Abstract
Macrophages in atherosclerotic lesions accumulate large amounts of cholesteryl-fatty acyl esters ("foam cell" formation) through the intracellular esterification of cholesterol by acyl-coenzyme A:cholesterol O-acyltransferase (ACAT). In this study, we sought to determine the subcellular localization of ACAT in macrophages. Using mouse peritoneal macrophages and immunofluorescence microscopy, we found that a major portion of ACAT was in a dense reticular cytoplasmic network and in the nuclear membrane that colocalized with the luminal endoplasmic reticulum marker protein-disulfide isomerase (PDI) and that was in a similar distribution as the membrane-bound endoplasmic reticulum marker ribophorin. Remarkably, another portion of the macrophage ACAT pattern did not overlap with PDI or ribophorin, but was found in as yet unidentified cytoplasmic structures that were juxtaposed to the nucleus. Compartments containing labeled beta-very low density lipoprotein, an atherogenic lipoprotein, did not overlap with the ACAT label, but rather were embedded in the dense reticular network of ACAT. Furthermore, cell-surface biotinylation experiments revealed that freshly harvested, non-attached macrophages, but not those attached to tissue culture dishes, contained approximately 10-15% of ACAT on the cell surface. In summary, ACAT was found in several sites in macrophages: a cytoplasmic reticular/nuclear membrane site that overlaps with PDI and ribophorin and has the characteristics of the endoplasmic reticulum, a perinuclear cytoplasmic site that does not overlap with PDI or ribophorin and may be another cytoplasmic structure or possibly a unique subcompartment of the endoplasmic reticulum, and a cell-surface site in non-attached macrophages. Understanding possible physiological differences of ACAT in these locations may reveal an important component of ACAT regulation and macrophage foam cell formation.
Highlights
Macrophages enter atherosclerotic lesions at an early stage and are found at all stages of lesion development thereafter [1]
A noticeable portion of A:cholesterol O-acyltransferase (ACAT) in macrophages, resides in a perinuclear cytoplasmic site that does not overlap with protein-disulfide isomerase (PDI) or ribophorin
While much of the ACAT pattern in adherent macrophages is similar to that of PDI and ribophorin, consistent with localization in the ER, a noticeable portion of ACAT resides in a perinuclear cytoplasmic site that does not overlap with PDI or ribophorin
Summary
Materials—Falcon and Corning tissue culture plasticware was purchased from Fisher. Tissue culture media and reagents and calf serum were obtained from Life Technologies, Inc., and fetal bovine serum was purchased from Gemini Bioproducts (Calabasas, CA). For colocalization studies involving -VLDL, macrophages were treated with 5 g/ml Texas Red-conjugated -VLDL in Dulbecco’s modified Eagle’s medium and 0.2% bovine serum albumin for 10 min at 37 °C, fixed and permeabilized with cold methanol (Ϫ20 °C) on ice, and labeled with the rabbit anti-ACAT serum. After washing twice with PBS, the cells were resuspended in RIPA buffer (0.5% sodium deoxycholate, 0.1% SDS, 1% Triton X-100, 20 mM Tris, 150 mM NaCl, and 5 mM EDTA, pH 8) containing 0.2% bovine serum albumin and protease inhibitors. The precipitates were centrifuged at 13,000 ϫ g for 10 s; the supernatants were removed; and the agarose was resuspended in RIPA buffer This washing step was repeated twice with RIPA buffer, twice with RIPA buffer containing 500 mM NaCl, and three times with PBS. To assess total membrane-bound ACAT in the macrophages for comparison on the immunoblot, the other half of the macrophages were disrupted at 4 °C by sonication (Model 450, Branson Ultrasonics Corp., Danbury, CT) in PBS containing 0.5 mg/ml NHS-SS-biotin and protease inhibitors
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