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
MolPharm articles become freely available 12 months after publication, and remain freely available for 5 years.Non-open access articles that fall outside this five year window are available only to institutional subscribers and current ASPET members, or through the article purchase feature at the bottom of the page.
|