title: Purinergic P2Y2 Receptor Control of Tissue Factor Transcription in Human Coronary Artery Endothelial Cells: NEW AP-1 TRANSCRIPTION FACTOR SITE AND NEGATIVE REGULATOR.
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.
authors.affiliation: From the Department of Drug Discovery and Development, Harrison School of Pharmacy, Auburn University, Auburn, Alabama 36849.
authors.forename: Yiwei
authors.initials: Y
authors.lastname: Liu
authors.affiliation: From the Department of Drug Discovery and Development, Harrison School of Pharmacy, Auburn University, Auburn, Alabama 36849.
authors.forename: Lingxin
authors.initials: L
authors.lastname: Zhang
authors.affiliation: From the Department of Drug Discovery and Development, Harrison School of Pharmacy, Auburn University, Auburn, Alabama 36849.
authors.forename: Chuan
authors.initials: C
authors.lastname: Wang
authors.affiliation: From the Department of Drug Discovery and Development, Harrison School of Pharmacy, Auburn University, Auburn, Alabama 36849.
authors.forename: Shama
authors.initials: S
authors.lastname: Roy
authors.affiliation: From the Department of Drug Discovery and Development, Harrison School of Pharmacy, Auburn University, Auburn, Alabama 36849. Electronic address: jzs0019@auburn.edu.
authors.forename: Jianzhong
authors.initials: J
authors.lastname: Shen
mesh_terms: D001483:Base Sequence; D002460:Cell Line; D003331:Coronary Vessels / Q000166:cytology* / Q000378:metabolism; D042783:Endothelial Cells / Q000378:metabolism*; D005786:Gene Expression Regulation; D006801:Humans; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D058473:Receptors, Purinergic P2Y2 / Q000235:genetics / Q000378:metabolism*; D013925:Thromboplastin / Q000235:genetics* / Q000378:metabolism; D018808:Transcription Factor AP-1 / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic
chemical_list: C546385:P2RY2 protein, human; D058473:Receptors, Purinergic P2Y2; D018808:Transcription Factor AP-1; D013925:Thromboplastin
keywords: Fra-1; G protein-coupled receptor; P2Y2 nucleotide receptor; endothelial cell; gene expression; gene transcription; tissue factor; vascular biology