Selective uptake of cholesteryl ester from low density lipoprotein is involved in HepG2 cell cholesterol homeostasis

Low density Lipoprotein (LDL) can follow either a holoparticle uptake pathw ay,initiated by the LDL receptor (LDLr), and be completely degraded, or it can deliver its cholesteryl eaters (CE) selectively to HepG2 cells. Althoug h high density Lipoprotein-CE selective uptake has been shown to be linked to cell. cholesterol homeostasis in nonhepatic cells, there is no available information on the effect of LDL-CE selective uptake on hepatic cell chole sterol homeostasis. In order to define the role of the LDL-CE selective upt ake pathway in hepatic cell cholesterol homeostasis, we used a cellular mod el that expresses constitutively a LDLr antisense mRNA and that shows LDLr activity at 31% the normal level (HepG2-all cells). The addition of a speci fic antibody anti-LDLr (IgG-C7) reduces LDL protein degradation (LDLr activ ity) to 7%. This cellular model therefore reflects, above all, LDL-CE selec tive uptake activity when incubated with LDL. The inactivation of LDLr redu ces LDL-protein association by 78% and LDL-CE association by only 43%. The LDL-CE selective uptake was not reduced by the inactivation of LDLr. The ac tivities of the various enzymes involved in cell cholesterol homeostasis we re measured in normal and LDLr-deficient cells during incubation in the abs ence or presence of LDL as a cholesterol source. Essentially, 3-hydroxy-3-m ethylglutaryl coenzyme A reductase and acyl coenzyme A:cholesterol acyltran sferase (ACAT) activities responded to LDL in LDLr-deficient cells as well as in normal HepG2 cells. Inhibition of lysosomal hydrolysis with chloroqui ne abolished the effect measured on ACAT activity in the presence of LDL, s uggesting that CE of LDL,but not free cholesterol, maintains cell cholester ol homeostasis. Thus,in HepG2 cells, when LDLr function is virtually abolis hed, LDL-CE selective uptake is coupled to cell cholesterol homeostasis.