P2Y2 Receptor Control of Tissue Factor Transcription in Human Coronary Artery Endothelial Cells: New AP‐1 Site and Negative Regulator

Abstract

Recently we 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), key initiator of the coagulation cascade. However, the molecular mechanism of this P2Y2R‐TF axis remains unclear. Here we report a role of a newly identified AP‐1 consensus sequence along with its new binding components in P2Y2R regulation of TF transcription. We identified with bioinformatics tools that a novel AP‐1 site at ‐1363 bp of 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 new distal AP‐1 site all significantly supressed 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, leading to Fra‐1 activation while JNK activated c‐Jun and ATF‐2. These findings reveal the basis for P2Y nucleotide receptor control of endothelial TF expression and indicate that targeting the P2Y2R‐Fra‐1‐TF pathway may be an attractive new strategy in control of vascular thrombogenicity and/or inflammation associated with endothelial dysfunction.