Non-platelet Purinergic Signaling and Tissue Factor Expression

Abstract

Objective—Our group found previously that human coronary artery endothelial P2Y2 receptor mediates tissue factor (TF) up-regulation at mRNA and protein levels. The exact mechanism remains unknown and can involve increased gene transcription, mRNA stability and protein stability. Other non-platelet tissues and cells, such as monocytes and microvascular endothelium, are also known to be actively involved in thrombosis and express inducible TF. Nevertheless, whether and how this induction can be regulated by purinergic stimulation is meagerly defined. The aim of this study was to determine how non-platelet purinergic stimulation affects TF transcription and mRNA stability.

Methods and Results—Here we show for the first time that endothelial P2Y2R mediated TF transcription up-regulation as well as mRNA stabilization. A new Evi-1 binding site was found but did not affect P2Y2R mediated endothelial TF expression as shown by luciferase assay. On the other hand, Evi-1 might play a role in monocyte differentiation and TNF-α signaling due to its changes shown by Western Blotting. New AP-1 binding site and subunits c-Jun, Fra-1 and ATF-2 were associated with TF transcription in human coronary artery endothelial cells (HCAEC) as demonstrated by luciferase assay, EMSA, Western Blotting, and ChIP assay. Of the three subunits, c-Jun and ATF-2 were under control of Rho/JNK pathway while Fra-1 was activated by Src/ERK1/2 axis. Among the three pathways, P2Y2R/Src/ERK1/2/Fra-1 played a negative role on TF transcription as reflected by real-time PCR of TF pre-mRNA. p38/MK-2 pathway was suggested to stabilize TF mRNA through AU-rich element binding proteins (AREBP) as shown by Western Blotting with pharmacological inhibitors. microRNA array screening yielded 44 microRNAs significantly changed by P2Y2R activation in HCAEC. Of them, down-regulation of microRNA-181a may contribute to higher TF mRNA stability. A primary profile of purinergic stimulation affecting induced TF expression in HCAEC, HUVEC, HMVEC, THP-1 cells and human primary monocytes was established by Western Blotting.

Conclusions—Purinergic induction of TF expression follows different patterns in various tissues. In HCAEC, under UTP stimulation, TF mRNA level is up-regulated due to both promoted transcription and improved mRNA stability. A novel AP-1 binding site was identified -1362 bp upstream of transcription starting site in TF promoter. Positive pathway P2Y2R/Rho/JNK/c-Jun/ATF-2 and negative pathway P2Y2R/Src/ERK1/2/Fra-1 orchestrate in the transcription up-regulation. Both AREBP and microRNAs are implied to contribute to improved mRNA stability. These insights into the TF induction mechanism shed lights on better understanding of nucleotide signaling and new antithrombotic therapy.