Early Growth Response Factor EGR-1 Regulates Gαq Gene in Megakaryocytic Cells.

Abstract Gαq (Gene GNAQ) plays a major role in platelet signal transduction but little is known regarding its transcriptional regulation. We studied Gαq promoter activity using luciferase reporter gene assays in human erythroleukemia (HEL) cells treated with 10 nM of phorbol 12-myristate 13-acetate (PMA) for 24 h to induce megakaryocytic (MK) transformation. RT-PCR analysis of HEL cell RNA revealed that Gαq mRNA was relatively low in untreated cells and it increased after PMA treatment with a peak at 5 h. Immunoblot analysis of HEL lysates showed enhanced Gαq expression with PMA. Luciferase reporter gene studies on full length construct (upto −1116 bp from ATG) and its serial 5′ truncations revealed a negative regulatory site at −238/−202 and two positive sites at −203/−138 and −1116/−731. In the region −238/−202 consensus sites were noted for two transcription factors PU.1 and GATA-1 that are known to regulate several megakaryocytic genes. Deletions of these sites alone or together revealed no change in the transcriptional activity of the gene in reporter studies. The positive region −203/−138 contained two overlapping Sp1/AP-2/EGR-1 consensus sites at −202/−189 and −164/−150. Gel shift studies were performed on oligonucleotides 1 (−203/−175) and 2 (−174/−152) using HEL cell extracts. Protein binding occured with Gαq oligonucleotides 1 and 2, which was competed with excess unlabeled oligos but not by unlabeled Sp1 or AP-2 consensus oligos. Supershift assay using antibody against Sp1 revealed neither competition nor supershift, suggesting that Sp1 does not bind to these oligonucleotides. No protein binding was noted when Gαq oligos 1 or 2 were incubated with extracts known to contain Sp1 or AP-2. These results indicate that Sp1 and AP-2 do not bind to the Gαq oligonucleotide regions. Protein binding to oligonucleotides 1 and 2 was abolished by excess unlabeled consensus EGR-1 oligo, and by immunodepletion of the EGR-1 protein from the nuclear extract with anti-EGR-1 antibody. These experiments reveal that EGR-1 binds to both Gαq oligonucleotides −203/−175 and −174/−152. In luciferase reporter studies mutations in EGR-1 sites present in both oligonucleotides 1 and 2 markedly decreased gene activity indicating functional relevance. In further studies, reduction in endogenous EGR-1 expression with antisense oligonucleotide to EGR-1 inhibited PMA induced Gαq transcription and protein in HEL cells. Lastly, EGR-1 deficient mouse platelets also showed ~50% reduction in the Gαq protein (immunoblotting) relative to wild type platelets. These studies suggest that Gαq gene is regulated during PMA induced differentiation by EGR-1, a transcription factor that regulates a wide array of genes involved in cellular proliferation, differentiation, and apoptosis, and in vascular response to injury and atherosclerosis.