Inducible expression of the megakaryocyte-specific gene glycoprotein IX is mediated through an Ets binding site and involves upstream activation of extracellular signal-regulated kinase.
Glycoprotein IX is a megakaryocyte-specific gene crucial for adequate and functional expression of the Glycoprotein Ib-IX complex. This study used phorbol 12-myristate 13-acetate (PMA) and thrombopoietin (TPO)-induced differentiation of Dami and UT-7 cells, respectively, to investigate the regulation of inducible Glycoprotein IX expression during megakaryocyte differentiation. PMA and TPO were able to modulate GPIX expression at mRNA and protein levels. Transient transfection studies using nested 5'-deletions and mutations of the GPIX promoter demonstrated the absolute requirement of an inverted Ets site 5'-ACTTCCT-3' for inducible reporter gene expression. The upstream signaling events associated with PMA and TPO-inducible expression of GPIX were also investigated. The mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase inhibitor PD98059 inhibited both PMA and TPO-inducible reporter activity in a dose-dependent manner, whereas inhibition of p38/MAPK had no significant effect. The protein kinase C inhibitor GF109203X failed to inhibit TPO-activation of the GPIX promoter in UT-7 cells. This study demonstrates that inducible expression in response to either PMA or TPO is mediated through the Ets site in the proximal promoter of GPIX and is dependent upon the upstream activation of MAPK/extracellular signal-regulated kinase.
Eisbacher
M
M
The Centre for Thrombosis and Vascular Research, School of Pathology, University of New South Wales, Sydney, NSW 2217, Australia.
Khachigian
L M
LM
Khin
T H
TH
Holmes
M L
ML
Chong
B H
BH
D020816:Amino Acid Motifs / Q000187:drug effects / Q000235:genetics; D001665:Binding Sites / Q000187:drug effects / Q000502:physiology; D002454:Cell Differentiation / Q000187:drug effects / Q000235:genetics*; D002478:Cells, Cultured; D004268:DNA-Binding Proteins / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005434:Flow Cytometry; D005786:Gene Expression Regulation / Q000187:drug effects / Q000502:physiology*; D017930:Genes, Reporter / Q000187:drug effects / Q000502:physiology; D006410:Hematopoiesis / Q000187:drug effects / Q000235:genetics*; D006801:Humans; D008156:Luciferases / Q000493:pharmacokinetics; D008533:Megakaryocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D020928:Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000187:drug effects / Q000378:metabolism*; D019038:Platelet Glycoprotein GPIb-IX Complex / Q000235:genetics* / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000187:drug effects / Q000502:physiology; D014176:Protein Biosynthesis; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism; D011506:Proteins / Q000187:drug effects; D051789:Proto-Oncogene Protein c-fli-1; D011518:Proto-Oncogene Proteins / Q000187:drug effects / Q000378:metabolism; D050783:Proto-Oncogene Proteins c-ets; D012333:RNA, Messenger / Q000187:drug effects / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013926:Thrombopoietin / Q000494:pharmacology; D015534:Trans-Activators / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D014157:Transcription Factors / Q000187:drug effects / Q000378:metabolism; D014162:Transfection; D048051:p38 Mitogen-Activated Protein Kinases
D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; D019038:Platelet Glycoprotein GPIb-IX Complex; D011506:Proteins; D051789:Proto-Oncogene Protein c-fli-1; D011518:Proto-Oncogene Proteins; D050783:Proto-Oncogene Proteins c-ets; D012333:RNA, Messenger; D015534:Trans-Activators; D014157:Transcription Factors; D013926:Thrombopoietin; D008156:Luciferases; D011493:Protein Kinase C; D020928:Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases; D013755:Tetradecanoylphorbol Acetate