Cloning and functional analysis of the promoter for KDR/flk-1, a receptor for vascular endothelial growth factor.
KDR/flk-1 is one of two receptors for vascular endothelial growth factor, a potent angiogenic peptide. KDR/flk-1 is an early marker for endothelial cell progenitors, and its expression is restricted to endothelial cells in vivo. To investigate the molecular mechanisms regulating expression of KDR/flk-1, we cloned and characterized the promoter of the human KDR/flk-1 gene. The transcription start site was localized by primer extension and ribonuclease protection to a nucleotide 303 base pairs (bp) 5' of the initiation methionine codon. The 5'-flanking sequence is rich in G and C residues and contains five Sp1 elements but no TATA consensus sequence. By reporter gene transfection experiments, we found that approximately 4 kilobases of KDR/flk-1 5'-flanking sequence directed high level luciferase activity in bovine aortic endothelial cells; further deletion analysis revealed positive regulatory elements between bp -225 to -164, -95 to -77, -77 to -60, and +105 to +127. Mutation of an atypical GATA sequence between bp +105 and +127 did not affect promoter activity, suggesting that GATA elements are not essential for the high level promoter activity of this gene. Consistent with endothelial cell-restricted expression of KDR/flk-1 mRNA, we found that the 4-kilobase flanking sequence directed high level promoter activity in endothelial cells but not in other cell types. To our knowledge this is the first report characterizing the KDR/flk-1 promoter. Understanding the KDR/flk-1 promoter will allow us to investigate endothelial cell-specific gene regulation and to uncover methods for targeting gene delivery specifically to endothelial cells.
Patterson
C
C
Cardiovascular Biology Laboratory, Harvard School of Public Health, Brigham and Women's Hospital, Boston, Massachusetts 02115, USA.
Perrella
M A
MA
Hsieh
C M
CM
Yoshizumi
M
M
Lee
M E
ME
Haber
E
E
D000818:Animals; D001483:Base Sequence; D002417:Cattle; D002478:Cells, Cultured; D003001:Cloning, Molecular; D016384:Consensus Sequence; D016228:Endothelial Growth Factors / Q000378:metabolism*; D004730:Endothelium, Vascular / Q000378:metabolism*; D015698:Genomic Library; D006801:Humans; D008156:Luciferases / Q000096:biosynthesis / Q000378:metabolism; D008222:Lymphokines / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D016296:Mutagenesis; D011401:Promoter Regions, Genetic; D020794:Receptor Protein-Tyrosine Kinases / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D017978:Receptors, Growth Factor / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D040262:Receptors, Vascular Endothelial Growth Factor; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D015183:Restriction Mapping; D017384:Sequence Deletion; D016329:Sp1 Transcription Factor / Q000378:metabolism; D016385:TATA Box; D014162:Transfection; D014471:Umbilical Veins; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors
D016228:Endothelial Growth Factors; D008222:Lymphokines; D017978:Receptors, Growth Factor; D011994:Recombinant Proteins; D016329:Sp1 Transcription Factor; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors; D008156:Luciferases; D020794:Receptor Protein-Tyrosine Kinases; D040262:Receptors, Vascular Endothelial Growth Factor