title: Phorbol 12-myristate 13-acetate (PMA) responsive sequence in Galphaq promoter during megakaryocytic differentiation. Regulation by EGR-1 and MAP kinase pathway.
abstract: Galphaq plays a major role in platelet signal transduction, but little is known regarding its transcriptional regulation. We have reported that Galphaq is upregulated during phorbol 12-myristate 13-acetate (PMA)-induced megakaryocytic transformation of human erythroleukemia (HEL) cells and regulated by EGR-1, an early growth transcription factor. These studies focused on the initial 238 bp of the 5' upstream region of the Galphaq gene. In the present studies we characterize a minimal region -1042/-1037 bp from ATG in the 5' upstream of the Galphaq promoter that is associated with PMA responsiveness. In luciferase reporter gene studies in HEL cells, Galphaq 5' upstream promoter sequence -1042/-1 showed an about four-fold increased activity in PMA-treated compared to untreated cells. Deletion of 6-nt -1042/-1037 eliminated the difference. Gel-shift studies on Galphaq probe (-1042/-1012 bp) revealed binding of EGR-1 with PMA-treated but not untreated nuclear extracts, and this was dependent on the sequence -1042/-1037. Silencing of endogenous EGR-1 inhibited Galphaq induction by PMA. MEK/ERK inhibitor U0126 blocked PMA effect on promoter activity of the -1042/-1 construct. In conclusion, EGR-1 binding to sequence -1042/-1037 bp in Galphaq promoter mediates the induction of Galphaq gene by PMA via the MEK/ERK signaling pathway. These studies provide the first evidence of a PMA-responsive element in Galphaq promoter, and new insights into regulation of Galphaq gene by EGR-1.
authors.affiliation: Sol Sherry Thrombosis Research Center, Temple University School of Medicine, 3400 N. Broad St., OMS-300, Philadelphia, PA 19140, USA.
authors.forename: Gauthami
authors.initials: G
authors.lastname: Jalagadugula
authors.forename: Danny N
authors.initials: DN
authors.lastname: Dhanasekaran
authors.forename: A Koneti
authors.initials: AK
authors.lastname: Rao
mesh_terms: D024506:5' Flanking Region / Q000187:drug effects; D002454:Cell Differentiation / Q000187:drug effects* / Q000235:genetics; D045744:Cell Line, Tumor; D051766:Early Growth Response Protein 1 / Q000235:genetics / Q000378:metabolism*; D024202:Electrophoretic Mobility Shift Assay; D043802:GTP-Binding Protein alpha Subunits, Gq-G11 / Q000235:genetics / Q000378:metabolism*; D017930:Genes, Reporter; D006801:Humans; D008533:Megakaryocytes / Q000187:drug effects* / Q000201:enzymology; D020928:Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D016376:Oligonucleotides, Antisense / Q000378:metabolism; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D020218:Response Elements / Q000187:drug effects*; D015398:Signal Transduction / Q000187:drug effects; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*; D015854:Up-Regulation
chemical_list: C497325:EGR1 protein, human; D051766:Early Growth Response Protein 1; D016376:Oligonucleotides, Antisense; D047428:Protein Kinase Inhibitors; D020928:Mitogen-Activated Protein Kinases; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D013755:Tetradecanoylphorbol Acetate