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Originally published In Press as doi:10.1074/jbc.M206127200 on September 30, 2002

J. Biol. Chem., Vol. 277, Issue 50, 48333-48341, December 13, 2002
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Cloning and Analysis of the Thrombopoietin-induced Megakaryocyte-specific Glycoprotein VI Promoter and Its Regulation by GATA-1, Fli-1, and Sp1*

Melissa L. HolmesDagger , Natalie BartleDagger , Michael EisbacherDagger , and Beng H. ChongDagger §

From the Dagger  Centre for Thrombosis and Vascular Research, and § Department of Medicine, St. George Clinical School, University of New South Wales, Sydney, New South Wales 2052, Australia

The exposure of collagen fibers at sites of vascular injury results in the adherence of platelets and their subsequent activation. The platelet collagen receptor glycoprotein (GP)1 VI plays a crucial role in platelet activation and thrombus formation and decreased levels or defective GPVI may lead to excessive bleeding. In addition, elevated levels of collagen receptors may predispose individuals to coronary heart disease or strokes. GPVI expression is restricted to platelets and their precursor cell, the megakaryocyte. In this study we investigate the regulation of GPVI expression and show that thrombopoietin induces its expression in the megakaryocytic cell line UT-7/TPO. A 5'-region flanking the transcription start point of the GPVI gene was cloned (-694 to +29) and we report that this putative GPVI promoter bestows megakaryocye-specific expression. Deletion analyses and site-directed mutagenesis identified Sp1227, GATA177, and Ets48 sites as essential for GPVI expression. We show that transcription factors GATA-1, Fli-1, and Sp1 can bind to and activate this promoter. Finally, GPVI mRNA was detected only in megakaryocytic cell lines expressing both Fli-1 and GATA-1, and we show that overexpression of Fli-1 in a stable cell line (which expresses endogenous GATA-1 and Sp1) results in expression of the endogenous GPVI gene.

* This work was supported by a grant from the Australian National Health and Medical Research Council and an infrastructure grant from the New South Wales Department of Health.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AF521646.

To whom correspondence should be addressed. Present address: Dept. of Medicine, St. George Clinical School, University of New South Wales, Sydney, New South Wales 2052, Australia. Tel.: 61-2-9350-2010; Fax: 61-2-9350-3998; E-mail: b.h.chong@unsw.edu.au.

Copyright © 2002 by The American Society for Biochemistry and Molecular Biology, Inc.
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