Abstract
BackgroundProtease-activated receptors (PAR) are seven transmembrane G-coupled receptors comprising four genes (PAR-1 ~ PAR-4). Mast cell has been identified to be able to express PARs and release an array of cytokines upon activation. Recently, it was reported that interleukin (IL)-12 could regulate the expression of PARs in mast cells, and tryptase could induce IL-4 and IL-6 release from mast cells. In order to further investigate the issues, RNA interference (RNAi) technique was employed and small interfering RNAs (siRNA) of PARs were transfected in P815 cells.ResultsThe results showed that siRNAs for PAR-1, PAR-2 and PAR-4 significantly downregulated expression of PAR-1, PAR-2 and PAR-4 mRNAs and proteins in P815 cells at 24, 48 and 72 h following transfection. siRNA PAR-1.2 and siRNA PAR-4.2 significantly reduced IL-12 induced upregulation of PAR-1 and PAR-4 expression, respectively when P815 cells were transfected with them for 48 h. siRNA PAR-2.3 blocked IL-12 induced downregulation of PAR-2 expression on both mRNA and protein levels. It was also observed that siRNA PAR-2.3 and siRNA PAR-1.2 reduced trypsin induced IL-4 release by approximately 92.6% and 65.3%, and SLIGKV-NH2 induced IL-4 release by 82.1% and 60.1%, respectively. Similarly, siRNA PAR-2.3 eliminated tryptase-induced IL-4 release by 75.3%, and siRNA PAR-1.2 diminished SFLLR-NH2 induced IL-4 release by 79.3%. However, siRNA PAR-1.2, siRNA PAR-2.3 and siRNA PAR-4.3 at 10 nM did not show any effect on tryptase-induced IL-6 release from P815 cells.ConclusionIn conclusion, siRNAs of PARs can modulate PAR expression and PAR related cytokine production in mast cells, confirming that PARs are likely to play a role in allergic reactions.
Highlights
Protease-activated receptors (PAR) are seven transmembrane G-coupled receptors comprising four genes (PAR-1 ~ PAR-4)
The aim of this study is to investigate the effect of PAR-1, PAR-2 and PAR-4 gene silencing on the expression of PARs and cytokine production in P815 cells by using RNA interference (RNAi)
Reduction of PAR-1 expression in P815 cells by small interfering RNAs (siRNA) Three target sequences in the coding region of PAR-1 mRNA were selected as siRNA sequences, namely siRNA PAR-1.1, siRNA PAR-1.2 and siRNA PAR-1.3
Summary
Protease-activated receptors (PAR) are seven transmembrane G-coupled receptors comprising four genes (PAR-1 ~ PAR-4). Mast cell has been identified to be able to express PARs and release an array of cytokines upon activation. It was reported that interleukin (IL)-12 could regulate the expression of PARs in mast cells, and tryptase could induce IL-4 and IL-6 release from mast cells. PAR-1, PAR-3 and PAR-4 serve as a receptor of thrombin[3,4,5]; PAR-1, PAR-2 and PAR-4 are receptors of trypsin and PAR-2 is a receptor of tryptase These serine proteinases have been discovered to play a crucial role in allergic inflammation. Tryptase was reported to be able to stimulate microvascular leakage in the skin of guinea pigs[6], to induce inflammatory cell accumulation in the peritoneum of mice[7], to elicit histamine release from mast cells and to enhance monocyte chemoattractant protein-1 (MCP-1) and IL-8 production in human endothelial cells[8]. Trypsin was found to be able to induce histamine release from human tonsil and skin mast cells[9] and to stimulate IL-8 and IL-6 release from human respiratory epithelial cells[10]
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