Abstract
We recently reported that the P2Y2 receptor (P2Y2R) is the predominant nucleotide receptor expressed in human coronary artery endothelial cells (HCAEC) and that P2Y2R activation by ATP or UTP induces dramatic up-regulation of tissue factor (TF), a key initiator of the coagulation cascade. However, the molecular mechanism of this P2Y2R-TF axis remains unclear. Here, we report the role of a newly identified AP-1 consensus sequence in the TF gene promoter and its original binding components in P2Y2R regulation of TF transcription. Using bioinformatics tools, we found that a novel AP-1 site at -1363 bp of the human TF promoter region is highly conserved across multiple species. Activation of P2Y2R increased TF promoter activity and mRNA expression in HCAEC. Truncation, deletion, and mutation of this distal AP-1 site all significantly suppressed TF promoter activity in response to P2Y2R activation. EMSA and ChIP assays further confirmed that upon P2Y2R activation, c-Jun, ATF-2, and Fra-1, but not the typical c-Fos, bound to the new AP-1 site. In addition, loss-of-function studies using siRNAs confirmed a positive transactivation role of c-Jun and ATF-2 but unexpectedly revealed a strong negative role of Fra-1 in P2Y2R-induced TF up-regulation. Furthermore, we found that P2Y2R activation promoted ERK1/2 phosphorylation through Src, leading to Fra-1 activation, whereas Rho/JNK mediated P2Y2R-induced activation of c-Jun and ATF-2. These findings reveal the molecular basis for P2Y G protein-coupled receptor control of endothelial TF expression and indicate that targeting the P2Y2R-Fra-1-TF pathway may be an attractive new strategy for controlling vascular inflammation and thrombogenicity associated with endothelial dysfunction.
Highlights
Nucleotides matter more than being the universal currency of energy transaction and the building blocks of genes
We recently reported that the P2Y2 receptor (P2Y2R) is the predominant nucleotide receptor expressed in human coronary artery endothelial cells (HCAEC) and that P2Y2R activation by ATP or UTP induces dramatic up-regulation of tissue factor (TF), a key initiator of the coagulation cascade
P2Y2R Activation Boosts TF Expression by Promoting Gene Transcription—Because our previous study demonstrated that P2Y2R is the only UTP-sensitive nucleotide receptor expressed in HCAEC [11], UTP was routinely used to activate the P2Y2R throughout this study
Summary
Nucleotides matter more than being the universal currency of energy transaction and the building blocks of genes. We recently reported that stimulation of human coronary artery endothelial cells (HCAEC) by ATP/UTP leads to dramatic up-regulation of tissue factor (TF) expression and activity through the P2Y2R [11]. Some are endogenous molecules, such as TNF-␣, interleukin-1, CD40 ligand [10], or UTP/ATP [11]; others are exogenous factors, including endotoxin [12] and caffeine [13] These inducers share some similar signal transduction pathways regulating TF induction in different cells. The PI3K/AKT pathway is shown to negatively regulate TF expression through an unknown mechanism [10] In this regard, our initial study already showed that endothelial P2Y2R activates ERK1/2, JNK, and p38 MAPK pathways without affecting the PI3K/AKT negative pathway [11]. P2Y2 Signaling and Tissue Factor Transcription transcriptional molecular mechanism in response to G proteincoupled receptor activation, i.e. P2Y2R, remains largely unknown. A negative regulator Fra-1 for the TF gene was identified for the first time
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
