Glucocorticoids repress transcription of phosphoenolpyruvate carboxykinase (GTP) gene in adipocytes by inhibiting its C/EBP-mediated activation.
The cytosolic form of the phosphoenolpyruvate carboxykinase (PEPCK-C) gene is selectively expressed in several tissues, primarily in the liver, kidney, and adipose tissue. The transcription of the gene is reciprocally regulated by glucocorticoids in these tissues. It is induced in the liver and kidney but repressed in the white adipose tissue. To elucidate which adipocyte-specific transcription factors participate in the repression of the gene, DNase I footprinting analyses of nuclear proteins from 3T3-F442A adipocytes and transient transfection experiments in NIH3T3 cells were utilized. Glucocorticoid treatment slightly reduced the nuclear C/EBP alpha concentration but prominently diminished the binding of adipocyte-derived nuclear proteins to CCAAT/enhancer-binding protein (C/EBP) recognition sites, without affecting the binding to nuclear receptor sites in the PEPCK-C gene promoter. Of members of the C/EBP family of transcription factors, C/EBP alpha was the strongest trans-activator of the PEPCK-C gene promoter in the NIH3T3 cell line. The glucocorticoid receptor (GR), in the presence of its hormone ligand, inhibited the activation of the PEPCK-C gene promoter by C/EBP alpha or C/EBP beta but not by the adipocyte-specific peroxisome proliferator-activated receptor gamma 2. This inhibition effect was similar using the wild type or mutant GR and did not depend on GR binding to the DNA. The glucocorticoid response unit (GRU) in the PEPCK-C gene promoter (-2000 to +73) restrained C/EBP alpha-mediated trans-activation, because mutation of each single GRU element increased this activation by 3-4-fold. This series of GRU mutations were repressed by wild type GR to the same percent as was the nonmutated PEPCK-C gene promoter. In contrast, the repression by mutant GR depended on the intact AF1 site in the gene promoter, whereby mutation of the AF1 element abolished the repression.
Olswang
Yael
Y
Department of Developmental Biochemistry, Hebrew University-Hadassah Medical School, Jerusalem, Israel 91120, USA.
Blum
Barak
B
Cassuto
Hanoch
H
Cohen
Hannah
H
Biberman
Yael
Y
Hanson
Richard W
RW
Reshef
Lea
L
D016475:3T3 Cells; D017667:Adipocytes / Q000201:enzymology*; D000818:Animals; D001483:Base Sequence; D022762:CCAAT-Enhancer-Binding Proteins / Q000037:antagonists & inhibitors*; D002454:Cell Differentiation; D018983:DNA Footprinting; D017931:DNA Primers; D003907:Dexamethasone / Q000494:pharmacology*; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects*; D005938:Glucocorticoids / Q000494:pharmacology*; D006801:Humans; D051379:Mice; D010729:Phosphoenolpyruvate Carboxykinase (GTP) / Q000235:genetics*; D011401:Promoter Regions, Genetic; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014158:Transcription, Genetic / Q000187:drug effects*; D015533:Transcriptional Activation
D022762:CCAAT-Enhancer-Binding Proteins; D017931:DNA Primers; D005938:Glucocorticoids; D003907:Dexamethasone; D010729:Phosphoenolpyruvate Carboxykinase (GTP)