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Early growth response transcription factor EGR‐1 regulates G α q gene in megakaryocytic cells
Author(s) -
JALAGADUGULA G.,
DHANASEKARAN D. N.,
KIM S.,
KUNAPULI S. P.,
RAO A. K.
Publication year - 2006
Publication title -
journal of thrombosis and haemostasis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.947
H-Index - 178
eISSN - 1538-7836
pISSN - 1538-7933
DOI - 10.1111/j.1538-7836.2006.02229.x
Subject(s) - biology , transcription factor , microbiology and biotechnology , reporter gene , signal transduction , transfection , gene expression , gene , ap 1 transcription factor , cell growth , biochemistry
Summary. Background: G α q (Gene GNAQ) plays a major role in platelet signal transduction but little is known regarding its transcriptional regulation. Objectives: We studied G α q promoter activity using luciferase reporter gene assays in human erythroleukemia (HEL) cells treated with phorbol 12‐myristate 13‐acetate (PMA) for 24 h to induce megakaryocytic transformation. Methods and results: PMA‐treated HEL cells showed enhanced G α q expression. Reporter (luciferase) gene studies on 5′ upstream construct (up to −116 bp from ATG) revealed a negative regulatory site at −238/−202 and two positive sites at −203/−138 and −1116/−731. The positive regulatory region −203/−138 contained overlapping Sp1/AP‐2/EGR‐1 consensus sites. Gel shift studies on G α q oligonucleotides 1 (−203/−175) and 2 (−174/−152) using HEL cell extracts demonstrated protein binding that was due to early growth response factor EGR‐1 at two sites. Mutations in either EGR‐1 site markedly decreased the gene activity, indicating functional relevance. Mutation of consensus E‐Box motif (−185/−180) had no effect. Reduction in the expression of endogenous EGR‐1 with antisense oligonucleotide to EGR‐1 inhibited PMA‐induced G α q transcription. Correspondingly, Egr‐1 deficient mouse platelets also showed ∼50% reduction in the G α q expression relative to wild‐type platelets. Conclusions: These studies suggest that G α q gene is regulated during PMA‐induced megakaryocytic differentiation by EGR‐1, an early growth response transcription factor that regulates a wide array of genes and plays a major role in diverse activities, including cell proliferation, differentiation and apoptosis, and in vascular response to injury and atherosclerosis.