Abstract 463: The Orphan G Protein-Coupled Receptor C5B is Regulated by Hyperglycemia and Activates Pro-Inflammatory Signaling in Endothelial Cells Via the Tyrosine Kinase Fyn

Abstract

Background: Atherosclerosis is driven by an inflammatory process of the vascular wall in which endothelial cells (EC) are decisively involved. The orphan G protein-coupled receptor GPRC5B activates inflammatory pathways in adipocytes and regulates the glucose and lipid metabolism in Drosophila melanogaster . Hypothesis: We hypothesize that GPRC5B is involved in inflammatory signaling of EC, particularly in hyperglycemia. Methods & Results: Stimulation of mouse brain endothelial cells (bEnd.3) and primary human umbilical vein endothelial cells (HUVEC) with high glucose concentration (25 mM), Tumor Necrosis Factor α (TNFα 0,1 μg/ml) or Lipopolysaccharides (LPS 1 μg/ml), respectively, leads to a significant upregulation of GPRC5B mRNA and protein level compared to unstimulated cells (mRNA / protein: gluc +254% / +86%, p<0.001; TNFα +203% / +124%, p<0.001; LPS +195%, p<0.05, n=6). Similar results are obtained in HUVECs after stimulation with high glucose and cytokines. Overexpression of GPRC5B in EC induced a significant increase in Interleukin 6 (IL6), Intercellular Adhesion Molecule 1 (ICAM1) and Vascular Cell Adhesion Molecule 1 (VCAM1) mRNA and protein expression. Furthermore, activation of NFκB (luciferase reporter assay) and phosphorylation of extracellular signal–regulated kinases 1/2 (Erk 1/2) were significantly increased compared to controls (mRNA: IL6 +42%, p<0.01; ICAM1 +37%, p<0.01; VCAM1 +40%, p<0.001, n=8. Protein: ICAM1 +48%, p<0.05; VCAM1 +52%, p<0.05; pErk 1/2 +67%, p<0.01, n=5. NFκB: +165%, p<0.001, n=18). Conversely, the siRNA-mediated GPRC5B knockdown resulted in diminished expression of IL6 and VCAM1 as well as decreased NFκB activity compared to controls (IL6 -55%, p<0.05, n=3, NFκB -36%, p<0.05, n=6) . The knockdown results were confirmed in HUVECs. Co-immunoprecipitations in EC revealed the tyrosine kinase Fyn as interaction partner of GPRC5B. Conclusion: Our data suggest that GPRC5B is not only regulated by glucose and pro-inflammatory cytokines but also activates pro-inflammatory and pro-atherogenic pathways in mouse and human endothelial cells. Therefore, GPRC5B might play an important role in hyperglycemia-accelerated vascular inflammation and pathogenesis of atherosclerosis.