Endothelial APC/PAR1 distinctly regulates cytokine-induced pro-inflammatory VCAM-1 expression

Cierra A Birch; Helen Wedegaertner; Lennis B Orduña-Castillo; Monica L Gonzalez Ramirez; Huaping Qin; JoAnn Trejo

doi:10.3389/fmolb.2023.1211597

Endothelial APC/PAR1 distinctly regulates cytokine-induced pro-inflammatory VCAM-1 expression

Front Mol Biosci. 2023 Aug 24:10:1211597. doi: 10.3389/fmolb.2023.1211597. eCollection 2023.

Authors

Cierra A Birch¹, Helen Wedegaertner^{1

2}, Lennis B Orduña-Castillo¹, Monica L Gonzalez Ramirez¹, Huaping Qin¹, JoAnn Trejo¹

Affiliations

¹ Department of Pharmacology, School of Medicine, University of California, San Diego, CA, United States.
² Biomedical Sciences Graduate Program, University of California, San Diego, CA, United States.

Abstract

Introduction: Dysfunction of the endothelium impairs its' protective role and promotes inflammation and progression of vascular diseases. Activated Protein C (APC) elicits endothelial cytoprotective responses including barrier stabilization, anti-inflammatory and anti-apoptotic responses through the activation of the G protein-coupled receptor (GPCR) protease-activated receptor-1 (PAR1) and is a promising therapeutic. Despite recent advancements in developing new Activated protein C variants with clinical potential, the mechanism by which APC/PAR1 promotes different cytoprotective responses remains unclear and is important to understand to advance Activated protein C and new targets as future therapeutics. Here we examined the mechanisms by which APC/PAR1 attenuates cytokine-induced pro-inflammatory vascular cell adhesion molecule (VCAM-1) expression, a key mediator of endothelial inflammatory responses. Methods: Quantitative multiplexed mass spectrometry analysis of Activated protein C treated endothelial cells, endothelial cell transcriptomics database (EndoDB) online repository queries, biochemical measurements of protein expression, quantitative real-time polymerase chain reaction (RT-qPCR) measurement of mRNA transcript abundance, pharmacological inhibitors and siRNA transfections of human cultured endothelial cells. Results: Here we report that Activated Protein C modulates phosphorylation of tumor necrosis factor (TNF)-α signaling pathway components and attenuates of TNF-α induced VCAM-1 expression independent of mRNA stability. Unexpectedly, we found a critical role for the G protein-coupled receptor co-receptor sphingosine-1 phosphate receptor-1 (S1PR1) and the G protein receptor kinase-2 (GRK2) in mediating APC/PAR1 anti-inflammatory responses in endothelial cells. Discussion: This study provides new knowledge of the mechanisms by which different APC/PAR1 cytoprotective responses are mediated through discrete β-arrestin-2-driven signaling pathways modulated by specific G protein-coupled receptor co-receptors and GRKs.

Keywords: GPCR; GRK; TNF-α; cytoprotection; thrombin.

Abstract

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