Characterization of Promoter 3 of the Human Thromboxane A Receptor Gene. A Functional AP-1 and Octamer Motif Are Required for Basal Promoter Activity

The TPalpha and TPbeta isoforms of the human thromboxane A(2) receptor (TP) arise by differential splicing but are under the transcriptional control of two distinct promoters, termed Prm1 and Prm3, respectively (Coyle et al. 2002 Eur J Biochem269, 4058-4073). The aim of the current study was to determine the key factors regulating TPbeta expression by functionally characterizing Prm3, identifying the core promoter and the cis-acting elements regulating basal Prm3 activity. Hence, the ability of Prm3 and a series of Prm3 deleted/mutated subfragments to direct reporter gene expression in human erythroleukemia 92.1.7 and human embryonic kidney 293 cells was investigated. It was established that nucleotides -118 to +1 are critical for core Prm3 activity in both cell types. Furthermore, three distinct regulatory regions comprising of an upstream repressor sequence, located between -404 to -320, and two positive regulatory regions required for efficient basal gene expression, located between -154 to -106 and -50 to +1, were identified within the core Prm3. Deletion and site-directed mutagenesis of consensus Oct-1/2 and AP-1 elements within the latter -154 to -106 and -50 to +1 regions, respectively, substantially reduced Prm3 activity while mutation of both elements abolished Prm3 activity. Electromobility shift and supershift assays confirmed the specificity of nuclear factor binding to the latter Oct-1/2 and AP-1 elements. Moreover, herein it was established that the core AP-1 element mediates phorbol myristic acid-induction of Prm3 activity hence providing a mechanistic explanation of phorbol ester up-regulation of TPbeta mRNA expression.