Zhao_2012_Am.J.Physiol.Cell.Physiol_303_C427

Cholesteryl ester CE hydrolysis is the rate-limiting step in the removal of free cholesterol FC from macrophage foam cells and several enzymes have been identified as intracellular CE hydrolases in human macrophages We have previously reported the antiatherogenic role of a carboxylesterase carboxylesterase 1 CES1 and the objective of the present study was to determine the contribution of CES1 to total CE hydrolytic activity in human macrophages Two approaches namely immune depletion and short hairpin sh)RNA-mediated knockdown were used Immuneprecipitation by a CES1-specific antibody resulted in a 70-80 decrease in enzyme activity indicating that CES1 is responsible for 70 of the total CE hydrolytic activity THP1-shRNA cells were generated by stably transfecting human THP1 cells with four different CES1-specific shRNA vectors Despite a significant 90 reduction in CES1 expression both at the 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 CES CES3 which is only expressed at 30 of the level of CES1 in human macrophages Transient overexpression of CES3 led to an increase in CE hydrolytic activity mobilization of intracellular lipid droplets and a reduction in cellular CE content establishing CES3 as a bona fide CE hydrolase This study provides the first evidence of functional compensation whereby increased expression of CES3 restores intracellular CE hydrolytic activity and FC efflux in CES1-deficient cells Furthermore these data support the concept that intracellular CE hydrolysis is a multienzyme process.