The t-PA -7351C>T enhancer polymorphism decreases Sp1 and Sp3 protein binding affinity and transcriptional responsiveness to retinoic acid.
We have previously identified a common polymorphism at the tissue-type plasminogen activator (t-PA) locus (-7351C>T), located within a GC-box in the retinoic acid (RA) and steroid hormone responsive t-PA enhancer. The aim of the present study was to functionally characterize this t-PA variant. Electrophoretic mobility shift assays (EMSAs) using crude nuclear extracts from human endothelial, HeLa, and NT2 neuronal cells revealed a 10-fold greater protein binding affinity to the wild-type C allele compared with the mutant T allele variant. Sp1 and Sp3 were identified as the GC-box binding proteins. Luciferase reporter assays showed that the C allele generated higher transcriptional activity after induction by RA, compared with the T allele variant. Further EMSAs showed that RA treatment enhanced Sp1/Sp3 binding to the GC-box. Formation of the Sp1/Sp3 containing complex was inhibited by anti-RA receptor (RAR) antibodies, suggesting that Sp1/Sp3 and RAR interact. The t-PA -7351C>T polymorphism is therefore functional at the level of transcription. The reduced binding affinity of Sp1/Sp3 to the T allele could explain our earlier observations of a reduced t-PA release and an increased risk of myocardial infarction in individuals carrying this allele.
Wolf
Anna Tjärnlund
AT
Institute of Clinical Neuroscience, Sahlgrenska University Hospital/Sahlgrenska, Göteborg University, S-413 45 Göteborg, Sweden.
Medcalf
Robert L
RL
Jern
Christina
C
D003596:Cytosine; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004742:Enhancer Elements, Genetic / Q000235:genetics*; D005312:Fetal Blood; D006801:Humans; D007136:Immunoglobulins / Q000378:metabolism*; D007231:Infant, Newborn; D007700:Kinetics; D011110:Polymorphism, Genetic; D011485:Protein Binding; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017384:Sequence Deletion; D051705:Sp3 Transcription Factor; D013941:Thymine; D010959:Tissue Plasminogen Activator / Q000235:genetics* / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism*; D014158:Transcription, Genetic / Q000187:drug effects*; D014212:Tretinoin / Q000494:pharmacology*
D004268:DNA-Binding Proteins; D007136:Immunoglobulins; C090698:SP1 antigen; C497121:SP3 protein, human; D014157:Transcription Factors; D051705:Sp3 Transcription Factor; D014212:Tretinoin; D003596:Cytosine; D010959:Tissue Plasminogen Activator; D013941:Thymine