Egr-1 activates basic fibroblast growth factor transcription. Mechanistic implications for astrocyte proliferation.
The mechanisms controlling the proliferation of astrocytes are of great interest but are not well defined. We have previously shown that the endogenous neuropeptides, endothelin-3 (ET-3), and atrial natriuretic peptide (ANP), modulate the proliferation of astrocytes through positively and negatively regulating the transcription of the immediate-early gene egr-1 which transactivates basic fibroblast growth factor (bFGF) by unknown mechanisms. In these studies, we determined the involvement of MAP kinase (Erk) activation by ET-3 in the transcription of egr-1, and the molecular determinants by which Egr-1 transactivates bFGF. Transfection of astrocytes with a mitogen-activated protein (MAP) kinase (MAPK) expression vector increased the transcription of a cotransfected egr-chloramphenicol acetyltransferase (CAT) construct 3-fold. This induction was totally abolished by a dominant negative MAPK mutant. A 3-fold induction of egr-CAT expression by ET-3 was significantly reduced by treatment with ANP, or a cotransfected dominant negative MAPK plasmid. Using mobility shift assays, we showed that ET-3 induced the expression of Egr-1 protein which bound specifically to several early growth-related protein (Egr-1) binding sites on the bFGF promoter, and that this effect was significantly reversed by treatment with ANP. We also found that the Sp1 transcriptional factor was bound at these same sites, but was not stimulated by ET-3. Deletion experiments indicated that only the site at -160 bp of the bFGF promoter was significant for bFGF transactivation by Egr-1. We conclude that the astrocyte mitogen, ET-3, stimulates egr-1 transcription through a MAP kinase (Erk) related mechanism, and that Egr-1 transactivates bFGF through a specific noncanonical, Egr-1 site on the promoter. ANP inhibits each of these steps, providing a pathway for its anti-proliferative action.
Biesiada
E
E
Department of Medicine, University of California, Irvine, Irvine, California 92716, USA.
Razandi
M
M
Levin
E R
ER
D000818:Animals; D001253:Astrocytes / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism*; D009320:Atrial Natriuretic Factor / Q000494:pharmacology; D001483:Base Sequence; D002455:Cell Division; D002478:Cells, Cultured; D004247:DNA / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D051766:Early Growth Response Protein 1; D016232:Endothelins / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D016222:Fibroblast Growth Factor 2 / Q000235:genetics*; D006801:Humans; D017874:Immediate-Early Proteins; D048369:MAP Kinase Kinase 1; D020929:Mitogen-Activated Protein Kinase Kinases; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000235:genetics; D011401:Promoter Regions, Genetic; D011485:Protein Binding / Q000187:drug effects; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000235:genetics / Q000378:metabolism; D051381:Rats; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D015533:Transcriptional Activation / Q000187:drug effects; D014162:Transfection
D004268:DNA-Binding Proteins; C497325:EGR1 protein, human; D051766:Early Growth Response Protein 1; C486110:Egr1 protein, rat; D016232:Endothelins; D017874:Immediate-Early Proteins; D009838:Oligodeoxyribonucleotides; D014157:Transcription Factors; D016222:Fibroblast Growth Factor 2; D009320:Atrial Natriuretic Factor; D004247:DNA; D011505:Protein-Tyrosine Kinases; D017346:Protein Serine-Threonine Kinases; D048369:MAP Kinase Kinase 1; C482125:MAP2K1 protein, human; D020929:Mitogen-Activated Protein Kinase Kinases