DEGRADATION OF MACROPHAGE APOE IN A NONLYSOSOMAL COMPARTMENT - REGULATION BY STEROLS

Macrophage derived apoE has been shown to play an important role in th e susceptibility of the vessel wall to atherosclerosis. Previous studi es have shown that macrophage sterol content modulates apoE synthesis and secretion, associated with a large transcriptional response of the apoE gene, The current studies were undertaken to evaluate the existe nce of additional posttranscriptional regulatory loci for the effect o f sterols on apoE synthesis and secretion, Using a macrophage cell lin e transfected to constitutively express an apoE cDNA to facilitate det ection of a post-transcriptional regulatory locus, we demonstrated tha t preincubations in 25-hydroxycholesterol and cholesterol lead to incr eased apoE secretion in pulse/chase experiments, Examination of cell l ysates in these experiments showed that apoE not secreted by control c ells was degraded and not detectable, suggesting that the preincubatio n in sterols increased secretion by decreasing degradation of newly sy nthesized apoE, The measurement of total protein and apoE degradation in cell fractions revealed an intermediate density fraction that degra ded significant amounts of newly synthesized total protein and newly s ynthesized apoE, In this fraction, degradation of total protein and ap oE was unaffected by chloroquine but was substantially reduced by N-ac etyl-Leu-Leu-nor-leucinal plus N-acetyl-Leu-Leu-methioninal or by lact a-cystin, suggesting the involvement of proteasomes, Preincubation in sterol/oxysterol or acetylated low density lipoprotein did not modify total protein degradation by this fraction but inhibited apoE degradat ion, Similar results were obtained using intermediate density fraction s isolated from human monocyte-derived macrophages, The results of our studies indicate that newly synthesized apoE in the macrophage can be degraded in an intermediate density nonlysosomal cellular compartment , which is sensitive to proteasomal inhibitors. Alteration of cellular lipid homeostasis by preincubation in sterol/oxysterol or acetylated low density lipoprotein inhibits apoE, but not total protein, degradat ion in this fraction, Inhibition of the degradation of apoE in this fr action likely contributes to the increased apoE secretion observed in sterol-enriched cells.