CYCLIC-AMP INDUCES APOLIPOPROTEIN-E BINDING-ACTIVITY AND PROMOTES CHOLESTEROL EFFLUX FROM A MACROPHAGE CELL-LINE TO APOLIPOPROTEIN ACCEPTORS

RAW 264 mouse macrophage cells were stably transfected with human apol ipoprotein E (apoE) expression vectors. Clonal derivatives were charac terized for expression of the human apoE2, apoE3, and apoE4 isoforms. An apoE4-expressing clonal cell line and a non-expressing clonal contr ol cell line were loaded overnight with either [H-3]cholesterol or [H- 3]choline, The cells were washed and incubated for 24 h in serum-free medium with or without the addition of 8-bromo-cyclic AMP (8-Br-cAMP), Only the apoE-secreting cells and only in the presence of 8-Br-cAMP r eleased large amounts of labeled cholesterol or phosphatidylcholine in to the medium, Mass analyses of cellular free and esterified cholester ol confirmed the results of the labeling studies; a decrease in cellul ar cholesterol content was observed in the 8-Br-cAMP-treated apoE-secr eting cells, concurrent with an increase in cholesterol found in the m edium. FPLC analysis of the medium demonstrated that 8-Br-cAMP treatme nt of the apoE-secreting cells led to an increased size fraction and a mount of a peak of secreted cholesterol which comigrated with apoE, Th e 8-Br-cAMP-mediated increase in cholesterol efflux was also observed in non-apoE-secreting cells incubated with exogenous apoE or apoAI, an d the effect of apoE was saturable, The apoE2, apoE3, and apoE4 isofor ms were equally efficient in promoting 8-Br-cAMP-dependent cholesterol efflux, Reductive methylation of apoE abolished its ability to promot e 8-Br-cAMP-dependent cholesterol efflux, Brefeldin A and monensin, in hibitors of protein processing through the Golgi, both blocked the 8-B r-cAMP stimulation of cholesterol efflux to exogenous apoE, 8-Br-cAMP induced specific apoE and apoAI binding, but not apoE degradation, by the RAW cells. We present a model wherein cAMP induces a membrane apol ipoprotein receptor that does not lead to endocytosis and degradation, but instead promotes the transfer of lipids to apolipoproteins, which can then be released from the cell.