Abstract 23: Identification of a Novel Intracellular Cholesteryl Ester Hydrolase (CES3) in Human Macrophages: Compensatory Increase Following CES1 Silencing

Abstract

Removal of free cholesterol (FC) from lesion-associated macrophage foam cells represents the major mechanism for reducing the lipid burden of atherosclerotic plaques and intracellular cholesteryl ester (CE) hydrolysis is recognized as the rate-limiting step in this process. We have earlier reported cloning of human macrophage CE hydrolase (gene symbol CES1) that associated with cytoplasmic lipid droplets and enhanced cellular CE mobilization. Macrophage-specific transgenic expression of CES1 attenuated diet-induced atherosclerosis and lesion necrosis in ldlr -/- mice. Several enzymes have since been identified as intracellular CE hydrolases in human macrophages and the objective of the present study was to determine the relative contribution of CES1 to the total CE hydrolytic activity in human macrophages. Immune-precipitation by CES1-specific antibody resulted in 70-80% decrease in enzyme activity indicating that CES1 is responsible for >70% of the total CE hydrolytic activity (Panel A). To examine the effects of CES1 depletion/knockdown on FC efflux, human monocyte/macrophages (THP1) were stably transfected with 4 different CES1-specific shRNA vectors (p81-p84) to generate THP1-shRNA cells; cells transfected with pRSL vector were used as controls. Despite significant reduction in CES1 expression both at mRNA and protein levels, CES1 knockdown neither decreased intracellular CE hydrolysis nor decreased FC efflux. Examination of the underlying mechanisms for the observed lack of effects of CES1 knockdown revealed a compensatory increase in the expression of a novel carboxylesterase, CES3 (Panel B). Human macrophage CES3 was identical to the reported CES3 except it lacked exon 8 and, in wild type THP1 cells it was only expressed at 29.2±6.1 percent of CES1. Transient over-expression of this novel variant of CES3 led to an increase in CE hydrolytic activity (>4 fold), mobilization of intracellular lipid droplets and reduction in cellular CE content (108.1±7.65 vs 160.9±7.00, P=0.0005), establishing CES3 as a bona fide CE hydrolase. Reciprocal backup circuits, where deficiency of a gene leads to an increase in another gene with similar function, have been described and this study provides first evidence of functional compensation in intracellular CE hydrolysis whereby increased expression of CES3 restores intracellular CE hydrolytic activity and FC efflux in CES1-deficient cells. To gain insight into the mechanisms underlying the observed increase in CES3 following CES1 deficiency in silico analyses of CES3 promoter was performed and several NF-kB binding sites were identified. Based on our earlier demonstration of a decrease in NF-kB activation by CES1 over-expression, it is speculated that conversely, deficiency of CES1 increases NF-kB activation leading to compensatory increase in CES3 expression.