EXPRESSION OF HETEROLOGOUS HUMAN APOLIPOPROTEIN-E BY J774 MACROPHAGESENHANCES CHOLESTEROL EFFLUX TO HDL(3)

Expression of apolipoprotein (apo) E by macrophages is tightly regulat ed by cellular cholesterol content. We have investigated a potential m odulating role for apoE on macrophage cholesterol homeostasis by stabl y transfecting the J774 macrophage, which does not express its endogen ous apoE gene, with a human apoE cDNA expression vector and comparing cholesterol homeostasis in this cell line with that of a control line transfected with the neomyocin resistance construct only. Incubation i n serum-free medium after cholesterol loading produced no difference i n cellular cholesterol content between apoE secreting and non-secretin g J774 cells. Similarly, in serum-free medium there was no difference in the amount of radiolabeled cholesterol effluxed. Addition of cAMP o r S58035 to cholesterol-loaded J774 cells did enhance efflux of radiol abeled cholesterol from apoE secreting compared to non-secreting macro phages but did not detectably alter cellular free cholesterol or chole steryl ester mass. Incubation with HDL(3) alone, however, significantl y decreased macrophage cholesteryl ester mass compared to a 24-h incub ation in serum-free medium from 10.5 +/- 3.9 to 3.2 +/- 2.0 (P < 0.01) in apoE-secreting J774 cells. During a 24-h incubation in HDL(3), cho lesteryl ester fell from 6.4 +/- 2.4 to 0.8 +/- 0.7 (Delta = 5.6 mu g/ mg) in apoE-secreting cells and from 9.3 +/- 2.2 to 7.7 +/- 1.9 mu g/m g (Delta = 1.6 mu g/mg) in non-secreting cells (P < 0.005 apoE-secreti ng vs. non-secreting cells). Drug-induced stimulation of cholesteryl e ster hydrolysis (with cAMP) or inhibition of cholesterol esterificatio n (with S58035) did not abolish the difference in cholesterol efflux t o HDL(3) between apoE-secreting and non-secreting cells indicating tha t the effect of apoE on efflux is not due to alteration of free choles terol-cholesteryl ester balance. jlr These data indicate that secretio n of endogenously synthesized apoE can enhance the loss of cellular ch olesterol from cholesterol-enriched macrophages. The reciprocal regula tion, represented by cholesterol modulation of apoE expression and apo E modulation of macrophage cholesterol balance, suggests a regulatory loop allowing macrophages to more efficiently function in order to mai ntain endogenous cellular and tissue cholesterol homeostasis.