EFFECT AND CELLULAR SITE OF ACTION OF CYSTEINE PROTEASE INHIBITORS ONTHE CHOLESTEROL ESTERIFICATION PATHWAY IN MACROPHAGES AND CHINESE-HAMSTER OVARY CELLS

Stimulation of intracellular cholesterol esterification, which is cata lyzed by the enzyme acyl CoA:cholesterol O-acyltransferase (ACAT), by atherogenic lipoproteins in macrophages is a key step in the developme nt of atheroma foam cells. Since other aspects of intracellular choles terol metabolism involve proteolytic reactions, we looked for evidence of intracellular proteolysis in the stimulation of the cholesterol es terification pathway. When macrophages and CHO cells were incubated wi th the cysteine protease inhibitor N-acetylleucylleucylnorleucinal (AL LN), the ability of beta-very-low-density lipoprotein (beta-VLDL) and free cholesterol-rich liposomes to stimulate cholesterol esterificatio n was inhibited by 60-90%. Epoxysuccinylleucylamido-3 -methylbutane et hyl ester (EST), a cysteine protease inhibitor structurally different from ALLN, also inhibited beta-VLDL-induced cholesterol esterification in CHO cells. The inhibitory effect of the protease inhibitors could not be explained by decreased net expansion of cellular cholesterol po ols, inhibition of lipoprotein cholesteryl ester hydrolysis, or blocka ge of cholesterol trafficking through the lysosomal pathway. Furthermo re, stimulation of cholesterol esterification by 25-hydroxycholesterol and sphingomyelinase was not inhibited by ALLN, indicating that ALLN is not acting as a direct ACAT inhibitor in the cells, and suggesting that the ALLN effect is specific for methods of stimulating cholestero l esterification that expand cellular cholesterol pools. Previous stud ies have shown that inhibition of protein synthesis (e.g., by cyclohex imide) stimulates cholesterol esterification in macrophages and CHO ce lls, suggesting the presence of a short-lived protein inhibitor of cho lesterol esterification. Herein, we show that, when added after cycloh eximide, ALLN does not inhibit cycloheximide-induced cholesterol ester ification in either cell type. The data in this report are consistent with a novel model in which a proteolytic reaction mediates the stimul ation of cholesterol esterification specifically by expanded cellular cholesterol pools. The apparent protease-dependent step is not depende nt upon lysosomal trafficking of cholesterol and is proximal to the AC AT enzyme itself; it may function by cleaving an endogenous inhibitor of the interaction of expanded cellular cholesterol pools with ACAT.