Abstract
BackgroundG protein-coupled receptors (GPCRs) are a major family of signaling molecules, central to the regulation of inflammatory responses. Their activation upon agonist binding is attenuated by GPCR kinases (GRKs), which desensitize the receptors through phosphorylation. G protein-coupled receptor kinase 2(GRK2) down-regulation in leukocytes has been closely linked to the progression of chronic inflammatory disorders such as rheumatoid arthritis and multiple sclerosis. Because leukocytes must interact with the endothelium to infiltrate inflamed tissues, we hypothesized that GRK2 down-regulation in endothelial cells would also be pro-inflammatory.ObjectivesTo determine whether GRK2 down-regulation in endothelial cells is pro-inflammatory.MethodssiRNA-mediated ablation of GRK2 in human umbilical vein endothelial cells (HUVECs) was used in analyses of the role of this kinase. Microscopic and biochemical analyses of Weibel-Palade body (WPB) formation and functioning, live cell imaging of calcium concentrations and video analyses of adhesion of monocyte-like THP-1 cells provide clear evidence of GRK2 function in histamine activation of endothelial cells.ResultsG protein-coupled receptor kinase 2 depletion in HUVECs increases WPB exocytosis and P-selectin-dependent adhesion of THP-1 cells to the endothelial surface upon histamine stimulation, relative to controls. Further, live imaging of intracellular calcium concentrations reveals amplified histamine receptor signaling in GRK2-depleted cells, suggesting GRK2 moderates WPB exocytosis through receptor desensitization.ConclusionsG protein-coupled receptor kinase 2 deficiency in endothelial cells results in increased pro-inflammatory signaling and enhanced leukocyte recruitment to activated endothelial cells. The ability of GRK2 to modulate initiation of inflammatory responses in endothelial cells as well as leukocytes now places GRK2 at the apex of control of this finely balanced process.
Highlights
To initiate an inflammatory response, both leukocytes and endothelial cells need to be activated by hormones and proinflammatory cytokines
To investigate a potential role for GRK2 in modulating endothelial behaviour, we examined whether GRK2depleted human umbilical vein endothelial cells (HUVECs) produce normal Weibel-Palade body (WPB), a key determinant of endothelial inflammatory function
A key pro-inflammatory stimulant of endothelial cells is histamine [38], which triggers calcium-mediated WPB exocytosis by binding to HRH1 [39,41], a G protein-coupled receptors (GPCRs) phosphorylated by GRK2 [37]
Summary
To initiate an inflammatory response, both leukocytes and endothelial cells need to be activated by hormones and proinflammatory cytokines. Many signaling pathways involved in both endothelial and leukocyte activation are initiated by stimulating G protein-coupled receptors (GPCRs) [2]. GPCR signal transduction is attenuated by GPCR kinase (GRK)-mediated phosphorylation of agonist-bound receptor. This promotes b-arrestin binding, which uncouples the receptor from its G proteins and mediates receptor internalization and recycling [3,4]. Disruption of this machinery alters the strength and/or duration of physiological responses to GPCR ligands [5]. Of seven GRK subfamilies, the ubiquitously expressed GRK2 has been most closely linked to inflammatory [2,6] and cardiovascular function [6,7]
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