Adenylyl cyclase 1 as a major isoform to generate cAMP signaling for apoA-1-mediated cholesterol efflux pathway.

HDL apoA-1-mediated cholesterol efflux pathway requires multiple cellular proteins and signal transduction processes, including adenylyl cyclase (AC)/cAMP signaling. Due to the existence of multiple transmembrane AC isoforms, it was not known how many AC isoforms are expressed and which ones are essential for cholesterol efflux in macrophage foam cells. These questions were investigated in THP-1 macrophages in this study. Quantitative RT-PCR detected mRNAs for all nine transmembrane AC isoforms, but only the mRNA and protein of the AC1 isoform were consistently upregulated by cholesterol loading and apoA-1. AC1 shRNA interference decreased AC1 mRNA and protein levels, resulting in reduction of apoA-1-mediated cAMP production and cholesterol efflux, while the intracellular cholesterol levels remained high. Confocal microscopy showed that apoA-1 promoted translocation of cholesterol and formation of cholesterol-apoA-1 complexes (protrusions) on the cholesterol-loaded macrophage surface. AC1 shRNA-interfered macrophages showed no translocation of cholesterol to the cell surface. AC1 shRNA interference also disrupted cellular localization of the intracellular cholesterol indicator protein adipophillin, and the expression as well as surface translocation of ABCA1. Together, our results show that AC1 is a major isoform for apoA-1-activated cAMP signaling to promote cholesterol transport and exocytosis to the surface of THP-1 macrophage foam cells.

ß-COP as a Component of Transport Vesicles for HDL Apolipoprotein-Mediated Cholesterol Exocytosis.

OBJECTIVE: HDL and its apolipoproteins protect against atherosclerotic disease partly by removing excess cholesterol from macrophage foam cells. But the underlying mechanisms of cholesterol clearance are still not well defined. We investigated roles of vesicle trafficking of coatomer ß-COP in delivering cholesterol to the cell surface during apoA-1 and apoE-mediated lipid efflux from fibroblasts and THP-1 macrophages. METHODS: shRNA knockout, confocal and electron microscopy and biochemical analysis were used to investigate the roles of ß-COP in apolipoprotein-mediated cholesterol efflux in fibroblasts and THP-1 macrophages. RESULTS: We showed that ß-COP knockdown by lentiviral shRNA resulted in reduced apoA-1-mediated cholesterol efflux, while increased cholesterol accumulation and formation of larger vesicles were observed in THP-1 macrophages by laser scanning confocal microscopy. Immunogold electron microscopy showed that ß-COP appeared on the membrane protrusion complexes and colocalized with apoA-1 or apoE during cholesterol efflux. This was associated with releasing heterogeneous sizes of small particles into the culture media of THP-1 macrophage. Western blotting also showed that apoA-1 promotes ß-COP translocation to the cell membrane and secretion into culture media, in which a total of 17 proteins were identified by proteomics. Moreover, ß-COP exclusively associated with human plasma HDL fractions. CONCLUSION: ApoA-1 and apoE promoted transport vesicles consisting of ß-COP and other candidate proteins to exocytose cholesterol, forming the protrusion complexes on cell surface, which were then released from the cell membrane as small particles to media.