormone response unit which provides an integrated response of the phosphoenolpyruvate carboxykinase gene to a variety of positive and negative signals.
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PMID:Integration of multiple signals through a complex hormone response unit in the phosphoenolpyruvate carboxykinase gene promoter. 805 68

We used the 3T3-F442A adipocytes and the FAO hepatoma cells to analyze the effect of oleate on phosphoenolpyruvate carboxykinase (PEPCK) gene expression. In serum-deprived, glucose-free medium, 1 mM oleate, bound to albumin in a 6:1 ratio, specifically stimulated PEPCK mRNA. In 3T3-F442A adipocytes, the maximum 5-fold increase occurred in 4 hours then rapidly declined to reach the basal level 20 hours later. This increase was cycloheximide-independent and actinomycin D-dependent, suggesting a direct, transcriptional effect of oleate. FAO cells also responded to oleate with a transient induction of PEPCK mRNA, although the extent of stimulation was lower. Thus, the PEPCK gene provides a useful molecular tool for studying the mechanisms by which fatty acids stimulate gene expression.
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PMID:Stimulation of phosphoenolpyruvate carboxykinase gene expression by fatty acids. 807 82

The gene for phosphoenolpyruvate carboxykinase (GTP) (EC 4.1.1.32) (PEPCK) is expressed in a tissue-specific manner in the liver, kidney, and adipose tissue and is regulated by hormones including cAMP and insulin. Previous studies have shown that the CCAAT/enhancer-binding protein alpha (C/EBP alpha) binds to several sites on the PEPCK promoter and activates transcription from the promoter in hepatoma cells. Here, we report that a second member of the C/EBP family, C/EBP beta, bound to the same sites on the PEPCK promoter. However, C/EBP beta stimulated transcription primarily through the cAMP-responsive element (CRE), which maps between positions -77 to -94, but not at the more 5'-binding sites. In addition, the nuclear factor-1 site, which is immediately adjacent to the CRE in the PEPCK promoter, was also required for the full response of the promoter to cotransfected C/EBP beta. In gel mobility assays, antibodies to both C/EBP beta and the cAMP regulatory element-binding protein (CREB), but not to C/EBP alpha, "supershifted" DNA-protein complexes formed between a synthetic CRE oligomer and proteins prepared from rat liver nuclei. C/EBP beta mRNA was expressed at low levels in both the periportal and pericentral regions of the liver lobule, whereas expression of the gene for C/EBP alpha was confined to the pericentral region of the liver lobule. PEPCK gene transcription is greatest in the periportal region of the liver. CREB also bound to the CRE and stimulated transcription of a PEPCK-CAT vector in the presence of an expression vector for the catalytic subunit of protein kinase A. C/EBP beta and CREB bound to the CRE with similar affinities, both of which were greater than the affinity of C/EBP alpha. Within 90 min after the administration of dibutyryl cAMP to rats, there was a marked increase in the hepatic concentration of C/EBP beta mRNA and a decrease in the level of mRNA for C/EBP alpha. These studies indicate that C/EBP beta can regulate PEPCK gene transcription by acting through the CRE and that C/EBP beta, together with CREB, may contribute to the cAMP responsiveness of the PEPCK promoter.
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PMID:Relative roles of CCAAT/enhancer-binding protein beta and cAMP regulatory element-binding protein in controlling transcription of the gene for phosphoenolpyruvate carboxykinase (GTP). 809 46

Despite detailed knowledge of the regulation of individual steps in the gluconeogenic pathway, the relative importance of each step to the overall control of gluconeogenesis by insulin is not known. The aim of this study was to determine the role of phosphoenolpyruvate carboxykinase (PEPCK) in the regulation of gluconeogenesis by insulin. Clones of the rat hepatoma cell line H4IIE-C3 were produced, overexpressing a PEPCK gene, driven by a promoter not responsive to insulin. In these cells basal gluconeogenesis from 2-[14C]pyruvate was increased 2.1-fold compared to controls (4.63 +/- 0.49 nmol/10(5) cells vs. 2.21 +/- 0.24 nmol/10(5) cells after 3 h, P < 0.05, n = 5). Increased gluconeogenesis was associated with an increase in basal PEPCK mRNA levels (1.9-fold) and enzyme activity (2.8-fold). Insulin (10(-7) M) suppressed basal gluconeogenesis, PEPCK mRNA levels, and enzyme activity in control cells, but no detectable decrease was observed in PEPCK-transfected cells. These experiments provide direct evidence in intact cells that PEPCK is the rate-limiting enzyme in gluconeogenesis from pyruvate and show that insulin's action to inhibit gluconeogenesis is predominantly on the inhibition of PEPCK transcription.
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PMID:Impaired suppression of gluconeogenesis induced by overexpression of a noninsulin-responsive phosphoenolpyruvate carboxykinase gene. 811 59

Transcription of the phosphoenolpyruvate carboxykinase (PEPCK) and PEPCK-chloramphenicol acetyltransferase (CAT) genes is induced by cAMP and glucocorticoids and is inhibited by insulin in H4IIE cells, as it is in liver. In contrast, PEPCK-CAT expression in HepG2 cells is not affected by insulin but is induced by cAMP, which in turn is repressed by glucocorticoids. Mutations were introduced into well defined transcription factor binding sites to investigate possible interactions between the cAMP regulatory element (CRE) binding protein (CREB) and glucocorticoid response unit (GRU) binding proteins. H4IIE rat hepatoma cells were transfected with PEPCK-CAT plasmids with or without an expression vector for protein kinase A (PKA). Glucocorticoid-induced CAT activity was dependent upon the GRU and was decreased in plasmids lacking the CRE. To determine the direct effects of CREB, the DNA binding and dimerization domain of GAL4 was substituted for that of CREB (CRG), and the PEPCK CRE was replaced with a GAL4 binding site (G4PEPCK-CAT). CRG elevated basal and glucocorticoid-induced activities of G4PEPCK-CAT equally and restored responsiveness to PKA. The basal activity of CRG was not diminished by concomitant treatment with PKA plus its inhibitor peptide, PKI, or by mutation of the PKA phosphorylation. Deletion of C-terminal regions of the CREB activation domain from CRG diminished basal activation without affecting induction by PKA. The glucocorticoid-induced level of CAT activity decreased in proportion to the reduced ability of CREB to activate basal transcription. Induction by glucocorticoid, in the absence or presence of PKA, was not affected by CRG, indicating that interaction of GRU-bound factors with CREB is not required for glucocorticoid induction of PEPCK. These results indicate that CREB is directly involved in basal and PKA-induced expression of PEPCK, and that CREB supports glucocorticoid-induced PEPCK expression through its positive effect on basal transcription.
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PMID:Involvement of 3',5'-cyclic adenosine monophosphate regulatory element binding protein (CREB) in both basal and hormone-mediated expression of the phosphoenolpyruvate carboxykinase (PEPCK) gene. 811 62

The minimal promoter/transcription factor requirements for induction of phosphoenolpyruvate carboxykinase (PEPCK) transcription by cAMP-activated protein kinase A (PKA) and inhibition of this induction by insulin were investigated. H4 hepatoma cells were treated with or without insulin following cotransfection with chloramphenicol acetyltransferase reporter genes and expression vectors coding for the cAMP response element-binding protein (CREB) activation domain fused to the GAL4 DNA binding domain (CRG) and the catalytic subunit of PKA. Mutation of the PEPCK CRE to a GAL4 binding site (G4-PEPCK) within the fully responsive PEPCK promoter (-600/+69) made induction by PKA dependent upon cotransfection of CRG and this induction by CRG+PKA was inhibited by insulin. Mutation of the insulin regulatory sequence (delta IRS-G4-PEPCK) did not prevent induction by cAMP or inhibition by insulin. Fusion of GAL4 binding sites to the PEPCK TATA region (-40/+1, G4-PT) allowed induction by CRG+PKA and inhibition by insulin. However, inhibition by insulin was not observed when the CREB activation domain in CRG was replaced with the activation domain of VP16 (G4-VP16) or when the PEPCK TATA region was replaced with TATA regions from other genes. Our results indicate that the minimal requirements for induction of PEPCK by PKA and inhibition by insulin include: 1) the CREB activation domain, 2) the PEPCK TATA sequence, and 3) insulin-responsive hepatoma cells. These data suggest that specific factors interacting with both the PEPCK TATA region and the CREB activation domain are required for insulin inhibition of PKA-induced transcription.
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PMID:Inhibition by insulin of protein kinase A-induced transcription of the phosphoenolpyruvate carboxykinase gene. Mediation by the activation domain of cAMP response element-binding protein (CREB) and factors bound to the TATA box. 818 41

Cyclic-AMP stabilizes phosphoenolpyruvate carboxykinase (GTP) (PEPCK) mRNA against degradation. To investigate the mechanism of this effect, RNA mobility shift assays were used to determine the interaction of cellular proteins with specific domains from the mRNA. We report here the identification of a protein with an affinity for sequences of PEPCK mRNA with a predicted stem-loop structure. RNA-protein complex formation was significantly reduced if the double-stranded RNA probe was preheated to 90 degrees C. The RNA-binding protein did not bind to the hairpin structure of poly(rI)-poly (rC), indicating some degree of sequence specificity and that the RNA-binding protein is not the interferon-induced double-stranded RNA-activated protein kinase. The binding activity was contained in the cytosolic fraction (100,000 x g) of rat hepatoma FTO-2B cells and was significantly enhanced by high concentrations of KCl. Chromatography on an anion exchanger separated the binding activity from a factor which, upon reconstitution, inhibited the interaction with the RNA probe. Incubation of cells with cAMP resulted in a 3-4-fold decrease in the activity of the RNA-binding protein. An inhibition in complex formation was observed with extracts as early as 60 min after exposure of cells to cAMP. Liver extracts from rats starved for 72 h also had reduced binding activity compared to extracts from fed animals. Cellular extracts treated with alkaline phosphatase exhibited an elevated level of complex formation. An analysis by SDS-polyacrylamide gel electrophoresis of the RNA-protein complex after ultraviolet light cross-linking demonstrated that the RNA-binding protein had a molecular mass of approximately 100 kDa. On the basis of these results, we suggest that liver cells contain a protein whose interaction with PEPCK mRNA is regulated by cAMP-dependent phosphorylation and which may be responsible for the cAMP-mediated control of PEPCK mRNA half-life.
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PMID:A cAMP-regulated RNA-binding protein that interacts with phosphoenolpyruvate carboxykinase (GTP) mRNA. 822 67

Gene transfer systems targeting the asialoglycoprotein receptor have been developed to introduce functional genes into cells in culture and livers of intact animals. A synthetic neoglycoprotein carrier was constructed and complexed to a chimeric gene containing the cDNA for human factor IX ligated to the promoter-regulatory region of the gene for phosphoenolpyruvate carboxykinase from the rat. The complex was used to transfect human hepatoma cells that express the asialoglycoprotein receptor. Human factor IX DNA sequences were found in cells 10 days after treatment. A 1.4 kB mRNA transcript was detected by Northern blot hybridization, which was inducible by treatment with dexamethasone or cAMP with theophylline. Western blot hybridization of proteins secreted into the culture medium detected human factor IX. The chimeric gene was also transferred into livers of rats using the neoglycoprotein carrier system after partial hepatectomy. Although the results were variable, the exogenous gene was transcribed in livers of several animals, and maximal levels of expression of the fully processed human factor IX were detected 30 days after introduction. The concentration of factor IX in the blood returned to control levels 60 days after transfection. Factor IX production was induced as late as 96 days after treatment by feeding transfected animals a diet high in protein but devoid of carbohydrates. This DNA carrier system can be used to introduce functional genes into the livers of rats, and may be a useful technique for gene therapy targeting the liver.
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PMID:Regulation of the phosphoenolpyruvate carboxykinase/human factor IX gene introduced into the livers of adult rats by receptor-mediated gene transfer. 837 Apr 79

Effects of tolbutamide on the activity of hepatic phosphoenolpyruvate carboxykinase (PEPCK), a rate limiting enzyme in gluconeogenesis, was examined using rat hepatoma (H4IIE) cells. Tolbutamide inhibited PEPCK activity induced by cAMP in a time- and dose-dependent manner. Tolbutamide effect was rapidly exerted and insulin-independent. The inhibitory effect of 5 mM tolbutamide corresponded with that of 10(-7) M insulin. These results suggest the possibility that tolbutamide plays a significant role on amelioration of the deranged glucose metabolism in the liver through repression of gluconeogenesis, primarily due to the inhibition of PEPCK activity.
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PMID:The inhibitory effect of tolbutamide on phosphoenolpyruvate carboxykinase activity in rat hepatoma H4IIE cells. 838 41

The specific binding sites for sulfonylureas in the rat liver membrane fraction were demonstrated and characterized. [3H]Glibenclamide binding to the liver membrane was specific, time- and temperature-dependent, and reversible. Scatchard analysis showed a single class binding site. The dissociation constant (Kd) for glibenclamide was 1.1 microM and the binding capacity (Bmax) was 50 pmol/mg protein. [3H]Glibenclamide binding could be displaced by other sulfonylureas. Half-maximal inhibition of binding (IC50) for glimepiride, gliclazide, acetohexamide, tolbutamide and chlorpropamide was 4.2 microM, 74 microM, 0.33 mM, 0.60 mM, 1.2 mM, respectively. Each value is close to the reported blood concentration when a therapeutic dose of each drug is administered orally. The order of IC50 values is coincident with the order of potency of the clinical hypoglycemic effect of these drugs. We had shown that these concentrations of sulfonylureas stimulate 6-phosphofructo-2-kinase in the liver or hepatocytes and inhibit phosphoenolpyruvate carboxykinase in the hepatoma cells. The specific binding sites demonstrated here may play some roles when sulfonylureas affect carbohydrate metabolism in the liver.
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PMID:Characterization of the binding sites for [3H]glibenclamide in rat liver membranes. 854 39

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