NOVEL ROLE FOR SP1 IN PHORBOL ESTER ENHANCEMENT OF HUMAN PLATELET THROMBOXANE RECEPTOR GENE-EXPRESSION

Expression of platelet thromboxane receptors is transcriptionally incr eased during megakaryocytic differentiation stimulated by phorbol 12-m yristate 13-acetate (PMA). We previously cloned and characterized the promoter region of the human thromboxane receptor gene and localized P MA-responsive elements to a region between 1.84 and 1.95 kilobase pair s (kb) 5' of the transcription initiation site (D'Angelo, D. D., Davis , M. G., Houser, W. A., Eubank, J. J., Ritchie, M. E., and Dorn, G. W. , II (1995) Circ. Res. 77, 466-474). Herein we report the localization of the PMA response element to a 14-nucleotide C-rich sequence, flank ed by an octanucleotide inverted repeat, located -1.938 to -1.925 kb 5 ' of the transcription start site of this gene. We further identify th e PMA-responsive enhancer factor that binds to this C-rich sequence as Sp1. Heterologous thromboxane receptor gene promoter/thymidilate kina se reporter constructs transfected into K562 cells exhibited PMA respo nsiveness when the C-rich element was included with additional 3' sequ ence from -1.924 to -1.84 kb. However, mutations of the C-rich element that disrupted a GC box located on the inverse strand eliminated PMA responsiveness and, in gel mobility shift assays, eliminated binding o f Sp1. PMA treatment of K562 cells significantly increased, by 5-fold, Sp1 binding to the C-rich element and increased both phosphorylated a nd nonphosphorylated Sp1 protein levels by 2-fold. Furthermore, PMA tr eatment transiently increased Sp1 mRNA levels prior to increasing thro mboxane receptor mRNA, suggesting that up-regulation of Sp1 contribute s to up-regulation of thromboxane receptors. Finally, we have detected an unidentified K562 nuclear protein that binds specifically to the s ense strand of the C-rich sequence overlapping the Sp1 binding site an d that, by stabilizing a double stem-loop conformation of this DNA seg ment, may also play a role in Sp1 regulation of this gene. These studi es are the first to describe regulatory and regulated roles for Sp1 in PMA-responsive gene expression and suggest that modulation of Sp1 lev els controls thromboxane receptor expression during megakaryocytic dif ferentiation.