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Protease‐activated receptor ‐3 signals independent of protease‐activated receptor‐1 to regulate endothelial nitric oxide synthase in human endothelial cells
Author(s) -
Tillery Lakeisha Carrie,
MotleyJohnson Evangeline
Publication year - 2013
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.27.1_supplement.1127.1
Subject(s) - enos , phosphorylation , signal transduction , microbiology and biotechnology , nitric oxide synthase type iii , nitric oxide synthase , nitric oxide , receptor , thrombin , endothelium , endothelial stem cell , biology , chemistry , biochemistry , endocrinology , immunology , platelet , in vitro
Protease‐activated receptor‐3 (PAR‐3) is widely recognized as a non‐signaling elusive member of a G protein‐coupled receptor family known to regulate the endothelial nitric oxide synthase (eNOS)/nitric oxide (NO) signaling pathway via phosphorylation of its regulatory sites. Failure of the eNOS/NO pathway leads to endothelial dysfunction, a precursor to cellular induced hypertension. Using human coronary artery endothelial cells (HCAECs) as a model for human endothelium, we demonstrated that PAR‐3 is highly co‐expressed with PAR‐1 and ‐2 and can be activated using a synthetic peptide ligand, SFNGGP‐NH 2 . We hypothesize that PAR‐3 can signal independently of PAR‐1 and ‐2 to regulate eNOS via site specific phosphorylation and induce proi‐nflammation via the NFkB pathway. Analysis using eNOS specific antibodies showed that 50uM of SFNGGP lead to the phosphorylation of the positive site of eNOS at ‐Ser1177 and the negative site ‐Thr495 in a time‐dependent manner. Pretreatment of HCAECs with 10uM SCH79797, a selective PAR‐1 inhibitor, did not block the increase in phosphorylation at both eNOS regulatory sites stimulated by SFNGGP. We created a stable PAR‐1 deficient cell line of HCAECs using lentiviral particles to further establish the signaling potential of PAR‐3 in the presence of thrombin and the PAR‐3 activating peptide. Our data suggests that PAR‐3 is able to regulate eNOS/NO signaling via site specific phosphorylation that is independent of PAR‐1 activation and cause pro‐inflammation with the secretion of cytokines. Understanding the signaling pathways responsible for eNOS‐induced NO and cytokine production will lead to a better understanding of endothelial dysfunction and vascular inflammation in cardiovascular diseases.