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:2.3.1.28 (chloramphenicol acetyltransferase)
5,100 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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 (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

Cytosolic phosphoenolpyruvate carboxykinase (PEPCK) plays a critical role in adipose tissue glyceroneogenesis. We have previously shown that transcription of the PEPCK gene was stimulated by isoprenaline and retinoic acid in 3T3-F442A adipocytes. We also showed that oleate increased PEPCK mRNA. Here, we analysed the effect that fatty acids of various chain lengths and unsaturation degrees exerted on PEPCK gene expression in 3T3-F442A adipocytes. When maintained in serum-free, glucose-free medium, differentiated cells responded to unsaturated long-chain fatty acids by a large increase in PEPCK mRNA whereas saturated fatty acids were inefficient. A maximum fivefold stimulation by oleate was attained at 4 h of treatment with 1 mM fatty acid bound to albumin in a 6:1 ratio. The poly-unsaturated very long-chain fatty acid all-cis-4,7,10,13,16,19-docosahexaenoic acid (C22:6) was even more potent and produced a tenfold increase. The expression of the genes encoding glycerol-3-phosphate dehydrogenase, hormone-sensitive lipase or actin remained unaffected by oleate exposure. A 4-h treatment by the hypolipidemic drug clofibrate, 0.5-2 mM, also produced a large (3-9-fold) increase in PEPCK mRNA. When used at non-saturating concentrations, oleate and clofibrate acted in an additive manner. At maximally effective concentrations, additivity was lost, suggesting that fatty acids and fibrates might act through similar mechanisms. Nuclear transcription experiments showed that oleate and clofibrate stimulated the transcription rate of the gene. 3T3-F442A cells were stably transfected with a plasmid containing the base pairs -2100 to +69 of the PEPCK gene promoter fused to the chloramphenicol acetyltransferase gene. These differentiated stable transfectants responded to oleate and clofibrate by a specific increase in chloramphenicol acetyltransferase activity. Adipocytes express various isoforms of peroxisome-proliferator-activated receptors that can be activated by fibrates and fatty acids. Potential recognition sequences for peroxisome-proliferator-activated receptors are present in the -2100 to +69 fragment of the PEPCK gene promoter. Thus, this gene represents an ideal molecular target for understanding the complex transcriptional control exerted by fatty acids and peroxisome proliferators.
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PMID:Fatty acids and fibrates are potent inducers of transcription of the phosphenolpyruvate carboxykinase gene in adipocytes. 853 80

Efficient transfer of genes maintaining a correct hormonal control in transfected cells is the prerequisite for gene regulation studies and for gene therapy. Differentiated cells, like adipocytes or hepatocytes, are difficult to transfect. In an attempt to improve gene transfer, we first transiently transfected cultured 3T3-F442A adipocytes with a construct containing the simian virus 40 (SV40) promoter fused to the chloramphenicol acetyltransferase (CAT) gene (pSV2-CAT), using various cationic liposomes. Among these, only lipofectAMINE was five times more efficient than the standard calcium phosphate procedure. To further augment efficiency, we transfected 3T3-F442A adipocytes and FAO hepatoma cells with the lipofectAMINE/pSV2-CAT complex in the presence of replication-deficient recombinant type-5 adenovirus at 200 pfu/cell. CAT activity of transiently transfected cells was increased about 50-fold when compared to the calcium phosphate procedure. To determine whether this methodology would be useful for obtaining stable transfectants and would not interfere with correct gene regulation, we used a construct containing -2100 to +69 bp of the phosphoenolpyruvate carboxykinase gene fused to the CAT gene (pPL1-CAT). This construct was shown previously to be cAMP-responsive after calcium-phosphate-mediated transfection of adipocytes and hepatoma cells. 3T3-F442A or FAO cells in which pPL1-CAT was either transiently or stably transferred by lipofectAMINE and adenovirus responded to isoproterenol or cAMP, respectively, with a 2-3-fold increase in CAT activity. Therefore the association of liposomes and adenovirus is an efficient method for transient or stable transfer of regulated genes in adipocytes and hepatoma cells.
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PMID:Efficient transfer of regulated genes in adipocytes and hepatoma cells by the combination of liposomes and replication-deficient adenovirus. 864 10

The onset of metabolic acidosis causes an increased transcription of the renal phosphoenolpyruvate carboxykinase (PCK) gene. When transgenic mice carrying a bovine growth hormone (bGH) gene driven by the -460 to +73 segment of the PCK promoter were made chronically acidotic, the bGH mRNA was increased twofold after 4 days. Confluent and well-differentiated cultures of LLC-PK1-F+ cells exhibit a 2.5-fold increase in PCK mRNA when transferred to acidic media (pH 6.9, 10 mM HCO3-) for 16 h. Confluent cultures transfected with PCK-490 CAT exhibit an increase (3.5-fold) in chloramphenicol acetyltransferase (CAT) activity when shifted to acidic medium for 48 h. Mutation or deletion of the P2 element causes a four- to fivefold decrease in basal CAT activity but does not affect the pH response. In contrast, mutations of the P3(II) element or the CRE-1 cAMP-response element have little effect on basal activity but cause a 50% decrease in the pH response. Other deletions or mutations have little effect on either activity. Thus changes in the activity or levels of the protein(s) in the renal proximal tubule that binds to the P3(II) and CRE-1 elements may mediate increased transcription of the PCK gene during metabolic acidosis.
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PMID:Promoter elements that mediate the pH response of PCK mRNA in LLC-PK1-F+ cells. 877 Jan 65

The phosphoenolpyruvate carboxykinase (PEPCK) gene promoter contains a glucocorticoid response unit (GRU) that includes, as a linear array, two accessory factor binding sites (AF1 and AF2) and two glucocorticoid receptor binding sites. All of these elements are required for a complete glucocorticoid response. AF1 and AF2 also partially account for the response of the PEPCK gene to retinoic acid and insulin, respectively. A second retinoic acid response element was recently located just downstream of the GRU. In this study we show that mutation of the 3' half-site of this element results in a 60% reduction of the glucocorticoid response of PEPCK promoter-chloramphenicol acetyltransferase (CAT) fusion constructs in transient transfection assays, thus the half-site is now termed AF3. A variety of assays were used to show that chicken ovalbumin upstream promoter transcription factor (COUP-TF) binds specifically to AF3 and that upstream stimulatory factor (USF) binds to an E-box motif located 2 base pairs downstream of AF3. Mutations of AF3 that diminish binding of COUP-TF reduce the glucocorticoid response, but mutation of the USF binding site has no effect. The functional roles of AF1, AF2, and AF3 in the glucocorticoid response were explored using constructs that contained combinations of mutations in all three elements. All three elements are required for a maximal glucocorticoid response, and mutation of any two abolish the response.
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PMID:The orphan receptor COUP-TF binds to a third glucocorticoid accessory factor element within the phosphoenolpyruvate carboxykinase gene promoter. 894 35

Glucocorticoids inhibit basal and hormone-induced phosphoenolpyruvate carboxykinase (PEPCK) gene transcription in adipocytes whereas beta-adrenergic agonists and fibrates are stimulatory. Here we show that dexamethasone inhibits the induction of PEPCK mRNA by isoprenaline or clofibrate in 3T3-F442A adipocytes. RU 38486 antagonizes dexamethasone effect, suggesting the involvement of the glucocorticoid receptor. In H4IIE hepatoma cells, glucocorticoids enhance PEPCK gene transcription through a complex region which encompasses an element, AF1, with a direct repeat 1-type sequence. Mutations in the AF1 sequence abolish binding of nuclear factors from liver and from 3T3-F442A adipocytes. We transiently transfected 3T3-F442A cells with a wild type or an AF1-mutated PEPCK-CAT construct comprising -2100 to +69 base pairs of the promoter fused to the chloramphenicol acetyltransferase (CAT) gene. With both constructs, CAT activity is decreased by dexamethasone and is increased by isoprenaline or by clofibrate. However, dexamethasone is unable to inhibit clofibrate induction of CAT activity in cells transfected with the AF1-mutated construct whereas it prevents isoprenaline action on both constructs. Hence, although a single hormone can repress stimulations originating from different intracellular routes, sites in the promoter which mediate inhibition of a specific stimulation are distinct.
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PMID:Glucocorticoids use a positive liver element to repress fibrate-induced adipose transcription of the phosphoenolpyruvate carboxykinase gene. 909 12

Glucose-6-phosphatase (G6Pase) catalyzes the final step in the gluconeogenic and glycogenolytic pathways. The transcription of the gene encoding the catalytic subunit of G6Pase is stimulated by glucocorticoids, whereas insulin strongly inhibits both basal G6Pase gene transcription and the stimulatory effect of glucocorticoids. To identify the insulin response sequence (IRS) in the G6Pase promoter through which insulin mediates its action, we have analyzed the effect of insulin on the basal expression of mouse G6Pase-chloramphenicol acetyltransferase (CAT) fusion genes transiently expressed in hepatoma cells. Deletion of the G6Pase promoter sequence between -271 and -199 partially reduces the inhibitory effect of insulin, whereas deletion of additional sequence between -198 and -159 completely abolishes the insulin response. The presence of this multicomponent IRS may explain why insulin potently inhibits basal G6Pase-CAT expression. The G6Pase promoter region between -198 and -159 contains an IRS, since it can confer an inhibitory effect of insulin on the expression of a heterologous fusion gene. This region contains three copies of the T(G/A)TTTTG sequence, which is the core motif of the phosphoenolpyruvate carboxykinase (PEPCK) gene IRS. This suggests that a coordinate increase in both G6Pase and PEPCK gene transcription is likely to contribute to the increased hepatic glucose production characteristic of patients with non-insulin-dependent diabetes mellitus.
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PMID:A multicomponent insulin response sequence mediates a strong repression of mouse glucose-6-phosphatase gene transcription by insulin. 911 20

Glucocorticoids exert pleiotropic effects, among which negative regulation of transcription has been recognized as of crucial importance. While glucocorticoids induce phosphoenolpyruvate carboxykinase (PEPCK) gene expression in liver cells, it represses gene activity in adipose cells. We used the 3T3-F442A adipocytes to analyze the underlying mechanisms in these cells, the synthetic glucocorticoid dexamethasone exerts a dominant repression either on basal or on beta-agonist stimulation of PEPCK gene expression. To determine whether glucocorticoid action required protein synthesis, we employed cycloheximide, anisomycin, and puromycin, three different translation inhibitors. None of these affected induction by isoprenaline or repression by dexamethasone of isoprenaline stimulation. In contrast, dexamethasone inhibitory action on basal PEPCK mRNA was totally prevented by the three translation inhibitors. Time courses of glucocorticoid action on basal and on induction by beta-agonist were similar. Half-maximal effect of dexamethasone on isoprenaline-induced PEPCK mRNA was obtained at about 10 nM, a tenfold higher concentration than that observed for the reduction of basal mRNA. Using the transcription inhibitor DRB, we showed that dexamethasone did not alter mRNA half-life, while isoprenaline strongly stabilized mRNA. In a 3T3-F442A stable transfectant bearing -2,100 base pairs of the PEPCK promoter fused to the chloramphenicol acetyltransferase (CAT) gene, isoprenaline stimulated CAT activity, whereas dexamethasone reduced basal and isoprenaline-induced CAT expression. Hence, beta-agonists exert both transcriptional and posttranscriptional regulation, while glucocorticoid action is purely transcriptional. However, mechanisms of glucocorticoid repression of basal and of beta-agonist stimulation appear different.
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PMID:Transcriptional and posttranscriptional mechanisms of glucocorticoid-mediated repression of phosphoenolpyruvate carboxykinase gene expression in adipocytes. 925 94


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