Abstract 12674: LDL Transcytosis by SR-BI Requires Signaling That is Distinct From Reverse Cholesterol Transport

Abstract

Introduction: How LDL crosses an intact endothelial layer to initiate atherosclerosis is poorly understood, although it is postulated to involve LDL transcytosis. To date, technical limitations have greatly limited the study of its molecular mechanisms. We have recently devised an assay for quantifying LDL transcytosis by primary human coronary artery endothelial cells and have reported an unexpected role for the scavenger receptor SR-BI. SR-BI is best known as the HDL receptor and mediates reverse cholesterol transport; its downstream signaling involves the adaptor PDZK1 and endothelial nitric oxide synthase (eNOS). However, whether SR-BI internalization and its canonical signaling pathway are required for LDL transcytosis is unknown. Hypothesis: We hypothesized that LDL transcytosis and reverse cholesterol transport require different signaling pathways. Methods: Transcytosis of DiI-labeled LDL by primary human coronary artery endothelial cells was studied by total internal reflection fluorescence microscopy. The role of lipid rafts, PDZK1, eNOS and MAP kinases was studied by siRNA and pharmacological inhibitors. Results: Incubation with LDL induced the internalization of SR-BI, which was enriched in lipid rafts. Knockdown of caveolin-1 significantly attenuated LDL transcytosis. Coronary endothelial cells expressed low levels of PDZK1 and knockdown by siRNA had no effect on transcytosis. Similarly, inhibition of eNOS or addition of exogenous nitric oxide (NO) by NO-donors did not affect LDL transcytosis. Over-expression of G420H-SR-BI, a mutant receptor which does not perform reverse cholesterol transport, actually induced LDL transcytosis while the SR-BI inhibitor BLT-1, which prevents reverse cholesterol transport, had no effect on LDL transcytosis. Finally, inhibition of c-Jun N-terminal kinase (JNK) significantly attenuated LDL transcytosis. Conclusions: SR-BI is internalized during LDL transcytosis but PDZK1 and nitric oxide are not required. Instead, JNK kinase is involved. These data raise the possibility that reverse cholesterol transport and LDL transcytosis by SR-BI are regulated separately. These findings suggest that LDL transcytosis by SR-BI may constitute a novel therapeutic target for atherosclerosis.