Phorbol 12-myristate 13-acetate (PMA) responsive sequence in Gαq promoter during megakaryocytic differentiation

Gauthami Jalagadugula; Danny N. Dhanasekaran; A. Koneti Rao

doi:10.1160/TH08-07-0496

Thrombosis and Haemostasis, Table of Contents

Thromb Haemost 2008; 100(05): 821-828
DOI: 10.1160/TH08-07-0496

Platelets and Blood Cells

Schattauer GmbH

Phorbol 12-myristate 13-acetate (PMA) responsive sequence in Gαq promoter during megakaryocytic differentiation

Regulation by EGR-1 and MAP kinase pathway

Gauthami Jalagadugula

¹Sol Sherry Thrombosis Research Center, Temple University School of Medicine, Philadelphia, Pennsylvania, USA

,

Danny N. Dhanasekaran

²Fels Institute for Cancer Research and Molecular Biology, Temple University School of Medicine, Philadelphia, Pennsylvania, USA

⁴Biochemistry, Temple University School of Medicine, Philadelphia, Pennsylvania, USA

,

A. Koneti Rao

¹Sol Sherry Thrombosis Research Center, Temple University School of Medicine, Philadelphia, Pennsylvania, USA

³Departments of Medicine, Temple University School of Medicine, Philadelphia, Pennsylvania, USA

› Author Affiliations

Abstract

Summary

Gαq plays a major role in platelet signal transduction, but little is known regarding its transcriptional regulation. We have reported that Gαq 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 Gαq gene. In the present studies we characterize a minimal region -1042/-1037 bp from ATG in the 5’ upstream of the Gαq promoter that is associated with PMA responsiveness. In luciferase reporter gene studies in HEL cells, Gαq 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 Gαq 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 Gαq 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 Gαq promoter mediates the induction of Gαq gene by PMA via the MEK/ERK signaling pathway. These studies provide the first evidence of a PMA-responsive element in Gαq promoter, and new insights into regulation of Gαq gene by EGR-1.

Keywords

EGR-1 - Gαq - MAP kinase - megakaryocytic differentiation - PMA responsive sequence

Full Text

References

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