Abstract
The alpha and beta isoforms of the human thromboxane A(2) (TXA(2)) receptor (TP) are encoded by a single gene but are transcriptionally regulated by distinct promoters, termed promoter 1 (Prm1) and Prm3, respectively. Herein, it was sought to identify factors regulating Prm1 within the megakaryocytic human erythroleukemia 92.1.7 cell line. Through gene deletion and reporter assays, the core Prm1 was localized to between nucleotides -6,320 and -5,895, proximal to the transcription initiation site. Furthermore, two upstream repressor and two upstream activator regions were identified. Site-directed mutagenesis of four overlapping Sp1/Egr1 elements and an NF-E2/AP1 element within the proximal region substantially reduced Prm1 activity. Deletion/mutation of GATA and Ets elements disrupted the upstream activator sequence located between -7,962 and -7,717, significantly impairing Prm1 activity. Electrophoretic mobility shift assays and chromatin immunoprecipitations confirmed that Sp1, Egr1, and NF-E2 bind to elements within the core promoter, whereas GATA-1 and Ets-1 factors bind to the upstream activator sequence (between -7,962 and -7,717). Collectively, these data establish that Sp1, Egr1, and NF-E2 regulate core Prm1 activity in the megakaryocytic-platelet progenitor cells, whereas GATA-1 and Ets-1 act as critical upstream activators, hence providing the first genetic basis for the expression of the human TXA(2) receptor (TP) within the vasculature.
Highlights
The a and b isoforms of the human thromboxane A2 (TXA2) receptor (TP) are encoded by a single gene but are transcriptionally regulated by distinct promoters, termed promoter 1 (Prm1) and Prm3, respectively
The recombinant plasmid pGL3b: Prm1 directed 7.83 6 0.70 relative luciferase units (RLUs) in human erythroleukemia (HEL) cells (Fig. 1A), compared with 23.9 6 1.1 RLUs directed by an SV40 promoter in the pGL3control vector, which acted as a reference
Imbalances in the levels of TXA2 and TXA2 receptor (TP) are implicated in a range of cardiovascular disorders [1,2,3,4], but the relative extent to which TPa and TPb contribute to such pathologies is unknown
Summary
The a and b isoforms of the human thromboxane A2 (TXA2) receptor (TP) are encoded by a single gene but are transcriptionally regulated by distinct promoters, termed promoter 1 (Prm1) and Prm, respectively. Electrophoretic mobility shift assays and chromatin immunoprecipitations confirmed that Sp1, Egr, and NF-E2 bind to elements within the core promoter, whereas GATA-1 and Ets-1 factors bind to the upstream activator sequence (between 27,962 and 27,717) These data establish that Sp1, Egr, and NF-E2 regulate core Prm activity in the megakaryocyticplatelet progenitor cells, whereas GATA-1 and Ets-1 act as critical upstream activators, providing the first genetic basis for the expression of the human TXA2 receptor (TP) within the vasculature.—Gannon, A. TPa, but not TPb, undergoes desensitization in response to the antiaggregatory/vasodilatory agents prostacyclin and nitric oxide involving direct phosphorylation of TPa by cAMPand cGMP-dependent protein kinase, respectively, within its unique C-tail domain [12, 13] The implication from the latter studies is that TPa is the main isoform involved. Consistent with that hypothesis, TPa and TPb show distinct patterns of mRNA and protein expression in a range of cell and tissue types of vascular origin [15], platelets exclusively express TPa [16]
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