HDL-MEDIATED EFFLUX OF INTRACELLULAR CHOLESTEROL IS IMPAIRED IN FIBROBLASTS FROM TANGIER DISEASE PATIENTS

To further elucidate the cellular mechanisms leading to HDL deficiency in Tangier disease, HDL-mediated cholesterol efflux was studied in cu ltured skin fibroblasts from Tangier patients. Both Tangier and contro l fibroblasts show specific saturable binding of HDL(3) to the cell me mbrane (B-max = 70 and 52 ng/mg protein, respectively; K-d = 8.8 and 1 0.6 mu g/mL, respectively). There was no appreciable uptake of HDL(3) by Tangier and control fibroblasts, indicating that cholesterol efflux from fibroblasts occurs at the cell membrane. When cellular cholester ol was labeled to equilibrium by [C-14]cholesterol incubation for 48 h ours at 37 degrees C, HDL(3)-mediated cholesterol efflux from Tangier fibroblasts was only 50% of control fibroblasts. To define this abnorm ality in HDL(3)-mediated cholesterol efflux more precisely, several ad ditional experiments were performed. First, membrane desorption of cho lesterol was determined after cell membranes were labeled with [C-14]c holesterol for 3 hours at 15 degrees C. With this labeling protocol, t here was no difference in HDL(3)-mediated cholesterol efflux between c ontrol and Tangier fibroblasts. Second, efflux of newly synthesized st erols was determined after incorporation of the precursor [C-14]mevalo nolactone. Under these conditions, specific HDL(3)-mediated efflux of sterols was almost absent in Tangier fibroblasts. Third, cells were la beled by incubation with reconstituted [H-3] cholesteryl-linoleate-LDL . Efflux of LDL-derived cholesterol was only slightly reduced for the first 4 hours of incubation. After 12 hours, there was no difference b etween control and Tangier cells. The combined data indicate that the reduced efflux of cholesterol from Tangier fibroblasts observed after homogeneous labeling is due to severely reduced efflux of newly synthe sized sterol. Since it has been shown previously that efflux of newly synthesized cholesterol depends on HDL-mediated activation of protein kinase C (PKC), the effect of pharmacological activation of PKC was an alyzed. incubation of Tangier fibroblasts in the presence of 1,2-dioct anoylglycerol (10(-5) mol/L), a membrane-permeable activator of PKC, l ed to normalization of HDL(3)-mediated efflux of newly synthesized cho lesterol. These data were interpreted to indicate that impaired activa tion of PKC rather than a defect in the transport mechanism of cellula r cholesterol leads to reduced HDL-mediated efflux of cholesterol from Tangier fibroblasts.