© 1995 American Heart Association, Inc.
Articles |
Characterization of 5' End of Human Thromboxane Receptor Gene
Organizational Analysis and Mapping of Protein Kinase C– Responsive Elements Regulating Expression in Platelets
From the University of Cincinnati (Ohio) Medical Center.
Correspondence to Gerald W. Dorn II, MD, University of Cincinnati Medical Center, ML 542, 231 Bethesda Ave, Cincinnati, OH 45267-0542.
Abstract Platelet thromboxane receptors are
acutely and reversibly upregulated after acute myocardial infarction.
To determine if platelet thromboxane receptors are under
transcriptional control, we isolated and characterized human genomic
DNA clones containing the 5' flanking region of the thromboxane
receptor gene. The exon-intron structure of the 5' portion of the
thromboxane receptor gene was determined initially by comparing
the nucleotide sequence of the 5' flanking genomic clone
with that of a novel human uterine thromboxane receptor cDNA
that extended the mRNA 141 bp further upstream than the previously
identified human placental cDNA. A major transcription initiation site
was located in three human tissues 
560 bp upstream from the
translation initiation codon and 380 bp upstream from any previously
identified transcription initiation site. The thromboxane
receptor gene has neither a TATA nor a CAAT consensus site. Promoter
function of the 5' flanking region of the thromboxane receptor
gene was evaluated by transfection of thromboxane receptor gene
promoter/chloramphenicol acetyltransferase (CAT) chimera plasmids into
plateletlike K562 cells. Thromboxane receptor promoter
activity, as assessed by CAT expression, was relatively weak but was
significantly enhanced by phorbol ester treatment. Functional
analysis of 5' deletion constructs in transfected K562 cells
and gel mobility shift localized the major phorbol ester–responsive
motifs in the thromboxane receptor gene promoter to a cluster
of activator protein-2 (AP-2) binding consensus sites located 1.8 kb
5' from the transcription initiation site. These studies are the first
to determine the structure and organization of the 5' end of the
thromboxane receptor gene and demonstrate that
thromboxane receptor gene expression can be regulated by
activation of protein kinase C via induction of an AP-2–like nuclear
factor binding to upstream promoter elements. These findings strongly
suggest that the mechanism for previously described upregulation of
platelet thromboxane receptors after acute myocardial
infarction is increased thromboxane receptor gene transcription
in platelet-progenitor cells.
Key Words: thromboxane receptor • platelets • gene expression • transcriptional regulation • protein kinase C
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