Summary. Background: Gaq (Gene GNAQ) plays a major role in platelet signal transduction but little is known regarding its transcriptional regulation. Objectives: We studied Gaq 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 Gaq 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 Gaq 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 Gaq transcription. Correspondingly, Egr-1 deficient mouse platelets also showed 50% reduction in the Gaq expression relative to wild-type platelets. Conclusions: These studies suggest that Gaq 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.
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