Kelley M. Argraves, PhD
Blood borne sphingosine-1-phosphate (S1P), carried on high density lipoprotein (HDL), is essential for endothelial barrier function. HDL-S1P promotes endothelial barrier function through interaction with the S1P receptor, S1PR1. An understudied aspect of HDL-S1P-S1PR1 signaling that appears to have profound importance to endothelial barrier homeostasis in physiological and pathophysiological state is regulation of S1PR1 turnover. Recent studies indicate that S1PR1 levels are downregulated after S1P stimulation by beta-arrestin-dependent endocytosis. Following endocytosis, a portion of the internalized S1PR1 pool undergoes proteasome-dependent degradation and a portion of the pool is recycled back to the cell surface. The S1P mimetic FTY720-P induces irreversible internalization of S1PR1, thereby suppressing S1PR1 signaling and inhibiting S1P-dependent egress of maturing T cells from the thymus, causing lymphopenia. Needed are new S1PR1 agonists that counteract loss of endothelial barrier function, as occurs in pulmonary edema, atherosclerosis, cancer and many inflammatory diseases. The ability to identify new drugs that promote S1P-S1PR1 signaling would benefit from a greater understanding of agonism-induced, beta-arrestin-dependent, S1PR1 internalization and endocytic vesicular trafficking.
Recently, we have shown that HDL-S1P increases the stability of S1PR1protein and increases levels of S1PR1 on the cell surface as compared to S1P carried on the other physiological S1P carrier, albumin. Thus, we hypothesize that drugs can be identified that reflect differences in carrier induced S1PR1 desensitization and recycling. We have developed a new quantitative approach for study of agonism-induced beta-arrestin-dependent internalization of S1PR1 that involves live cell real time fluorescent imaging of endothelial cells expressing EGFP-labeled S1PR1. This approach allows for unprecedented analysis of the kinetics of endocytosis, endocytic vesicle size and direction of the subcellular trafficking of endocytic vesicles containing beta-arrestin and S1PR1. Employing this novel approach our hypothesis will be addressed in the following Specific Aim: Use live cell, real time fluorescent imaging to define the internalization and trafficking patterns and kinetics of endothelial cell-S1PR1 in response to natural S1P, commercially available S1PR1 agonists and S1P derivatives synthesized by the COBRE Lipidomics Synthetic Core. Information from these studies is expected to advance the long-term goal of identifying new S1PR1 agonist drugs that act similar to HDL-S1P in mediating sustained S1PR1 signaling on the endothelium and thus ameliorate endothelial barrier pathologies such as edema and atherosclerosis.
Project Description 