A cAMP Response Element and an Ets Motif Are Involved in the Transcriptional Regulation of flt-1 Tyrosine Kinase (Vascular Endothelial Growth Factor Receptor 1) Gene*
- From the Department of Genetics, Institute of Medical Science, University of Tokyo, Minato-ku, Tokyo 108, Japan and
- ‡ Unité d'Oncologie Moléculaire-CNRS URA 1160, Institut Pasteur, 1 rue Calmette, 59019 Lille Cédex, France
- § To whom correspondence and requests for reprints should be addressed: Dept. of Genetics, Inst. of Medical Science, University of Tokyo, Minato-ku, Tokyo 108, Japan. Tel.: 81-3-5449-5550; Fax: 81-3-5449-5425.
Abstract
The flt-1 gene encodes a transmembrane tyrosine kinase, Flt-1, a receptor for vascular endothelial growth factor. The expression of flt-1 gene is restricted to endothelial cells in vivo. To understand the molecular mechanism underlying endothelial-specific expression of this gene, we studied the functional significance of transcriptional motifs in the 200-base pair region of the human flt-1 gene promoter, which has been identified to confer cell type specificity. By point mutation analysis using chloramphenicol acetyltransferase plasmids in 293E1 cells, which express significant levels of flt-1 mRNA, we found that an Ets motif, E4, at −54 to −51 and a cAMP response element (CRE) at −83 to −76 are involved in the transcriptional regulation of this gene. Disruption of either this CRE or E4 within the promoter sequence of 90 base pairs resulted in a decrease in chloramphenicol acetyltransferase activity of 90%, indicating that co-existence of both of CRE and Ets motif E4 is necessary for transcription of the flt-1 gene. Co-transfection of an expression vector containing c-ets-1, c-ets-2, or c-erg cDNA with this 90-base pair sequence yielded a 5-8-fold elevation of chloramphenicol acetyltransferase activity, further supporting the idea that Ets family protein(s) participates in the regulation of the flt-1 gene. Gel shift assays using nuclear extracts of 293E1 and endothelial cells demonstrated the existence of protein factor(s) that specifically binds to CRE and Ets motif E4, respectively. Taken together, our results strongly suggest cooperation of a CRE and an Ets motif for the function of the flt-1 gene promoter.
Footnotes
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↵* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵1 The abbreviations used are:
- VEGF
-
vascular endothelial growth factor
- Flt-1
-
fms-like tyrosine kinase
- KDR
-
kinase insert domain-containing receptor
- Flk-1
-
fetal liver kinase
- CRE
-
cAMP response element
- HUVEC
-
human umbilical vein endothelial cells
- CAT
-
chloramphenicol acetyltransferase
- bp
-
base pair(s).
-
- Received August 7, 1996.
- Revision received September 9, 1996.
- © 1996 by The American Society for Biochemistry and Molecular Biology, Inc.
