Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:4.1.1.32 (phosphoenolpyruvate carboxykinase)
4,204 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Dexamethasone rapidly stimulated transcription of the tyrosine aminotransferase and metallothionein-I genes--but not of the phosphoenolpyruvate carboxykinase gene--in rat hepatocytes cultured in serum-free medium. This differential response was not observed for cyclic AMP. The results suggest that the phosphoenolpyruvate carboxykinase gene--but not the tyrosine aminotransferase and metallothionein-I genes--requires a factor which is permissive for stimulation of transcription by the glucocorticoid receptor.
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PMID:Differential induction of transcription for glucocorticoid-responsive genes in cultured rat hepatocytes. 196 35

The minimal DNA sequence required for glucocorticoid induction of the phosphoenolpyruvate carboxykinase (PEPCK) gene in H4IIE rat hepatoma cells was defined. This novel glucocorticoid response unit (GRU) spans about 110 base pairs (bp) and includes two receptor-binding elements plus two accessory factor-binding elements. Purified glucocorticoid receptor bound to two regions (GR1 and GR2) between -395 and -349 bp relative to the transcription start site. Factors in crude rat liver nuclear extract bound to DNA in the regions -455 to -431 and -420 to -403 bp, which are designated accessory factor 1 (AF1) and accessory factor 2 (AF2) elements, respectively. Gel retardation analysis revealed that at least two proteins bound to AF1 and that they were distinct from the protein(s) that bound to AF2. Various combinations of GR1, GR2, AF1, and AF2 were fused to the chloramphenicol acetyltransferase (CAT) reporter gene and cotransfected with a glucocorticoid receptor expression plasmid (pSVGR1) into H4IIE cells to identify the functional GRU. Neither the glucocorticoid receptor binding region nor the accessory factor binding region alone was sufficient to confer glucocorticoid responsiveness. The two components of the glucocorticoid receptor binding region functioned independently, and each accounted for half of the maximal response, provided the accessory factor elements were present. Similarly, deletion of either AF1 or AF2 diminished glucocorticoid induction of the PEPCK gene to approximately half of the maximum. We propose that the complex PEPCK gene GRU provides the stringent regulation required of this critical enzyme in liver.
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PMID:Characterization of a complex glucocorticoid response unit in the phosphoenolpyruvate carboxykinase gene. 238 23

Exposure of Reuber hepatoma cells (RHC) to 30 and 300 fM human rIL-1 (hurIL-1) for 4 h significantly decreased cytosolic glucocorticoid binding. Scatchard analysis indicated that the 30 and 300 fM doses of hurIL-1 significantly decreased the Bmax (maximum number of available binding sites), but did not alter the Kd (affinity of the glucocorticoid receptor for ligand). The decrease in cytosolic glucocorticoid binding, expressed relative to cytosol protein, did not result from increased intracellular protein in hurIL-1-treated RHC. In addition, the receptor binding reaction in RHC treated with 300 fM hurIL-1 could be resolved only by computer application of a three-parameter model. Sucrose density gradient ultracentrifugation analysis confirmed significantly less untransformed (8 to 10S) receptor-ligand complexes in hurIL-1-treated RHC, which is biologically significant because hurIL-1 (300 fM) also inhibited the glucocorticoid induction of the gluconeogenic enzyme, phosphoenolpyruvate carboxykinase (PEPCK). Altered transformation of the receptor-ligand complex, a possible mechanism of action for hurIL-1-mediated inhibition of PEPCK induction, was examined. However, receptor transformation, verified by in vitro activation by high salt (0.3 M KCl) of glucocorticoid receptor-ligand complexes and subsequent sucrose density gradient ultracentrifugation analysis, was not affected by hurIL-1. Furthermore, cytoplasmic glucocorticoid binding, determined in intact cell dexamethasone uptake experiments, was decreased in hurIL-1-treated RHC. The decrease in cytoplasmic glucocorticoid binding was reflected subsequently in decreased nuclear binding. The results support our hypothesis that, during acute infection and inflammation, mediators alter metabolic pathways in the liver by interfering with glucocorticoid action.
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PMID:Human recombinant IL-1 alters glucocorticoid receptor function in Reuber hepatoma cells. 284 96

Bacterial endotoxin inhibits the glucocorticoid induction of several hepatic enzymes including phosphoenolpyruvate carboxykinase (PEPCK). Experiments were performed to elucidate the mechanism of this inhibition by examination of the early events in the glucocorticoid induction process. At a dose of endotoxin 2-to 10-fold greater than that required to inhibit the induction of PEPCK activity, no effect on the entry of glucocorticoids into hepatocytes or their ability to form complexes with glucocorticoid receptors could be measured. Binding data showed no effect of endotoxin treatment on the association or dissociation kinetics of the steroid-receptor binding reaction. Scatchard analysis revealed no effect on the affinity and number of hepatic glucocorticoid receptor binding sites, indicating that down-regulation of receptors is not responsible for inhibition of induction. Finally, activation of receptor complexes was unaffected as well by endotoxin treatment. We conclude from these data that endotoxin does not act at the early events in the glucocorticoid induction process and must therefore intervene at a subsequent step.
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PMID:Endotoxin treatment inhibits glucocorticoid induction of hepatic enzymes at a late induction step. 351 9

The mechanism by which endotoxin administration results in hypoglycemia was evaluated by characterizing [3H]dexamethasone binding and phosphoenolpyruvate carboxykinase activity in hepatic cytosol preparations from treated and control mice. Starved mice were given Escherichia coli O111:B4 endotoxin or saline intraperitoneally on day 3 after bilateral adrenalectomy. [3H]dexamethasone binding was measured by the charcoal method after the incubation of cytosol preparations with [3H]dexamethasone in the presence or absence of unlabeled dexamethasone. Changes in [3H]dexamethasone binding were found to be time and dose dependent in treated mice. When mice given different doses of endotoxin reached the same stage of morbidity, as indicated by the average time of death, significantly lower glucocorticoid binding was measured. Scatchard analysis of binding isotherms defined a single class of binding sites. Association and dissociation rate constants and the equilibrium dissociation constant (Kd) were not altered, but the maximum number of binding sites was depressed by endotoxin. The rank order of potency of competitors for [3H]dexamethasone binding, dexamethasone greater than hydrocortisone = corticosterone greater than deoxycorticosterone greater than progesterone greater than testosterone = estradiol, was consistent with a glucocorticoid receptor, although the competition was not altered by endotoxin. Endotoxin treatment prevented the glucocorticoid-induced increase in hepatic phosphoenolpyruvate carboxykinase activity. We conclude that the hypoglycemia of endotoxin poisoning is effected, in part, by the inhibition of the glucocorticoid-mediated induction of phosphoenolpyruvate carboxykinase via the down regulation of hepatic glucocorticoid receptors.
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PMID:Down regulation of hepatic glucocorticoid receptors after endotoxin treatment. 684 Aug 56

Insulin-like growth factor-binding protein-1 (IGFBP-1) modulates the action of IGFs on target cells. IGFBP-1 transcription is highly regulated by hormonal and metabolic factors. In rat H4-II-E hepatoma cells, IGFBP-1 messenger RNA is stimulated by dexamethasone, cAMP, and phorbol esters, and dominantly inhibited by insulin. To identify the cis-elements that determine transcriptional regulation by these agents, we have coupled rat IGFBP-1 promoter fragments to a luciferase reporter gene and transfected H4-II-E cells using the cationic liposome procedure. Promoter fragments whose 5'-end was at nucleotide (nt) -925 or -327 (with respect to the transcription initiation site, 1) conferred positive regulation of promoter activity by dexamethasone, cAMP, and phorbol esters. Insulin inhibited promoter activity in the presence of any of the three stimulatory agents. Stimulation by cAMP or phorbol esters was abolished when the region between nt -327 and -235 was deleted. Although this region contains potential activating protein-2 and activating protein-1 sites, the sites responsible for this regulation have not yet been identified. By contrast, stimulation by dexamethasone was retained in deletion constructs whose 5'-end was at nt -92, but was abolished by site mutagenesis of either the left or right half-sites of a potential glucocorticoid response element (GRE) located between nt -91 and -77. Surprisingly, substitution mutations in an up-stream region, -108 to -99 (M4), also decreased dexamethasone-stimulated promoter activity despite the presence of an intact GRE. We postulate that a positive factor that binds to the wild-type M4 region neutralizes factors that inhibit interaction of the glucocorticoid receptor with the GRE. The M4 region also is involved in inhibition by insulin. Insulin inhibition of dexamethasone-stimulated promoter activity was lost after deletion of nt -135 to -92 or mutation of the region between nt -108 and -99. This insulin response element is conserved in the human IGFBP-1 promoter and is homologous to the insulin response element of the phosphoenolpyruvate carboxykinase gene, which also is rapidly inhibited by insulin in H4-II-E cells. The rat IGFBP-1 promoter provides a valuable model system for studying the multihormonal regulation of transcription.
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PMID:Identification of cis-elements mediating the stimulation of rat insulin-like growth factor-binding protein-1 promoter activity by dexamethasone, cyclic adenosine 3',5'-monophosphate, and phorbol esters, and inhibition by insulin. 752 64

The phosphoenolpyruvate carboxykinase (PEPCK) gene is regulated at the transcriptional level by a variety of effectors in a tissue-specific fashion. In order to study the parameters involved in the tissue-specific hormonal regulation of the PEPCK gene, we have used a transient expression test in well-differentiated rat hepatoma cells as well as in dedifferentiated variants. In this test, the PEPCK promoter is induced by glucocorticoids in well-differentiated FGC4 cells, but not in H5 dedifferentiated variants, in spite of the presence in H5 cells of the glucocorticoid receptor. Study of the PEPCK promoter using electrophoretic mobility shift assays reveals binding sites for the liver-enriched transcription factors HNF1, vHNF1, HNF3, HNF4, and CAAT/enhancer binding protein members. Overexpression of the liver-enriched transcription factors absent in the dedifferentiated variants, such as HNF1 and HNF4, is not sufficient to restore glucocorticoid response of the PEPCK promoter in the variants. Moreover, systematic analysis of the PEPCK promoter reveals that the presence of a region covering a cAMP-responsive element (CRE1 at -80) and a CAAT box is necessary for full response of the PEPCK promoter to glucocorticoids in well-differentiated rat hepatoma cells. In a cotransfection test, overexpression of the regulatory subunit of protein kinase A (PKA), causing sequestering of PKA, abolishes the glucocorticoid response of the promoter in well-differentiated cells. On the other hand, in dedifferentiated variants, overexpression of the catalytic subunit of PKA restores the response to glucocorticoids. The action of PKA on the glucocorticoid response requires the presence of the CRE1 element and is promoter specific because it does not concern nonhepatic promoters such as the long terminal repeats of the mouse mammary tumor virus. These results suggest that the full response of the PEPCK promoter to glucocorticoids requires activation of another signal transduction pathway, the cAMP-mediated pathway.
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PMID:Response of the phosphoenolpyruvate carboxykinase gene to glucocorticoids depends on the integrity of the cAMP pathway. 781 33

Glucocorticoids stimulate hepatic phosphoenolpyruvate carboxykinase (PEPCK; EC 4.1.1.32) gene expression, thereby increasing the rate of gluconeogenesis. The effect of glucocorticoids on PEPCK gene expression is mediated by a set of promoter elements collectively referred to as the glucocorticoid response unit. The response unit spans a 100-bp segment and includes two glucocorticoid receptor binding sites (GR1 and GR2) and two accessory factor binding sites (AF1 and AF2), all of which are required for a maximal glucocorticoid response. The AF1 element also serves as a retinoic acid response element and may be involved in developmental and tissue-specific expression of the gene. In this study we report that COUP-TF and HNF-4, two orphan members of the nuclear receptor superfamily, bind to the AF1 element and function as accessory factors for the glucocorticoid response of the PEPCK gene.
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PMID:The orphan receptors COUP-TF and HNF-4 serve as accessory factors required for induction of phosphoenolpyruvate carboxykinase gene transcription by glucocorticoids. 783 1

Cytosolic phosphoenolpyruvate carboxykinase (GTP) (PEPCK) is a key glyceroneogenic enzyme in adipose tissue. The regulation of PEPCK gene expression by retinoic acid (RA) and dexamethasone (DEX) was studied in 3T3-F442A adipocytes maintained in a serum-free medium. RA induced whereas DEX reduced PEPCK mRNA steady-state level. RA stimulation was about 4-fold and DEX repression was of 80% in 4 hrs. In addition to reducing basal mRNA level, DEX was able to counteract RA induction in a dominant manner. The use of the glucocorticoid antagonist RU 38486 indicated that the DEX effect was mediated by the glucocorticoid receptor. Stable transfectants bearing the region of the PEPCK promoter from -2100 to +69 fused to the chloramphenicol acetyltransferase (CAT) gene (pPL1-CAT) were used to study PEPCK gene regulation in differentiated adipocytes. In such cells, RA stimulated CAT expression 3 to 5.5 fold. DEX had no effect on basal CAT activity whereas it inhibited the stimulation induced by RA. Thus, in adipocytes, the PEPCK gene regulatory region between -2100 and +69 bp mediates both stimulation by RA and repression by DEX of RA action.
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PMID:Glucocorticoids antagonize retinoic acid stimulation of PEPCK gene transcription in 3T3-F442A adipocytes. 798 26

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


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