Egr-1 Activates Basic Fibroblast Growth Factor Transcription: MECHANISTIC IMPLICATIONS FOR ASTROCYTE PROLIFERATION

Elzbieta Biesiada,Mahnaz Razandi,Ellis R Levin

doi:10.1074/jbc.271.31.18576

Abstract

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.

Highlights

From the Departments of ‡Medicine and ¶Pharmacology, University of California, Irvine, Irvine, California 92716 and §Long Beach Veterans Affairs Medical Center, Long Beach, California 90822
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 by unknown mechanisms
We compared the level of egr-chloramphenicol acetyltransferase (CAT) expression in cells stimulated with ET-3 alone, and with cells treated with ET-3 ϩ ANP or cells cotransfected with Dom Neg MAPK plasmid prior to ET-3 exposure

Summary

Introduction

From the Departments of ‡Medicine and ¶Pharmacology, University of California, Irvine, Irvine, California 92716 and §Long Beach Veterans Affairs Medical Center, Long Beach, California 90822. 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. We were interested in determining whether Egr-1 regulates activation of the bFGF promoter through direct interaction with DNA and what are the proximal signals which lead to the positive or negative modulation of egr-1 expression by these neuropeptides These issues are potentially important beyond our model because Egr-1 is the first immediate early protein/ transcriptional factor identified to activate bFGF transcription [11]

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Conclusion