Early growth response transcription factor EGR-1 regulates Galphaq gene in megakaryocytic cells.
BACKGROUND
Galphaq (Gene GNAQ) plays a major role in platelet signal transduction but little is known regarding its transcriptional regulation.


OBJECTIVE
We studied Galphaq 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.


RESULTS
PMA-treated HEL cells showed enhanced Galphaq 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 Galphaq 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 Galphaq transcription. Correspondingly, Egr-1 deficient mouse platelets also showed approximately 50% reduction in the Galphaq expression relative to wild-type platelets.


CONCLUSIONS
These studies suggest that Galphaq 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.
Jalagadugula
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G
Sol Sherry Thrombosis Research Center, Temple University School of Medicine, Philadelphia, PA 19140, USA.
Dhanasekaran
D N
DN
Kim
S
S
Kunapuli
S P
SP
Rao
A K
AK
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D001792:Blood Platelets / Q000378:metabolism; D002454:Cell Differentiation / Q000187:drug effects; D045744:Cell Line, Tumor; D051766:Early Growth Response Protein 1 / Q000235:genetics / Q000378:metabolism*; D024202:Electrophoretic Mobility Shift Assay; D043802:GTP-Binding Protein alpha Subunits, Gq-G11 / Q000235:genetics / Q000378:metabolism*; D017353:Gene Deletion; D017930:Genes, Reporter; D006801:Humans; D008156:Luciferases; D049408:Luminescent Agents; D008533:Megakaryocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics; D012333:RNA, Messenger / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000031:analogs & derivatives / Q000494:pharmacology; D014158:Transcription, Genetic; D014162:Transfection
C497325:EGR1 protein, human; D051766:Early Growth Response Protein 1; C486109:Egr1 protein, mouse; D049408:Luminescent Agents; D009841:Oligonucleotides; D012333:RNA, Messenger; C016658:phorbolol myristate acetate; D008156:Luciferases; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D013755:Tetradecanoylphorbol Acetate