HDL-Mediated Efflux of Intracellular Cholesterol Is Impaired in Fibroblasts From Tangier Disease Patients

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Abstract
Abstract To further elucidate the cellular mechanisms leading to HDL deficiency in Tangier disease, HDL-mediated cholesterol efflux was studied in cultured skin fibroblasts from Tangier patients. Both Tangier and control fibroblasts show specific saturable binding of HDL 3 to the cell membrane (B max = 70 and 52 ng/mg protein, respectively; K d =8.8 and 10.6 μg/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 cholesterol was labeled to equilibrium by [ 14 C]cholesterol incubation for 48 hours at 37°C, HDL 3 -mediated cholesterol efflux from Tangier fibroblasts was only 50% of control fibroblasts. To define this abnormality in HDL 3 -mediated cholesterol efflux more precisely, several additional experiments were performed. First, membrane desorption of cholesterol was determined after cell membranes were labeled with [ 14 C]cholesterol for 3 hours at 15°C. With this labeling protocol, there was no difference in HDL 3 -mediated cholesterol efflux between control and Tangier fibroblasts. Second, efflux of newly synthesized sterols was determined after incorporation of the precursor [ 14 C]mevalonolactone. Under these conditions, specific HDL 3 -mediated efflux of sterols was almost absent in Tangier fibroblasts. Third, cells were labeled by incubation with reconstituted [ 3 H]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 between 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 synthesized 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 analyzed. Incubation of Tangier fibroblasts in the presence of 1,2-dioctanoylglycerol (10 −5 mol/L), a membrane-permeable activator of PKC, led to normalization of HDL 3 -mediated efflux of newly synthesized cholesterol. These data were interpreted to indicate that impaired activation of PKC rather than a defect in the transport mechanism of cellular cholesterol leads to reduced HDL-mediated efflux of cholesterol from Tangier fibroblasts.