HDL-Mediated Efflux of Intracellular Cholesterol Is Impaired in Fibroblasts From Tangier Disease Patients
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 HDL3 to the cell membrane (Bmax= 70 and 52 ng/mg protein, respectively; Kd=8.8 and 10.6 μg/mL, respectively). There was no appreciable uptake of HDL3 by Tangier and control fibroblasts, indicating that cholesterol efflux from fibroblasts occurs at the cell membrane. When cellular cholesterol was labeled to equilibrium by [14C]cholesterol incubation for 48 hours at 37°C, HDL3-mediated cholesterol efflux from Tangier fibroblasts was only 50% of control fibroblasts. To define this abnormality in HDL3-mediated cholesterol efflux more precisely, several additional experiments were performed. First, membrane desorption of cholesterol was determined after cell membranes were labeled with [14C]cholesterol for 3 hours at 15°C. With this labeling protocol, there was no difference in HDL3-mediated cholesterol efflux between control and Tangier fibroblasts. Second, efflux of newly synthesized sterols was determined after incorporation of the precursor [14C]mevalonolactone. Under these conditions, specific HDL3-mediated efflux of sterols was almost absent in Tangier fibroblasts. Third, cells were labeled by incubation with reconstituted [3H]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 HDL3-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.
- Received October 19, 1994.
- Accepted February 22, 1995.
Tangier disease is a rare, autosomal recessive disorder of cellular lipid and lipoprotein metabolism. Clinically, it is characterized by severe reduction of serum HDL and the major apolipoproteins (apo) of HDL, apo A-I and apo A-II. Concomitant with the reduction of HDL plasma levels, there is cholesteryl ester deposition in various tissues, particularly in the reticuloendothelial system.1 2 3 Most Tangier patients also have decreased levels of LDL and low total serum cholesterol, whereas serum triglycerides are elevated in many cases. The diagnosis of Tangier disease is supported by the combination of the lipoprotein abnormalities described above with hyperplastic yellow-orange tonsils and hepatosplenomegaly.3 Whether there is an increased cardiovascular risk in Tangier disease is still a matter of debate.3 4 However, patients with classic Tangier disease with no additional risk factors such as the patients reported in this article do not show signs of premature atherosclerosis.5
Metabolic studies in Tangier patients showed that the reduced levels of HDL, apo A-I (<1% of normal), and apo A-II (5% to 10% of normal)6 are due to rapid catabolism of HDL and its apolipoproteins in Tangier patients, whereas synthetic rates are within the normal range.7 Structural defects of apo A-I and apo A-II have been excluded as the cause for hypercatabolism of HDL.4 8 9
These data suggest an abnormality in the interaction of cells with HDL leading to hypercatabolism. A key experiment showed that Tangier mononuclear phagocytes (MNPs) degrade internalized HDL completely in lysosomes, rather than resecrete the internalized HDL particles as observed in control MNPs.10 11 12 This was the first evidence for an abnormality of cellular lipid metabolism in Tangier disease. Further analysis of cellular lipid metabolism showed that Tangier MNPs have increased rates of synthesis for phospholipids, triglycerides, and cholesteryl esters compared with normal.13 At the same time, catabolism of cellular phospholipids is enhanced, whereas the catabolism of triglycerides and cholesteryl esters is normal. This may account for the observed lipid storage in these cells.
The biochemical abnormalities of Tangier MNPs are accompanied by distinct morphological abnormalities. These affect mainly the Golgi apparatus and the lysosomal compartment and are found in MNPs as well as in fibroblasts.14
Tangier MNPs erroneously target HDL to a lysosomal compartment. If the genetic defect specifically disturbs retroendocytosis of HDL, one would expect that Tangier fibroblasts, which do not internalize HDL,15 would interact normally with HDL. Interaction of normal skin fibroblasts with HDL results in a net cholesterol efflux if the cells have not been cholesterol depleted. This effect is apparently related to a pathway involving activation of protein kinase C (PKC). Evidence exists that PKC mediates translocation of cholesterol to the cell membrane after specific binding of HDL3 to the cell membrane.16 HDL thereby increases the amount of newly synthesized sterol in the membrane, which will increase the effective concentration gradient for desorption. This effect of HDL appears to be mediated by its protein moiety.16 The same investigators showed that PKC activators such as 1,2-diacylglycerol and phorbol myristate induce translocation of intracellular cholesterol to the plasma membrane and cholesterol efflux, whereas inhibition of PKC by sphingosine reduces cholesterol efflux.16
In the present investigation, HDL3-mediated efflux of cholesterol from Tangier fibroblasts was analyzed. The experiments were designed to study efflux from different cellular cholesterol pools to determine whether specific transport routes are affected in Tangier disease.
Cutaneous fibroblasts were obtained from two patients homozygous for Tangier disease: Patient 1 (E.G.) was a 60-year-old woman (triglycerides, 2.94 to 4.89 mmol/L; cholesterol, 2.02 to 2.67 mmol/L); patient 2 (J.S.) was a 57-year-old man, brother of patient 1 (triglycerides, 1.58 to 2.24 mmol/L; cholesterol, 1.16 to 1.50 mmol/L). Niemann-Pick type C (NPC) fibroblasts were obtained from an 11-year-old male patient (D.O.). Four lines of control fibroblasts (G.M., T.L., N.F., R.W.) were cultured from the cutis of normolipemic individuals who underwent abdominal surgery.
Cell culture media were obtained from Gibco-BRL. [14C]Cholesterol (51 mCi/mmol), [14C]mevalonolactone (54.1 mCi/mmol), and [3H]cholesteryl linoleate (71.4 Ci/mmol) were purchased from NEN. All other chemicals and solvents were from Merck. All other biochemicals, including antibodies, were from Sigma.
Fibroblasts were cultured according to standard conditions in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with l-glutamine, nonessential amino acids, and 10% fetal calf serum in a humidified 5% CO2 atmosphere at 37°C. All fibroblast cultures were used between passages 5 and 15 and cultured for 7 days after splitting 1:2 to ensure that they were confluent for at least 2 days.
Human LDL (d=1.006 to 1.063 g/mL), HDL3 (d=1.125 to 1.21 g/mL), and lipoprotein-deficient serum (LPDS, d>1.23 g/mL) were isolated from serum of individual normolipemic volunteers by sequential ultracentrifugation in a Beckman L-70 ultracentrifuge equipped with a 70-Ti rotor at 4°C.17 Serum was prepared from recalcified plasma to prevent release of growth factors and cytokines by white blood cells into the serum during clotting. The lipoprotein fractions were extensively dialyzed against a buffer containing 0.15 mol/L NaCl and 5 mmol/L Na2EDTA (pH 7.4) at 4°C. The final dialysis step was performed against a 0.15 mol/L NaCl without EDTA.
Determination of HDL3-Binding to Cultured Fibroblasts
Binding of HDL3 to fibroblasts was determined by use of 125I-labeled HDL3 as described previously.11 Binding was performed at 4°C in DMEM containing 1 mg/mL bovine serum albumin and the labeled ligand. After the incubation period, cells were washed five times and lysed by addition of 600 μL of 0.3 mol/L NaOH. Cell-associated radioactivity was determined in an LKB-Pharmacia gamma-counter.
Flow Cytometric Determination of Uptake of DiI-Labeled Lipoproteins
Labeling of lipoproteins with the fluorescent dye 1,1′-dioctadecyl-3,3,3′3′-tetramethyl-indocarbocyanine perchlorate [DiI(3)-C18] was carried out as described earlier.18 Fibroblasts were incubated with DiI-labeled HDL3 for 2 hours at 37°C. DiI-lipoprotein labeled cells were analyzed by flow cytometry in a Becton Dickinson FACScan. Cellular accumulation of DiI was measured at 580 nm in samples containing at least 10 000 cells. Autofluorescence of unlabeled cells was subtracted.
Labeling of LDL With [3H]Cholesteryl Linoleate
Aliquots of human LDL (1.9 mg protein) were lyophilized in the presence of potato starch (ratio of starch to LDL protein, 12:1 wt/wt) in Siliclad-treated glass tubes. Neutral lipids were removed by two extractions with 5 mL heptane at −18°C. The heptane-extracted LDL was then mixed with 200 μL heptane containing 6 mg (9.2 μmol, 51 μCi) [3H]cholesteryl linoleate and kept at −18°C for 2 hours with intermittent vortexing.19 Finally, heptane was evaporated, and the radiolabeled LDL was resuspended in 1 mL of 10 mmol/L tricine buffer, pH 8.4, for 36 hours at 4°C. Soluble reconstituted LDL was separated from the potato starch by centrifugation at 2000 rpm for 10 minutes at 4°C.
Metabolic Labeling of Fibroblasts
After reaching confluence for at least 2 days, fibroblasts were rinsed and then incubated with DMEM containing 10% LPDS for 48 hours to deplete the cells of cholesterol. Thereafter, cells were rinsed again and incubated for 3 hours with either 0.5 μCi/mL [14C]cholesterol to label membrane cholesterol or 2.0 μCi/mL [14C]mevalonolactone to label newly synthesized cholesterol. Incubation was performed at 15°C to minimize intracellular cholesterol transport. For homogeneous labeling of cellular cholesterol, fibroblasts were incubated without prior incubation in LPDS in the presence of 10% fetal calf serum with 2.0 μCi/mL [14C]cholesterol for 48 hours at 37°C.20 To label lysosomal cholesterol, cells were incubated with reconstituted LDL (0.29 μCi/mL containing 52 nmol cholesteryl linoleate) for 3 hours at 37°C.21 After incubation with the radioactive tracer, the medium was removed and cells were rinsed five times with 3 mL phosphate-buffered saline (PBS). Cells from three dishes were harvested to determine mevalonate uptake and cholesterol synthesis in cells pulsed with [14C]mevalonolactone or cholesterol incorporation in cells pulsed with [14C]cholesterol or reconstituted LDL.
Determination of Cholesterol Efflux From Fibroblasts
To determine sterol efflux, cells were incubated in DMEM containing 1% BSA supplemented with increasing concentrations of HDL3 as indicated. Aliquots of the medium were taken at the time points specified. Radioactivity in the medium was determined by liquid scintillation counting. Specific HDL3-mediated efflux is defined as the difference between efflux in the presence of HDL3 and 1% BSA minus the efflux in the presence of 1% BSA only. After 24 hours, the cells were carefully rinsed three times with PBS, harvested, and resuspended in 1 mL PBS. The cell suspension was centrifuged at 10 000 rpm for 10 minutes, the supernatant was removed, and the cells were resuspended in 800 μL PBS. Finally, cells were sonicated on ice for 15 seconds with a Branson sonifier. Aliquots were taken for protein determination and lipid extraction.
Activation of PKC
Activation of PKC during the efflux experiments was achieved by incubation of cells in the presence of 10−5 mol/L of the membrane-permeable 1,2-dioctanoylglycerol (1,2-DOG) as described by Mendez et al.16 This was added after the 3-hour labeling period and again after 4 and 8 hours of incubation.
Determination of Cellular Lipids
Lipid extractions were performed according to the method of Bligh and Dyer.22 Cellular lipids were separated by high-performance thin-layer chromatography (HPTLC) with cholesteryl formate (Sigma) used as an internal standard.23 Samples were dissolved in 30 μL of the solvent used for chromatography. External standards containing free cholesterol, cholesteryl stearate, and cholesteryl formate and the samples were applied to 10×20-cm silica gel HPTLC plates (Merck) with a capillary dispenser (Camag). Separation conditions for neutral lipids have been described previously.23 HPTLC plates were developed with manganese chloride/sulfuric acid. Quantification of cellular free cholesterol and cholesteryl esters was carried out by scanning the plates with a fluorescence scanner (Camag). The amount of radioactivity in specific cellular lipids was determined by scraping the respective spots from the HPTLC plates, solubilization in scintillation liquid, and counting in a beta-counter.
Protein was determined according to the method described by Smith et al.24
Analysis of HDL3 Binding and Uptake by Fibroblasts
Binding of HDL3 to cultured fibroblasts was determined by use of 125I-labeled HDL3. Tangier fibroblasts have a slightly higher specific binding of HDL3 than control fibroblasts (Bmax, 70 versus 52 ng/mg cell protein) and a similar affinity (Kd, 8.8 versus 10.6 μg/mL) (Fig 1⇓). This indicates that HDL binding capacity and affinity of Tangier fibroblasts are not reduced.
Uptake of HDL3 was determined with DiI-labeled HDL3. Neither by fluorescence flow cytometry nor by confocal laser scan microscopy could appreciable uptake of DiI-labeled HDL3 into Tangier or control fibroblasts be demonstrated. Hepatocytes, which were used as control, internalized HDL3 as expected (data not shown). This confirms previous data on fibroblasts15 16 and indicates that cholesterol efflux from control and Tangier fibroblasts must take place at the cell surface.
Cholesterol Efflux After Homogeneous Labeling of Fibroblasts
Fibroblasts were labeled by incubation with [14C]cholesterol for 48 hours at 37°C. Under these conditions, cellular cholesterol pools are homogeneously labeled.25 Efflux to BSA increased from 3.14±0.29% of uptake after 1 hour to 5.84±0.39% after 8 hours in Tangier cells and from 3.28±0.46% of uptake after 1 hour to 6.44±0.36% after 8 hours in control fibroblasts, with no significant difference between the two cell types. With 200 μg/mL HDL3 in the medium, HDL3-specific efflux increased from 2.63±1.27% of uptake after 1 hour to 12.34±1.59% after 8 hours in Tangier fibroblasts. In control fibroblasts, there was an increase from 3.43±0.36% after 1 hour to 30.65±2.21% after 8 hours. Specific HDL3-mediated efflux of cellular cholesterol from Tangier fibroblasts was only about 50% compared with controls for almost all concentrations and time points (Fig 2⇓).
Efflux of Plasma Membrane Cholesterol From Fibroblasts
The plasma membrane cholesterol pool was labeled with [14C]cholesterol for 3 hours at 15°C, and cholesterol efflux was measured by incubation of the cells with increasing amounts of HDL3. Under these conditions, a concentration- and time-dependent efflux of cholesterol from the cell membrane occurred (Fig 3⇓). HDL3-mediated cholesterol efflux of Tangier fibroblasts was similar to that of control fibroblasts for all concentrations. This indicates that membrane desorption of cholesterol is not disturbed in Tangier disease and does not account for the reduced efflux of cholesterol.
Efflux of LDL-Derived Cholesterol From Fibroblasts
Efflux of LDL-derived cholesterol was measured in control fibroblasts, Tangier fibroblasts, and as an internal control in NPC fibroblasts, which are known to have a defect in the release of lysosomal cholesterol. Cells were labeled by incubation with reconstituted [3H]cholesteryl linoleate-LDL for 3 hours at 37°C. To exclude differences in the activity of the LDL-receptor pathway, uptake of labeled cholesterol and cholesteryl esters was measured. Total uptake after the pulse period was similar in control and Tangier fibroblasts, whereas it was significantly lower in NPC fibroblasts (Table 1⇓). Incubation with different concentrations of HDL3 increased cholesterol efflux compared with 1% BSA in both control and Tangier fibroblasts in a concentration-dependent manner. Only during the first 4 hours was efflux from Tangier fibroblasts apparently lower than that from control fibroblasts. Thereafter, effluxes from control and Tangier fibroblasts were similar (Fig 4⇓). As expected, in NPC fibroblasts, HDL3-mediated efflux was low. After 24 hours of chase, 32% of the cholesterol taken up into control cells was recovered in the medium. In Tangier cells, efflux was 31%, which is not significantly different from normal. Efflux from NPC fibroblasts was reduced significantly, to 17% of the total radioactivity taken up (Table 1⇓). These data suggest that efflux of cholesterol incorporated into fibroblasts by uptake of reconstituted LDL for 3 hours is similar between control and Tangier fibroblasts.
Efflux of Newly Synthesized Sterol From Fibroblasts
To analyze efflux of newly synthesized sterols, cells were labeled with radioactive mevalonolactone. Since differences in sterol synthesis between control and Tangier fibroblasts might lead to apparent differences in sterol efflux, de novo cholesterol synthesis was determined after incubation for up to 24 hours with [14C]mevalonolactone (0.5 μCi/mL). There were no statistically significant differences between the two cell types. This indicates that uptake of mevalonolactone and synthesis of cholesterol are similar in the two cell types.
When control fibroblasts were labeled at 15°C for 3 hours with [14C]mevalonolactone as precursor of endogenous sterol synthesis, HDL3 increased sterol efflux at 37°C in a concentration-dependent manner. Specific HDL3-mediated efflux was calculated as the increase over efflux in the presence of 1% BSA only. The medium was analyzed after 4 hours by lipid extraction and HPTLC to identify the radioactive sterols or sterol precursors. By this procedure, only late precursors after the lanosterol step cannot be separated from cholesterol. Of the unspecific efflux to BSA, 88±6% was water soluble. This could represent either nonmetabolized mevalonolactone or early nonsterol precursors of sterol synthesis. Total efflux to medium containing 100 μg/mL HDL3 was composed of 75±7% water-soluble cholesterol precursors (60±5% of uptake). The water-insoluble radioactive products all migrated in the cholesterol position, indicating that they were cholesterol or potentially late precursors of cholesterol, such as zymosterol and desmosterol. Specific HDL3-mediated efflux was completely accounted for by cholesterol (or cholesterol and desmosterol/zymosterol). This demonstrates that the reduction in specific HDL3-mediated efflux is due to newly synthesized cholesterol (or cholesterol and zymosterol/desmosterol) and not to any other precursors.
In control fibroblasts, specific HDL3-mediated sterol efflux ranged between 3% and 18% for HDL3 concentrations between 10 and 100 μg/mL (Fig 5⇓). Most of the HDL3-specific efflux occurred within the first 4 hours of incubation. In Tangier fibroblasts, the efflux to 1% BSA was not appreciably different from control fibroblasts (data not shown). Specific HDL3-mediated efflux of sterol was almost nonexistent (0% to 2.5%) during the whole incubation time in the cell cultures of both patients. In fact, there was no overlap between HDL3-specific efflux between the four control and two Tangier fibroblast cultures with 50 and 100 μg/mL of HDL3 (Fig 4⇑). This indicates that the lack of HDL3-mediated efflux of newly synthesized sterols is specific for Tangier fibroblasts.
Effect of PKC Stimulation on Efflux of Newly Synthesized Cholesterol
HDL3 has been shown to induce sterol translocation to the cell membrane by activation of PKC. In parallel experiments, we observed that incubation with HDL3 does not lead to normal activation of PKC in Tangier fibroblasts (W. Drobnik, MD, et al, unpublished data). The lack of HDL3-induced activation of PKC might be responsible for the reduced HDL3-mediated efflux of newly synthesized sterol observed here. Therefore, PKC was activated by addition of 10−5 mol/L 1,2-DOG to the medium, and the effect on sterol efflux to BSA and HDL3 was determined. In unstimulated Tangier fibroblasts (patient J.S.), HDL3-mediated sterol efflux was again virtually nonexistent. PKC stimulation considerably increased specific HDL3-mediated efflux of newly synthesized sterols (Fig 6⇓). In fact, specific HDL3-mediated sterol efflux from Tangier fibroblasts after PKC stimulation was similar to that from control cells without or with additional PKC stimulation. BSA-mediated sterol efflux was also increased after PKC stimulation. Therefore, in control cells, PKC stimulation resulted in a slight net reduction of specific HDL3-mediated sterol efflux (data not shown).
Cholesterol and Cholesteryl Ester Content of Tangier and Control Cells
To investigate whether the reduced efflux of newly synthesized sterol leads to an enrichment of cellular cholesterol, lipids were extracted from Tangier and control cells and quantified. Cells were incubated for 48 hours with DMEM containing 10% LPDS to reduce cellular cholesterol stores and to induce de novo sterol synthesis. Cells were incubated consecutively in DMEM supplemented with 100 μg/mL HDL3 for 24 hours. Tangier fibroblasts showed a significant enrichment in cholesteryl esters under these conditions (Table 2⇓). This indicates that all or part of the intracellular cholesterol pool is not available for transport to the cell membrane but rather is esterified by acyl coenzyme A:cholesterol acyltransferase.
It has been shown previously by our group that fibroblasts from Tangier patients similar to Tangier MNPs have an abnormal Golgi apparatus and vesicular compartment.14 This was taken as evidence that the genetic defect in cellular lipid metabolism and traffic shown in Tangier MNPs10 is also expressed in fibroblasts. To test this hypothesis, a detailed analysis of cholesterol traffic was performed in Tangier fibroblasts.
Analysis of cellular cholesterol traffic in normal cells has revealed that there are different transport mechanisms for cholesterol from cholesterol stores or cholesterol-poor intracellular membranes, eg, the endoplasmic reticulum, to the cell membrane. These obviously depend on the origin of cholesterol (for review see References 25 and 2625 26 ). DeGrella and Simoni27 showed that when cells are pulsed with precursors of sterol synthesis, newly synthesized cholesterol is labeled within minutes. Transport of newly synthesized cholesterol from the endoplasmic reticulum is energy dependent. It is completely abolished by temperatures <15°C. At 37°C, transport takes between 10 and 60 minutes.
In contrast to these findings, transport of cholesterol to the cell membrane taken up via the LDL-receptor and the lysosomal route is not inhibited by energy poisons, indicating that it is not energy dependent. Lysosomal cholesterol appears to be somewhat faster in the cell membrane than newly synthesized cholesterol. However, transport time (2 to 40 minutes) is similar to that for newly synthesized cholesterol.25 28 29 Evidence for a specific transport route of lysosomal cholesterol to the cell membrane also derives from NPC fibroblasts, in which the transport of lysosomal cholesterol to the cell membrane is disturbed, whereas the transport of newly synthesized cholesterol appears to be normal.21 25 Thus, there is evidence for two, at least in part independent, transport routes of cellular cholesterol to the cell membrane. Defects in either of these pathways might affect cholesterol homeostasis of the cell and reverse cholesterol transport.
In the present study, sterol transport was determined by measuring efflux to an extracellular acceptor. Cellular cholesterol pools were labeled in four different ways: (1) homogeneous labeling of cellular cholesterol by long-term incubation with [14C]cholesterol, (2) incorporation of labeled cholesterol into the cell membrane lipid pool by diffusion, (3) uptake of labeled cholesteryl esters by the LDL-receptor pathway, and (4) incorporation of labeled mevalonolactone into newly synthesized sterols.
Homogeneous labeling of all cellular cholesterol pools showed a reduction of specific HDL3-mediated cholesterol efflux to approximately 50% of control. In further experiments, it could be shown that this reduction is not caused by disturbances in membrane desorption or transport of lysosomal cholesterol, which were shown to be normal. The slight reduction of efflux of LDL-derived cholesterol observed during the first 4 hours disappears after longer incubation (Fig 4⇑) and cannot account for the overall reduction in cholesterol efflux. The normal efflux of LDL-derived cholesterol after the relatively short labeling procedure is perhaps because cholesterol taken up via LDL rapidly exchanges with other cholesterol pools, particularly the cell membrane, before it becomes accessible to acyl coenzyme A:cholesterol acyltransferase, as has been shown previously.30 These data imply that under the labeling conditions used, most of the radioactive cholesterol will be in the cell membrane rather than in intracellular pools and, in particular, in intracellular cholesteryl esters.
The major result of this investigation is the almost complete absence of the concentration-dependent specific HDL3-mediated efflux of newly synthesized sterol from Tangier fibroblasts. The most likely explanation for this observation is a defect in the transport of sterols from the endoplasmic reticulum to the cell membrane. The reason for the disturbed cholesterol translocation could be either a defect in one or more steps in the transport process itself or a defect in the regulation of transport.
It has been shown recently that HDL apolipoproteins induce sterol transport to the cell membrane for desorption by activating PKC.16 31 Desorption itself appears to depend solely on the physicochemical properties of the lipid acceptor available.32 33 That means that HDL3 serves a dual function as cholesterol acceptor and activator of transport processes that provide cholesterol to the membrane for desorption. This suggested to us that PKC activation or another signal induced by HDL leading to translocation of cellular cholesterol to the cell membrane might be defective in Tangier fibroblasts. Therefore, the effects of PKC activation on HDL3-mediated efflux of newly synthesized cholesterol were analyzed. When PKC was activated by 1,2-DOG, there was no difference in HDL3-mediated efflux between control and Tangier fibroblasts. This is evidence that the genetic defect in Tangier disease leads to an inadequate stimulation of PKC by HDL3, resulting in retention of cholesterol in cellular pools. The reduced PKC activation could not be correlated to a reduction of specific binding sites for HDL on Tangier fibroblasts. This may be interpreted in two ways: (1) HDL binding to the signal-transducing receptor is not affected by the genetic defect or (2) binding to the signal-transducing receptor is defective but responsible for only a minor fraction of specific binding of HDL.
The present study supports the concept that Tangier disease is caused by a cellular defect leading to abnormal regulation of lipid transport. We show for the first time that the interaction of HDL3 with Tangier fibroblasts is not followed by normal efflux of newly synthesized sterol and that this defect can be overcome by pharmacological PKC activation. Further studies are needed to identify the cellular defect at the molecular level. Skin fibroblasts, even though not a major player in lipoprotein metabolism, will be a useful tool for these studies, as has been the case for other disorders of lipid metabolism.
This study was supported in part by a grant to Prof Dr Schmitz by the Deutsche Forschungsgemeinschaft within the SFB 310. The expert technical assistance of Renate Glätzl is greatly appreciated. The authors are indebted to Dr David Bowyer, Cambridge, for critically reading and commenting on the manuscript. This study was possible only with the continuing cooperation of the patients.
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