Association of ACAT1-positive vesicles with late endosomes/ lysosomes in cholesterol-rich human macrophages.
OBJECTIVE
Acyl-coenzyme A: cholesterol acyltransferase1 (ACAT1) is an endoplasmic reticulum (ER)-resident enzyme that catalyzes the conversion of cholesterol into cholesteryl esters. We previously showed that cholesterol-loaded macrophages produce numerous ER-derived vesicles with elevated ACAT1 enzyme activity; some of these vesicles were shown to be closely associated with Golgi-related organelle(s). The aim of this study was to investigate the translocation of ACAT1 vesicle in cholesterol-loaded macrophages.


METHODS
To demonstrate association of ACAT1 with late endosomes/lysosomes (LE/LS), primary human macrophages with or without cholesterol-loading was subjected to confocal microscopy, immunoelectron microscopy, subcellular fractionation, and immunoadsorption assay. Furthermore, cholesterol esterification assay was also carried out to investigate function of ACAT1 associated LE/LS.


RESULTS
Confocal fluorescence microscopy revealed that no significant ACAT1 signal was associated with the signal for LAMP2, a marker protein for LE/LS, in cholesterol non-loaded macrophages; however, approximately 20% of the total ACAT1 signals colocalized with the LAMP2 signal in cholesterol-loaded macrophages. ACAT1-positive membranes isolated by immunoadsorption using ACAT1-specific antibody contained LAMP2, demonstrating the association of ACAT1 and LE/LS. In addition, in macrophages phagocytosing latex beads, the close association of ACAT1 with LE/LS can be demonstrated in phagosomes isolated from cholesterol-loaded macrophages, not from non-loaded macrophages. Furthermore, cholesterol-loaded macrophages re-esterified aggregated LDL-derived cholesteryl ester even in the presence of U18666A, a reagent known to block egression of cholesterol from LE/LS.


CONCLUSIONS
Our results indicated that cholesterol-loaded human macrophages produce LE/LS in close association with ACAT1, and may promote efficient esterification of modified LDL-derived free cholesterol on LE/LS.
Lei
XiaoFeng
X
Department of Cell Pathology, Graduate School of Medical Sciences, Kumamoto University, 1-1-1 Honjo, Kumamoto, Japan.
Fujiwara
Yukio
Y
Chang
Catherine C Y
CC
Chang
Ta-Yuan
TY
Takeya
Motohiro
M
Sakashita
Naomi
N
D000101:Acetyl-CoA C-Acetyltransferase / Q000378:metabolism*; D000818:Animals; D002478:Cells, Cultured; D002784:Cholesterol / Q000378:metabolism*; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D011992:Endosomes / Q000378:metabolism*; D004951:Esterification; D006801:Humans; D015151:Immunoblotting; D052119:Lysosomal-Associated Membrane Protein 2 / Q000378:metabolism; D008247:Lysosomes / Q000378:metabolism*; D008264:Macrophages / Q000378:metabolism*; D051379:Mice; D013347:Subcellular Fractions
D052119:Lysosomal-Associated Membrane Protein 2; D002784:Cholesterol; C506639:ACAT1 protein, human; C506641:Acat1 protein, mouse; D000101:Acetyl-CoA C-Acetyltransferase