Abstract 16554: Sphingosine-1-Phosphate Activates the Mitochondrial Unfolded Protein Response in the Pulmonary Vasculature

Abstract

Introduction: Aberrant activation of pathways that promote pulmonary arterial smooth muscle (PASMC) and endothelial cell (PAEC) proliferation causes vascular remodeling and increased vascular resistance that leads to the deadly disease Pulmonary Arterial Hypertension (PAH). Sphingosine-1-phosphate (S1P) induces PASMCs proliferation and disruption of its signaling mitigates vascular remodeling and PAH. Mitochondria regulate proliferation and can promote or inhibit it in a signal dependent manner. The mitochondrial unfolded protein response (UPR mt ) is a mechanism by which mitochondria preserve cell survival due to stress. Mitochondria are important mediators of PASMC proliferation and PAH development as both mitochondrial (mt) fission and aerobic glycolysis contribute to disease progression. Studies in C. elegans suggest a link between regulation of mt fission and activation of the UPR mt as suppression of fission leads to S1P dependent activation of the UPR mt . Hypothesis: We hypothesized that S1P stimulation leads to activation of the UPR mt to promote proliferation of hPASMCs and hPAECs. Methods: hPASMCs were stimulated with S1P (1 μM) for up to 24h. Cell extracts were analyzed by immunoblotting for proteins (eIF2α, ATF-5, HSP70 and Lonp1) that regulate the UPR mt . Mitochondrial dynamics was assessed by immunoblotting or immunofluorescence for mediators of fission (Drp-1) and fusion (mitofusin-2). SPHK1, the lipid kinase that catalyzes S1P generation, was overexpressed in hPAECs followed by immunoblotting for UPR mt and mt fission activation. PCNA expression was assessed to measure cell proliferation. Results: S1P stimulation of hPASMCs enhanced proliferation and caused rapid and sustained activation of the UPR mt as phosphorylation of eIF2α and expression of ATF-5 and HSP70 were increased. S1P also enhanced mt fission in hPASMCs as Drp-1 phosphorylation was increased concomitant with decreased mitofusin-2 expression. Overexpression of SPHK1 in hPAECs increased proliferation and activation of both the UPR mt and mt fission. Conclusions: S1P promotes functional and phenotypic changes of mitochondria that leads to activation of pathways that promote PASMC and PAEC proliferation which could contribute to vascular remodeling in PAH.