Cyclic AMP-induced transcriptional repression of the insulin-responsive glucose transporter (GLUT4) gene: identification of a promoter region required for down-regulation of transcription.
The mechanism(s) by which cyclic AMP represses transcription of the GLUT4 gene was investigated. 3T3-L1 preadipocytes were stably transfected with a series of 5' deletion mutants of the mouse GLUT4 gene promoter fused to the bacterial CAT gene and then were induced to differentiate into adipocytes. A method based on reverse transcription/polymerase chain reaction (PCR) amplification was developed and optimized to quantitate expression of CAT mRNA transcripts. Treatment with 8-bromo-cAMP down-regulated the level of CAT mRNA in adipocytes transfected with the -7000/CAT, -785/CAT and -469/CAT constructs, but not the -78/CAT construct. Thus, the regulatory element(s) which mediates transcriptional repression by cAMP resides in the proximal promoter of the GLUT4 gene between positions -469 and -78. Since down-regulation of GLUT4 mRNA is unaffected by inhibitors of protein synthesis, cAMP (and insulin) may activate phosphorylation or dephosphorylation of an existing transcription factor that interacts with the GLUT4 proximal promoter.
Flores-Riveros
J R
JR
Department of Biological Chemistry, Johns Hopkins University, School of Medicine, Baltimore, MD 21205.
Kaestner
K H
KH
Thompson
K S
KS
Lane
M D
MD
D016475:3T3 Cells; D015124:8-Bromo Cyclic Adenosine Monophosphate / Q000494:pharmacology*; D000273:Adipose Tissue / Q000378:metabolism; D000818:Animals; D001483:Base Sequence; D015500:Chloramphenicol O-Acetyltransferase / Q000096:biosynthesis / Q000235:genetics; D004252:DNA Mutational Analysis; D015536:Down-Regulation; D051275:Glucose Transporter Type 4; D007739:L Cells; D051379:Mice; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins / Q000096:biosynthesis / Q000235:genetics*; D009124:Muscle Proteins; D011401:Promoter Regions, Genetic / Q000235:genetics; D012333:RNA, Messenger / Q000032:analysis; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D017384:Sequence Deletion; D013234:Stem Cells / Q000378:metabolism; D014158:Transcription, Genetic / Q000187:drug effects*; D014162:Transfection
D051275:Glucose Transporter Type 4; D009004:Monosaccharide Transport Proteins; D009124:Muscle Proteins; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; C495693:Slc2a4 protein, mouse; D015124:8-Bromo Cyclic Adenosine Monophosphate; D015500:Chloramphenicol O-Acetyltransferase