Regulated expression of the alpha isoform of the human thromboxane A2 receptor during megakaryocyte differentiation: a coordinated role for WT1, Egr1, and Sp1.

Thromboxane plays an essential role in hemostasis, regulating platelet aggregation and vessel tone. In humans, it signals through the TPalpha and TPbeta isoforms that are transcriptionally regulated by distinct promoters Prm1 and Prm3, respectively. Herein, the consequence of megakaryocytic differentiation on Prm1-directed TPalpha expression was investigated. Phorbol 12-myristate 13-acetate (PMA) treatment substantially increased TPalpha mRNA and Prm1-directed gene expression in human erythroleukemia and K562 cells. Deletional analyses localized the major responsive element(s) to the upstream -8500 to -7504 region while mutation of four WT1/Egr1/Sp1 cis elements therein established that each contributes to the induction. Moreover, PMA increased Egr1, but not WT1 or Sp1, expression while the NGFI-A-binding protein 1 co-repressor impaired PMA induction of Egr1- and Prm1-directed gene expression. Chromatin immunoprecipitations established that WT1 is predominantly bound in vivo to the 5' Prm1 region in non-differentiated human erythroleukemia cells. In response to PMA, there was initial induction in Egr1 and associated reduction in WT1 binding to Prm1 in vivo, which was displaced by Sp1 following sustained treatment. Collectively, data establish that regulated WT1 followed by sequential Egr1 and Sp1 binding to elements within Prm1 mediate repression and subsequent induction of TPalpha during differentiation into the megakaryocytic phenotype, shedding significant insights into factors regulating TPalpha expression therein.