Polymorphisms in the 5'-UTR of the tissue factor gene are associated with altered expression in human endothelial cells.
BACKGROUND
Enhanced tissue factor (TF) expression mediates many disease processes. Recently, four completely concordant polymorphisms were detected in the 5'-UTR of the TF gene. Three were single base changes and one was an 18-bp insertion/deletion at -1208.


OBJECTIVE
This study was undertaken to determine if the I-allele or the D-allele would associate with elevated TF expression in human umbilical vein endothelial cells (HUVEC).


METHODS
HUVEC were genotyped by polymerase chain reaction for 18-bp insert status. TF expression was induced by interleukin (IL)-1 or phorbol 12-myristate 13-acetate (PMA). Total TF activity was determined by a one-stage clotting assay and surface TF activity by a chromogenic assay. Protein binding differences between the I- and D-alleles were examined by gel shift assays.


RESULTS
IL-1- or PMA-induced total TF activity in D-allele HUVEC was increased 2.0-2.5-fold above that seen in II HUVEC. Surface clotting activity in D-allele cells was 1.3-1.7-fold greater than in II-allele cultures. Experiments with consensus site mutation oligos suggested that the 18-bp insert creates GATA and CCAAT-enhancer binding protein (C/EBP) transcription factor recognition sites.


CONCLUSIONS
The D-allele is associated with enhanced TF activity in HUVEC. The differences in TF expression between the alleles may be due to variant transcription factor binding in the -1208 region. Further studies are warranted to investigate whether the D-allele is associated with increased incidence of pathological processes that involve TF.
Terry
C M
CM
Department of Internal Medicine, Pulmonary and Hematology Divisions, Virginia Commonwealth University, Richmond, VA, USA. christi.terry@hsc.utah.edu
Kling
S J
SJ
Cheang
K I
KI
Hoidal
J R
JR
Rodgers
G M
GM
D020121:5' Untranslated Regions; D000483:Alleles; D020816:Amino Acid Motifs; D001777:Blood Coagulation; D002478:Cells, Cultured; D004247:DNA / Q000378:metabolism; D042783:Endothelial Cells / Q000166:cytology*; D004730:Endothelium, Vascular / Q000166:cytology* / Q000473:pathology; D017353:Gene Deletion; D005838:Genotype; D006801:Humans; D008957:Models, Genetic; D009154:Mutation; D011110:Polymorphism, Genetic; D012333:RNA, Messenger / Q000378:metabolism; D012307:Risk Factors; D013755:Tetradecanoylphorbol Acetate; D013925:Thromboplastin / Q000235:genetics*
D020121:5' Untranslated Regions; D012333:RNA, Messenger; D004247:DNA; D013925:Thromboplastin; D013755:Tetradecanoylphorbol Acetate