脂肪細胞におけるレチノール結合タンパク質4(RBP4)遺伝子発現調節機構の解析 : RBP4遺伝子プロモーター上に存在するGLUT4ノックダウン依存的転写活性化配列の同定と転写活性化に必要な転写因子のクローニング

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Retinol binding protein 4 (RBP4) is the transport protein that carries retinol in blood. RBP4 was recently described as a new adipokine that reduced insulin sensitivity. Although mice lacking glucose transporter 4 (GLUT4) in adipocytes enhance RBP4 gene expression, its molecular mechanisms are unknown. We demonstrate promoter analysis of RBP4 in 3T3-L1 adipocytes, using a stable knockdown of GLUT4 (G4KD-L1) by a lentiviral shRNA-mediated system. We found a GLUT4 knockdown-dependent transcriptional activation (G4KA) element 〜1.3kb upstream of the RBP4 promoter. Mutations within the G4KA sequence significantly reduced expression of the RBP4 promoter-reporter construct in G4KD-L1 cells. In a yeast one-hybrid screen of a G4KD-L1 cell cDNA library, using the G4KA element as bait, we identified subunits of 20 S proteasome, PSMB1 and PSMA4, as binding partners. In ChIP assays, both subunits bound to the G4KA element, however, only PSMB1 tightly bound in response to GLUT4 knockdown. PSMB1 RNA interference, but not PSMA4, significantly inhibited RBP4 transcription. Nuclear transportation of PSMB1 was increased in G4KD-L1. These results provide evidence for an exclusive proteasome subunit-related mechanism for transcriptional activation of RBP4 by GLUT4 knockdown.
2010-07-25