Transcription factors Sp1 and Sp3 alter vascular endothelial growth factor receptor expression through a novel recognition sequence.
Kinase domain receptor (KDR) is a high affinity, endothelial cell-specific, autophosphorylating tyrosine kinase receptor for vascular endothelial growth factor. This transcriptionally regulated receptor is a critical mediator of endothelial cell (EC) growth and vascular development. In this study, we identify a DNA element modulating KDR promoter activity and evaluate the nuclear binding proteins accounting for a portion of the cell-type specificity of the region. KDR promoter luciferase activity was retained within -85/+296 and was 10-30-fold higher in EC than non-EC. Electrophoretic mobility shift assays demonstrated specific nuclear protein binding to -85/-64, and single point mutations suggested important binding nucleotides between -79/-68 with five critical bases between -74/-70 (5'-CTCCT-3'). DNA-protein complexes were displaced by Sp1 consensus sequence oligodeoxynucleotides and supershifted by Sp1- and Sp3-specific antibodies. Sp1 and Sp3 protein in EC nuclear extracts bound the -79/-68 region even when all surrounding classic Sp1 recognition sites were removed. Sp1 protein in nuclear extracts was 4-24-fold higher in EC than non-EC, whereas Sp3 was 3-7-fold higher. Sp1/Sp3 ratios in EC were 2-10-fold higher. Overexpression of Sp1 protein increased KDR promoter activity 3-fold in both EC and non-EC, whereas simultaneous co-expression of Sp3 attenuated this response. An Sp1 consensus sequence cis element "decoy" reduced EC KDR promoter activity and mRNA expression by 85 and 69%, respectively. An antisense phosphorothioate oligodeoxynucleotide to Sp1 inhibited Sp1 and KDR protein expression by 66 and 68%, respectively, without changing Sp3 protein expression. These data illustrate that Sp1 and Sp3 modulate KDR promoter activity through a novel recognition binding sequence. However, since Sp1-mediated promoter activation is attenuated by Sp3, endothelial selective KDR promoter activity may be partially regulated by variations in the Sp1/Sp3 ratio.
Research Division, Joslin Diabetes Center, Boston, Massachusetts 02215, USA.
Y
Y
Hata
E
E
Duh
K
K
Zhang
G S
GS
Robinson
L P
LP
Aiello
D001483:Base Sequence; D002478:Cells, Cultured; D004268:DNA-Binding Proteins / Q000502:physiology*; D005786:Gene Expression Regulation; D006801:Humans; D008969:Molecular Sequence Data; D016376:Oligonucleotides, Antisense / Q000494:pharmacology; D011401:Promoter Regions, Genetic; D020794:Receptor Protein-Tyrosine Kinases / Q000235:genetics*; D017978:Receptors, Growth Factor / Q000235:genetics*; D040262:Receptors, Vascular Endothelial Growth Factor; D016329:Sp1 Transcription Factor / Q000235:genetics / Q000502:physiology*; D051705:Sp3 Transcription Factor; D013873:Thionucleotides / Q000494:pharmacology; D014157:Transcription Factors / Q000502:physiology*; D016335:Zinc Fingers
D004268:DNA-Binding Proteins; D016376:Oligonucleotides, Antisense; D017978:Receptors, Growth Factor; C497121:SP3 protein, human; D016329:Sp1 Transcription Factor; D013873:Thionucleotides; D014157:Transcription Factors; D051705:Sp3 Transcription Factor; D020794:Receptor Protein-Tyrosine Kinases; D040262:Receptors, Vascular Endothelial Growth Factor