Novel Role for Sp1 in Phorbol Ester Enhancement of Human Platelet Thromboxane Receptor Gene Expression

Abstract

Expression of platelet thromboxane receptors is transcriptionally increased during megakaryocytic differentiation stimulated by phorbol 12-myristate 13-acetate (PMA). We previously cloned and characterized the promoter region of the human thromboxane receptor gene and localized PMA-responsive elements to a region between 1.84 and 1.95 kilobase pairs (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, flanked by an octanucleotide inverted repeat, located ؊1.938 to ؊1.925 kb 5 of the transcription start site of this gene. We further identify the PMA-responsive enhancer factor that binds to this Crich sequence as Sp1. Heterologous thromboxane receptor gene promoter/thymidilate kinase reporter constructs transfected into K562 cells exhibited PMA responsiveness when the C-rich element was included with additional 3 sequence 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 of Sp1. PMA treatment of K562 cells significantly increased, by 5-fold, Sp1 binding to the C-rich element and increased both phosphorylated and nonphosphorylated Sp1 protein levels by 2-fold. Furthermore, PMA treatment transiently increased Sp1 mRNA levels prior to increasing thromboxane receptor mRNA, suggesting that up-regulation of Sp1 contributes to up-regulation of thromboxane receptors. Finally, we have detected an unidentified K562 nuclear protein that binds specifically to the sense strand of the C-rich sequence overlapping the Sp1 binding site and that, by stabilizing a double stem-loop conformation of this DNA segment, may also play a role in Sp1 regulation of this gene. These studies are the first to describe regulatory and regulated roles for Sp1 in PMA-responsive gene expression and suggest that modulation of Sp1 levels controls thromboxane receptor expression during megakaryocytic differentiation.Thromboxane A 2 is one of the most potent platelet-aggregating and vasoconstricting substances known and is crucial for maintenance of normal hemostasis (1). While considerable attention has been directed toward measuring increased thromboxane production in various disease states including acute coronary ischemic syndromes (2, 3), relatively little is known about how target tissue responses to thromboxane may be modulated. Two intriguing studies have reported increased numbers of platelet thromboxane receptors, with enhanced platelet aggregation, in acute myocardial infarction and unstable angina pectoris (4, 5). The observation that this thromboxane receptor abnormality was reversible after acute myocardial infarction (4) and the discovery of an identical abnormality in patients with unstable angina (the clinical precursor of completed myocardial infarct) (5), strongly sugg...

P38 mitogen-activated protein kinase mediates hexosamine-induced TGFβ1 mRNA expression in human mesangial cells

“…These results complement previous observations suggesting a PKC-dependent mechanism in the regulation of hexosamine-induced TGFβ1 mRNA expression [24]. The cellular content of the transcription factor Sp1, involved in the regulation of TGFβ1 transcription [31], are up-regulated by PKC activation [32]. Furthermore hexosamines promote Sp1 O-linked N-acetylglucosamine glycosylation [33], which stabilizes this transcription factor, increasing its cellular concentrations [34].…”

Sp1 recognition sites in the proximal promoter of the human vascular endothelial growth factor gene are essential for platelet-derived growth factor-induced gene expression

“…In contrast to the situation in endothelial cells, in megakaryocytes and in osteosarcoma cells constitutive binding of Sp1 and/or Sp3 was necessary for basal as well as for phorbol ester-induced transcription of the PDGF-B gene. Increased binding of Sp1 to its respective recognition sites was reported as a further mechanism by which Sp1 mediates enhanced gene expression, for instance in PDGF-induction of the human low density lipoprotein receptor gene (Basheeruddin et al, 1995) and in phorbol ester-induction of the human platelet thromboxane receptor gene (D'Angelo et al, 1996).…”

Glucosamine activates the plasminogen activator inhibitor 1 gene promoter through Sp1 DNA binding sites in glomerular mesangial cells.

“…However, there is no evidence yet that Sp1 protein stability is altered by glucosamine in the presence of physiological concentrations of glucose or by increasing glucose concentrations above normal. Other potential mechanisms of glucosamine-induced changes in Sp1 DNA binding, which are also relevant in the case of high glucose, include dephosphorylation of residues in the Sp1 DNA binding domain (61), release of Sp1 from an inhibitor (36), and increased Sp1 mRNA levels induced by PKC (64).…”

“…Although Sp1 is constitutively expressed (57), its activity is regulated by diverse factors. Indeed, the activity of Sp1 varies during development (58,59) and is modulated by growth factors (57), retinoblastoma protein (60), high glucose (61), cAMP (62), PKC (63,64), and extracellular signal-regulated kinase (ERK) (65).…”