Role for Early Growth Response-1 Protein in α1-Adrenergic Stimulation of Fibroblast Growth Factor-2 Promoter Activity in Cardiac Myocytes
- Gene Technology and Departments of Physiology, and Pharmacology and Therapeutics, University of Manitoba, Winnipeg, Manitoba, Canada
Abstract
Fibroblast growth factor-2 (FGF-2), a mitogenic, angiogenic, and cardioprotective agent, is released from the postnatal heart by a mechanism of transient remodelling of the sarcolemma during contraction. Both release of FGF-2 and its synthesis can be increased with adrenergic stimulation. We reported previously that FGF-2 synthesis can be regulated at the transcriptional level by α-adrenergic stimulation of cultured neonatal rat cardiac myocytes as well as in the adult mouse heart. Examination of the proximal promoter region of both human and rat FGF-2 gene sequences revealed binding sites for the early growth response-1 (Egr-1) protein. Using gel mobility shift assays, we observed a transient increase in a complex between nuclear extracts from neonatal rat cardiac myocytes treated with inducers of Egr-1, including the α-adrenergic agonist phenylephrine, angiotensin II, and phorbol ester, and a consensus Egr-1 DNA element. A similar complex was seen with the FGF-2 promoter region −7/+42 as the DNA probe, but not when the Egr-1 element at nucleotides +3/+31 was disrupted. Participation of Egr-1 protein in the complex was confirmed by competition with Egr-1 DNA elements and antibodies. With deletion analysis and transfection of neonatal rat cardiac myocytes, the α-adrenergic response was localized to nucleotides −110/+42 of the FGF-2 gene in the context of a hybrid FGF-2/luciferase reporter gene, −110FGFp.luc. Overexpression of Egr-1 increased −110FGFp.luc gene expression, whereas mutation of its Egr-1 element at nucleotides +3/+31 abolished α-adrenergic responsiveness. These data indicate that Egr-1 is involved in the α-adrenergic stimulation of the FGF-2 promoter region in neonatal cardiac myocytes.
Footnotes
-
Send reprint requests to: Peter A. Cattini, Department of Physiology, University of Manitoba, 730 William Ave., Winnipeg, Manitoba, Canada R3E 3J7. E-mail: Peter_Cattini{at}UManitoba.CA
-
This study was supported by a grant from the Medical Research Council of Canada (to M.R.C.). F.S. was the recipient of an Medical Research Council Studentship, K.A.D. was the recipient of a Heart and Stroke Foundation Studentship, and P.A.C. was the recipient of a Medical Research Council Scientist award.
- Abbreviations:
- NE
- norepinephrine
- FGF-2
- fibroblast growth factor-2
- Egr-1
- early growth response-1
- FBS
- fetal bovine serum
- CMV
- cytomegalovirus
- SV40
- simian virus 40
- EMSA
- electrophoretic gel mobility shift assay
- PMA
- phorbol-12-myristate-13-acetate
- ATII
- angiotensin II
- WT
- wild type
- MUT
- mutation
- PE
- phenylephrine
- DMEM
- Dulbecco's modified Eagle's medium
- Praz
- prazosin
-
- Received November 29, 1999.
- Accepted February 2, 2000.
- The American Society for Pharmacology and Experimental Therapeutics
