Identification of adipocyte differentiation-related regulatory element for adrenomedullin gene repression (ADRE-AR) in 3T3-L1 cells.
Adrenomedullin (AM), a potent vasodilator peptide, has been suggested to act against cardiovascular complications and insulin resistance in the metabolic syndrome. We have already reported the AM gene repression in the early phase of adipocyte differentiation of NIH 3T3-L1 cells. Here we show adipocyte differentiation-related regulatory element for AM gene repression (ADRE-AR) in 36-bp region (-2135/-2100) of the AM gene. 3T3-L1 cells were induced to differentiate to adipocytes by insulin, dexamethasone and 3-isobutyl-1-methylxanthine. On the third day of differentiation, the promoter function was analyzed using the reporter plasmids, which contain the promoter region of AM gene (-4616/+108) in pGL3-basic luciferase reporter vector. The promoter activity decreased to about 20% in 3T3-L1 adipocytes when compared with 3T3-L1 preadipocytes, and a 36-bp region (-2135 to -2100) upstream from the transcription initiation site of the AM gene was necessary for higher AM gene expression in preadipocytes. This 36-bp ADRE-AR contains three copies of G/AAAA sequence (5'-GAAATGAAAGTAAAA-3') (-2124/-2110), which are conserved between mouse and human, and the introduction of mutations in each copy of G/AAAA sequence decreased the promoter activity in preadipocytes and adipocytes. Electrophoretic mobility shift assay showed that the full-length ADRE-AR was specifically bound by a certain nuclear protein(s). The present study has raised the possibility that ADRE-AR may play important roles in the AM gene expression in preadipocytes, and that the AM gene may be repressed through the ADRE-AR in adipocytes.
Li
Yin
Y
Department of Molecular Biology and Applied Physiology, Tohoku University School of Medicine, Sendai, Miyagi 980-8575, Japan.
Zhang
Yan
Y
Furuyama
Kazumichi
K
Yokoyama
Satoru
S
Takeda
Kazuhisa
K
Shibahara
Shigeki
S
Takahashi
Kazuhiro
K
D041721:3T3-L1 Cells; D017667:Adipocytes / Q000166:cytology* / Q000378:metabolism; D053607:Adrenomedullin; D000818:Animals; D001483:Base Sequence; D002454:Cell Differentiation; D015870:Gene Expression; D008156:Luciferases / Q000378:metabolism; D051379:Mice; D010455:Peptides / Q000235:genetics*; D010957:Plasmids / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D011863:Radioimmunoassay; D014158:Transcription, Genetic
D010455:Peptides; D053607:Adrenomedullin; D008156:Luciferases