Plasma membrane calcium ATPase 4 (PMCA4) and inflammation of the aortic endothelium

Abstract

Background: The inflammatory response relies on the well-coordinated trafficking of leukocytes from the circulation to sites of tissue injury. The endothelial expression of cell adhesion molecules, namely the selectin family, mediates the initial adhesive interactions between leukocytes and the endothelium. Immune cell infiltration is often involved in the initiation and progression of several vascular diseases, including atherosclerosis. Plasma Membrane Calcium ATPase 4 (PMCA4) belongs to a family of transmembrane ion transporters that extrude calcium from the cytosol to the extracellular environment (Strehler, 2015). They are the only high-affinity Ca2+ extrusion system in mammalian cells encoded by the ATP2B1-4 genes (PMCA1–4) (Strehler, 2015). PMCA4 acts as a structural protein providing a scaffold to interacting proteins at the plasma membrane of endothelial cells. Several of these interactions demonstrate functional significance, accounting for its emerging role in signal transduction. This work contributes to the delineation of PMCA4 in the physiology of the vascular endothelium and characterises a role in leukocyte adhesion and trafficking. Methods and Results: The effect of the pro-inflammatory cytokine IL-1β on the expression of PMCA4 was examined. A time and dose-dependent downregulation of PMCA4 mRNA and protein expression in human Aortic Endothelial Cells (AoEC) was observed. Differential gene expression was determined in PMCA4 silenced AoEC using an ECM-CAM array and RNA-sequencing. The siRNA-mediated knockdown of PMCA4 induced the expression of cell adhesion molecules, Selectin P (SELP) and SELL. The knockdown enhanced the IL-1β-induced expression of the metalloproteinases ADAMTS1 and -4. Ingenuity pathway analysis (IPA) revealed a role in immune cell trafficking with a propensity to develop aneurysms (activation z-score,-1.177). Similarly, silencing PMCA4 enhanced the VEGF-induced expression of ADAMTS1, VCAM-1 and SELE. Cyclosporin A partially ablated this enhancement through VEGF, implicating CN/NFAT transcriptional activity. Consistent with the IPA, a significant decrease in PMCA4 mRNA expression was observed in abdominal aortic lesions from hypercholesterolaemic ApoE-deficient mice infused with angiotensin II. Conclusion: The downregulation of PMCA4 is associated with a state of endothelial activation, indicated by the induction of cell adhesion molecules. Our results demonstrate that PMCA4 regulates intracellular signalling in the endothelium in response to leukocyte-derived inputs. Reinforced by bioinformatic analysis, our findings suggest that PMCA4 may influence leukocyte adhesion and trafficking on the endothelium.