Identification of glucose transporter 4 knockdown-dependent transcriptional activation element on the retinol binding protein 4 gene promoter and requirement of the 20 S proteasome subunit for transcriptional activity.
Retinol binding protein 4 (RBP4) is the transport protein that carries retinol in blood. RBP4 was described recently as a new adipokine that reduced insulin sensitivity. Mice lacking glucose transporter 4 (GLUT4) in adipocytes have enhanced Rbp4 gene expression; however, the molecular mechanism is unknown. We found a G4KA (GLUT4 knockdown-dependent transcriptional activation) element located approximately 1.3 kb upstream of the Rbp4 promoter. Mutations within the G4KA sequence significantly reduced expression of the Rbp4 promoter-reporter construct in G4KD-L1 (GLUT4 knockdown 3T3-L1) adipocyte cells. In a yeast one-hybrid screen of a G4KD-L1 cell cDNA library, using the G4KA element as bait, we identified subunits of the 20 S proteasome, PSMB1 and PSMA4, as binding partners. In chromatin immunoprecipitation assays, both subunits bound to the G4KA element; however, only PSMB1 was tightly bound in the GLUT4 knockdown model. PSMB1 RNA interference, but not PSMA4, significantly inhibited Rbp4 transcription. Nuclear transportation of PSMB1 was increased in G4KD-L1 cells. These results provide evidence for an exclusive proteasome subunit-related mechanism for transcriptional activation of RBP4 within a GLUT4 knockdown model.
National Institute of Health and Nutrition, 1-23-1 Toyama, Shinjyuku, Tokyo 162-8636, Japan.
Erina
E
Inoue
Aoi
A
Yamashita
Hirofumi
H
Inoue
Mariko
M
Sekiguchi
Asuka
A
Shiratori
Yuji
Y
Yamamoto
Tadahiro
T
Tadokoro
Yoshiko
Y
Ishimi
Jun
J
Yamauchi
D041721:3T3-L1 Cells; D000818:Animals; D015153:Blotting, Western; D047369:Chromatin Immunoprecipitation; D024202:Electrophoretic Mobility Shift Assay; D051275:Glucose Transporter Type 4 / Q000235:genetics / Q000502:physiology*; D016086:Lentivirus / Q000235:genetics; D051379:Mice; D011401:Promoter Regions, Genetic / Q000235:genetics*; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism*; D034622:RNA Interference / Q000502:physiology; D054839:Retinol-Binding Proteins, Plasma / Q000235:genetics*; D014158:Transcription, Genetic / Q000235:genetics / Q000502:physiology; D015533:Transcriptional Activation / Q000235:genetics / Q000502:physiology*
D051275:Glucose Transporter Type 4; C502487:Rbp4 protein, mouse; D054839:Retinol-Binding Proteins, Plasma; D046988:Proteasome Endopeptidase Complex