Transcriptional regulation of the human thromboxane synthase gene expression

Abstract

Thromboxane synthase (TS) catalyzes the conversion of prostaglandin H{dollar}\sb2{dollar} into thromboxane A{dollar}\sb2{dollar} (TxA{dollar}\sb2),{dollar} which is a potent mediator of platelet aggregation and vasoconstriction. A deficiency of platelet TS or mutations in the TxA{dollar}\sb2{dollar} receptor gene have been shown to result in bleeding disorders, while an elevated level of TxA{dollar}\sb2{dollar} may be associated with cardiovascular and renal diseases. Several post-transcriptional events have been demonstrated to curtail the level of TS in vivo, presumably for preventing over-production of the autacoid. At present, little is known about the transcriptional regulation of TS gene expression. To address this, a genomic DNA containing the human TS promoter was cloned and characterized. 5{dollar}\sp\prime{dollar}-RACE (rapid amplification of cDNA ends) and ribonuclease (RNase) A/T1 protection assays revealed multiple transcription initiation sites. Deletion analysis indicated that TS transcription is mainly TATA-independent. A proximal positive regulatory sequence (PPRS, {dollar}-{dollar}90 to {dollar}-{dollar}25 bp) and several distal repressive elements, including a silencer, were also identified in the promoter. The PPRS worked in an orientation-independent but position-dependent manner, and could be further divided into two independent elements, PPRS{dollar}\sb1{dollar} ({dollar}-{dollar}90 to {dollar}-{dollar}50 bp) and PPRS{dollar}\sb2{dollar} ({dollar}-{dollar}50 to {dollar}-{dollar}25 bp). While similar amounts of nuclear factor(s) from different cell types may interact with PPRS{dollar}\sb2,{dollar} those interacting with PPRS{dollar}\sb1{dollar} exhibit cell specificity. Internal sequence deletion and oligonucleotide competition established that a binding sequence for NF-E2 in PPRS{dollar}\sb1{dollar} ({dollar}-{dollar}60 tgctgattcat {dollar}-{dollar}50) was important for enhancing TS promoter activity in HL-60 cells. The presence of NF-E2 mRNA in HL-60 cells was demonstrated by RT-PCR amplification of the cDNA and Northern analysis. A 9-fold trans-activation of luciferase (luc) reporter gene expression was detected when NF-E2 cDNA was co-expressed with a TS promoter/luc construct. Despite that NF-E2 and the cis-elements could alter the level of TS transcription, they were not sufficient for restricting cell-specific TS expression. Analysis of the methylation status of the TS promoter in several human cell lines revealed cell-specific patterns of methylation that might correlate with TS expression. Taken together, these results suggest that the expression of human TS gene is modulated by multiple factors including cis-elements, trans-activator(s), and possibly genomic methylation.