RvE1 Stimulates Calcium Mobilization Through the ERV-1 Receptor

Objectives: The lipid mediator Resolvin E1 is known to activate G-protein coupled receptors, such as ERV-1 and BLT-1. Upon binding to the receptor, downstream biological agonist functions are activated. The initial steps of receptor signaling are not known. This study investigated calcium mobilization stimulated by 2 ERV1 agonists, Resolvin E1 and chemerin.
Methods: ERV-1 was overexpressed in Chinese Hamster Ovary (CHO) cells by transfection. To investigate calcium mobilization, cells were labeled with the fluorescent probe, Calcium5 FLIPR. After agonist treatment, detection of changes in fluorescence were detected a in microplate reader and expressed as change in relative fluorescence units (∆RFU). The CHO cells were treated in vitro with RvE1 (1nM, 100nM, final concentration) and Chemerin (1nM, 100nM, final concentation) and read at intervals of 1, 15 and 30 minutes. Regulation of ERV1 mRNA expression after RvE1 binding was assessed by qPCR.
Results: Binding of RvE1 induced an increase in free calcium as indicated by an increase in fluorescence (1nM, ∆RFU = 0.083, and 100nM, ∆RFU =8.33) which continued for 30 minutes (1nM, ∆RFU = 3.17, and 100nM, ∆RFU =11.67). By contrast, chemerin, a peptide agonist of ERV1 with 10E5 lower binding constant, did not induce calcium signaling until 30 minutes (1nM, ∆RFU = 1.83): increasing the stimulus to 100nM had little impact. Gene expression after ligand binding was decreased.
Conclusions: ERV-1 receptor activation by RvE1 is calcium dependent. RvE1 induces calcium mobilization as early as 1 minute after stimulation. Receptor expression is not upregulated by RvE1 binding suggesting that calcium immobilization is regulated by internalization of the receptors.