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Abstract
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.
Highlights
(GTP) (EC 4.1.1.32)(PEPCK) is expressed ian tissue- phoenolpyruvate carboxykinase (GTP) (PEPCK)’ from the specific manner in the liver, kidney, and adipose tisrastueis stimulated by glucagon,glucocortiand is regulated byhormonesincluding cAMP and coids, and thyroid hormone and is inhibited by insulin and insulin
C/EBPa.Within 90 min after the administration of dibutyryl cAMP to rats, there wasa marked increase in the hepatic concentration of C/EBPB mRNA and a carrying a chimeric PEPCK-bovine growth hormongeene, we have shown that P3 is important for directing expression of the gene for PEPCK in theliver.’
We suggest that bind to the CRE of the PEPCK promoter in vitro and can C/EBPP contributes to the expression of the PEPCK gene activate gene transcription when cotransfected into hepatoma and possibly other genes involved in energy metabolism
Summary
(GTP) (EC 4.1.1.32)(PEPCK) is expressed ian tissue- phoenolpyruvate carboxykinase (GTP) (PEPCK)’ from the specific manner in the liver, kidney, and adipose tisrastueis stimulated by glucagon (acting via CAMP),glucocortiand is regulated byhormonesincluding cAMP and coids, and thyroid hormone and is inhibited by insulin and insulin. Constructionof CAT Vectors, CellTransfections,and CATAssaysThe construction of the serial deletions of the PEPCK promoter ligated to the CAT reporter gene has been described (Park et al, 1990).For details of the design of the block mutations placed in the protein-binding domains of the PEPCK promoter and thesequences changed, see Liu et al (1990) These mutations introduced random sequence in place of the nucleotides a t CRE-1 (-87/-74), P1 (-123/ -87), P3 (-248/-230), and P4 (-320/-269) and eliminated protein binding to the mutated site (Liu et al, 1990). Affinity Determination of CIEBPP, C/EBPa, andCREB-Gel shift analyses were conducted using the end-labeled CRE (-94/-77) oligonucleotide and the individual transcription factors essentially as described by Bokar et al (1988).The DNA-protein hybridization was conducted a t room temperature in the presence of 1 Fg/Fl bovine serum albumin. DNA probes prepared from the cDNAs of glutamine synthetase (Moorman et al, 1988) and C/EBP/3 were labeled with a-35S-dCTPand used at a concentration of 0.1 ng/ml
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