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 apolipoprotein E (apoE) expression vectors. Clonal derivatives were characterized for expression of the human apoE2, apoE3, and apoE4 isoforms. An apoE4-expressing clonal cell line and a non-expressing clonal control cell line were loaded overnight with either 3Hcholesterol or 3Hcholine. 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 released large amounts of labeled cholesterol or phosphatidylcholine into the medium. Mass analyses of cellular free and esterified cholesterol confirmed the results of the labeling studies; a decrease in cellular cholesterol content was observed in the 8-Br-cAMP-treated apoE-secreting cells, concurrent with an increase in cholesterol found in the medium. FPLC analysis of the medium demonstrated that 8-Br-cAMP treatment of the apoE-secreting cells led to an increased size fraction and amount of a peak of secreted cholesterol which comigrated with apoE. The 8-Br-cAMP-mediated increase in cholesterol efflux was also observed in non-apoE-secreting cells incubated with exogenous apoE or apoAI, and the effect of apoE was saturable. The apoE2, apoE3, and apoE4 isoforms were equally efficient in promoting 8-Br-cAMP-dependent cholesterol efflux. Reductive methylation of apoE abolished its ability to promote 8-Br-cAMP-dependent cholesterol efflux. Brefeldin A and monensin, inhibitors of protein processing through the Golgi, both blocked the 8-Br-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 apolipoprotein 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.