Suppression of EDAG gene expression by phorbol 12-myristate 13-acetate is mediated through down-regulation of GATA-1.
EDAG, a hematopoietic tissue-specific protein, is involved in the regulation of proliferation, differentiation and apoptosis of hematopoietic cells. In this study, a dose-dependent inhibition of EDAG expression by PMA was observed in K562 cells. The responsive element for the PMA-induced inhibition was contained in the region between -211 and +32bp of the EDAG gene promoter. By oligonucleotide-directed mutagenesis, EMSA, ChIP and transient transfection assays, we found that two tandem repeat GATA-1 sites in the promoter of EDAG gene played an important role in the PMA-mediated down-regulation of the EDAG gene expression in K562 cells. The kinetics of EDAG expression during PMA induction showed that the levels of EDAG expression were down-regulated concomitantly with GATA-1 down-expression. Decreased GATA-1 expression by siRNA reduced expression of EDAG in K562 cells, and restored expression of GATA-1 significantly rescued EDAG expression from PMA-mediated suppression. Overexpression of EDAG in K562 cells inhibited the megakaryocytic differentiation induced by PMA which raised the interesting possibility that PMA induced K562 cells differentiation toward megakaryocytic phenotype through, at least in part, the inhibition of EDAG expression. In vivo analysis confirmed that EDAG was highly expressed in primitive progenitor cells and down-regulated in megakaryocytes which was consistent with the expression pattern of GATA-1. Furthermore, PKC and MAPK specific inhibitors treatment attenuated the down-regulation of EDAG induced by PMA. Taken together, these results suggest that the inhibition of the EDAG gene by PMA is mediated through down-regulation of transcription factor GATA-1 and involved the PKC/MAPK signaling pathway.
Li
Chang-Yan
CY
Beijing Institute of Radiation Medicine, 27 Taiping Road, Beijing, China.
Fang
Fang
F
Xu
Wang-Xiang
WX
Xu
Cheng-Wang
CW
Zhan
Yi-Qun
YQ
Wang
Zhi-Dong
ZD
Ding
Ya-Li
YL
Li
Yong-Hui
YH
Sun
Hui-Bin
HB
Yang
Xiao-Ming
XM
D001483:Base Sequence; D001665:Binding Sites; D002454:Cell Differentiation / Q000187:drug effects; D002478:Cells, Cultured; D003609:Dactinomycin / Q000494:pharmacology; D015536:Down-Regulation / Q000187:drug effects; D050982:GATA1 Transcription Factor / Q000378:metabolism* / Q000502:physiology; D020868:Gene Silencing / Q000187:drug effects*; D006801:Humans; D020014:K562 Cells; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000235:genetics*; D011401:Promoter Regions, Genetic; D011485:Protein Binding / Q000187:drug effects; D012333:RNA, Messenger / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*; D014162:Transfection
D050982:GATA1 Transcription Factor; C494688:GATA1 protein, human; C435745:HEMGN protein, human; D009687:Nuclear Proteins; D012333:RNA, Messenger; D003609:Dactinomycin; D013755:Tetradecanoylphorbol Acetate