Abstract
Abstract only
Recently we reported that in human coronary artery endothelial cells (HCAEC), the P2Y2 nucleotide receptor (P2Y2R) regulates the pro-thrombotic gene tissue factor (TF) expression through positive and negative signaling mechanisms. Here, we report the identification of a P2Y2R ligand JZS0312 which selectively activates the negative, but not the positive pathways, leading to decreased TF expression. Unlike the endogenous agonist UTP, JZS0312 alone had no effect on TF mRNA transcription, but it unexpectedly inhibited UTP-induced TF expression. Mechanistic study showed that JZS0312 induced intracellular Ca
2+
mobilization in HCAEC which exhibited the same efficacy as UTP and was inhibited by AR-C118925, a P2Y2R-selective antagonist. However, when evaluated in their activation of the MAPK pathways, JZS0312 only activated the ERK1/2, but not JNK and p38, whereas UTP activated all the three, suggesting JZS0312 as a biased ligand for the P2Y2R. This notion was supported by the fact that JZS0312-induced ERK1/2 activation was inhibited by AR-C1189258 and it had no effect on ERK1/2 in P2Y2R-null cells. In addition, JZS0312 selectively activated the TF gene repressor Fra-1, but not the positive AP-1 subunits c-Jun and ATF-2, whereas UTP activated all of them. Furthermore, luciferase activity assay indicates that JZS0312 suppressed TF gene promoter activity via ERK1/2 activation, while UTP increased overall TF promoter activity. These findings reveal that JZS0312 acts as a biased agonist in human endothelial P2Y2R, in which it selectively activates the ERK1/2-Fra1 pathway, leading to suppression of TF gene transcription. Thus, JZS0312 represents the first identified biased ligand in the P2Y receptor family, opening new avenue for potential drug discovery in relevant inflammatory and thrombotic diseases.