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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1995;15:1369-1377.)
© 1995 American Heart Association, Inc.

Articles

Activation of Phosphatidylinositol-Specific Phospholipase C in Response to HDL₃ and LDL Is Markedly Reduced in Cultured Fibroblasts From Tangier Patients

Wolfgang Drobnik; Christoph Möllers; Therese Resink; Gerd Schmitz

From the Institute for Clinical Chemistry and Laboratory Medicine, University of Regensburg, Regensburg, Germany, and the Department of Research (T.R.), University Hospital, Basel, Switzerland.

Correspondence to Prof Dr G. Schmitz, Institut für Klinische Chemie und Laboratoriumsmedizin, Universität Regensburg, Franz-Josef-Strauß Allee 11, D-93042 Regensburg, Germany.

	Abstract

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Abstract We compared HDL₃- and LDL-induced signal transduction in normal and Tangier fibroblasts to elucidate whether impaired signal transduction responses to lipoproteins might contribute to disturbed cellular lipid and lipoprotein metabolism in Tangier disease, a rare autosomal disorder of cellular lipid and lipoprotein metabolism. In several cell types HDL and LDL activate a currently unknown isoform of phosphatidylinositol-specific phospholipase C (PI-PLC) that results in the generation of 1,2-diacylglycerol and inositol 1,4,5-trisphosphate. Compared with normal fibroblasts, Tangier fibroblasts stimulated with HDL₃ or LDL resulted in a significantly reduced accumulation of inositol phosphates and 1,2-diacylglycerol formation. Furthermore, in Tangier fibroblasts both lipoproteins failed to mobilize calcium from internal pools, and the cytosol-to-membrane redistribution of protein kinase C (in both the and isoforms) was markedly reduced. Thus, the data indicate an impaired PI-PLC activation in response to lipoproteins in Tangier fibroblasts.

Key Words: signal transduction • Tangier disease • lipoproteins • protein kinase C • calcium

	Introduction

Top Abstract Introduction Methods Results Discussion References

 
Both HDL₃ and LDL activate signal transduction cascades in several cell types. The activation of PI-PLC in response to HDL₃ and LDL occurs in vascular smooth muscle cells and type II pneumocytes.^{1 2} PI-PLC–catalyzed hydrolysis of PIP₂ yields two second messengers, InsP₃ and 1,2-DAG. In accordance with the known second-messenger functions of InsP₃³ and 1,2-DAG,⁴ both lipoproteins induce the mobilization of Ca²⁺ from internal pools⁵ and the activation of serine/threonine-specific PKC.^{2 6 7} Growing evidence suggests that each of the several isoforms of PKC influences different cellular functions,⁸ although it is not clear which isoforms are activated by HDL₃ and LDL. In human platelets HDL₃ also stimulates phosphatidylcholine-specific phospholipase C.⁹ Evidence for a role of lipoprotein-induced signal transduction in the regulation of lipid and lipoprotein metabolism comes from studies in fibroblasts⁶ and adipose cells,⁷ which show that HDL₃-mediated efflux of newly synthesized cholesterol depends on HDL₃-promoted PKC activation. The removal of excess cholesterol from peripheral cells is the first step of reverse cholesterol transport, and many studies have demonstrated such a net removal of cellular cholesterol in response to HDL₃.^{10 11} This function may account for the inverse correlation between coronary heart disease risk and plasma levels of HDL cholesterol.¹² In contrast to nonspecific cholesterol desorption from the plasma membrane,¹³ HDL-induced translocation of cholesterol from internal stores to the plasma membrane^{14 15} has been proposed as depending on HDL₃-induced signal transduction and PKC activation.^{6 7}

Tangier disease is a rare autosomal recessive disorder of cellular lipid and lipoprotein metabolism. Concomitant with a severe reduction of HDL plasma levels, there is cholesteryl ester deposition in various tissues.^{16 17 18} Metabolic studies in Tangier patients have shown that the reduced levels of HDL are most likely due to an increased catabolism of HDL or its precursors.^{19 20} In contrast to normal mononuclear phagocytes, Tangier mononuclear phagocytes degrade internalized HDL completely in lysosomes.^{21 22} In addition to the defective formation of mature HDL particles from HDL precursor molecules, other abnormalities in cellular lipid metabolism and traffic have been demonstrated.²³ Upon cholesterol loading with acetylated LDL, Tangier mononuclear phagocytes accumulate two types of unusual lysosomes and numerous dilated Golgi cisternae appear.²⁴ Similar dilated Golgi cisternae have also been detected in Tangier fibroblasts, thus providing evidence that the genetic defect of Tangier disease is expressed in fibroblasts.²⁴ Reduced HDL₃-mediated efflux of newly synthesized cholesterol and phospholipids from Tangier fibroblasts can be normalized by the addition of 1,2-DAG.²⁵ This particular ability of 1,2-DAG to normalize reduced HDL₃-mediated cholesterol efflux in Tangier fibroblasts led us to consider whether impaired lipoprotein-induced signal transduction might contribute to the observed disorder of cellular lipid and lipoprotein metabolism. Therefore, we compared HDL₃- and LDL-induced signal transduction between normal and Tangier fibroblasts. The results presented here indicate that the activation of PI-PLC in response to HDL₃ and LDL is impaired in Tangier fibroblasts.

	Methods

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Subjects
Cutaneous fibroblasts were obtained from three patients homozygous for Tangier disease. Patient 1, a 60-year-old woman (triglycerides, 2.94 to 4.89 mmol/L; cholesterol, 2.02 to 2.67 mmol/L); patient 2, a 57-year-old man, brother of patient 1 (triglycerides, 1.58 to 2.24 mmol/L; cholesterol, 1.16 to 1.5 mmol/L); and patient 3, a 42-year-old Pakistani man unrelated to patients 1 and 2 (triglycerides, 3.42 to 3.88 mmol/L; cholesterol, 1.50 to 2.02 mmol/L). Three lines of control fibroblasts were cultured from the cutis of normolipidemic individuals who underwent abdominal surgery (a 38-year-old man, a 53-year-old woman, and a 59-year-old man).

Materials
Cell culture media were obtained from GIBCO-BRL. myo-[2-³H]Inositol (24.4 Ci/mmol) and [³H]palmitic acid were obtained from New England Nuclear. [-³²P]ATP (3000 Ci/mmol) and PKC enzyme assay systems were purchased from Amersham. Bradykinin, thapsigargin, aprotinin, leupeptin, antipain, digitonin, and fura 2-AM were purchased from Sigma Chemical Co. All other chemicals were reagent grade or of superior quality. The goat anti-mouse rabbit IgG antibody was purchased from Amersham. With exception of PKC (from Amersham) all other PKC antibodies were obtained from Dr Doriano Fabbro, Ciba-Geigy.

Cell Culture
Fibroblasts were routinely maintained in Dulbecco's modified Eagle's medium supplemented with L-glutamine, nonessential amino acids, and 10% fetal calf serum under 5% CO₂ in a humidified incubator at 37°C. The experiments used cells at passages 5 through 15. For all experimental protocols fibroblasts were grown to confluence. Cell numbers were routinely determined by enzymatic dissociation with 0.04% trypsin in PBS and counting in a Coulter counter.

Lipoproteins
LDL (d=1.006 to 1.063 g/mL) and HDL₃ (d=1.125 to 1.21 g/mL) were isolated from sera of individual normolipidemic volunteers by sequential centrifugation in a Beckman L-70 ultracentrifuge equipped with a 70 Ti rotor at 4°C. Serum was prepared from recalcified plasma to prevent release of growth factors and cytokines by blood cells during clotting. The lipoprotein fractions were dialyzed against 0.15 mol/L NaCl at 4°C. Concentrations of lipoproteins are given in terms of their protein with albumin as a standard. Lipoproteins were stored at 4°C and used within 1 week. The purity of lipoprotein preparations was determined by the analysis of apolipoprotein composition, applying free flow isotachophoresis^{26 27} and isoelectric focusing–polyacrylamide gel electrophoresis.

Determination of [Ca²⁺]_i
Confluent fibroblasts (in 75-cm² flasks) were incubated for 1 hour in serum-free medium, detached by incubation with 0.04% trypsin/PBS solution for 5 to 10 minutes at 37°C, and collected by centrifugation. Cells were subsequently loaded with 1 µmol/L fura 2–pentaacetoxy–methyl ester at 37°C for 30 minutes in HEPES–buffer A (in mmol/L: HEPES 10, NaCl 135, KCl 5, CaCl₂ 1, MgCl₂ 1, and glucose 6, pH 6.9). Cells were then washed and resuspended in HEPES–buffer B (as described above, but pH 7.4) at a density of 10⁶ cells/mL. Fluorescence was measured at 20°C while the buffer was stirred in a Hitachi F-2000 spectrofluorometer at excitation wavelengths of 340 nm and 380 nm and an emission wavelength of 505 nm. Fluorescence signals were calibrated by using 200 µmol/L digitonin for measurement of maximum fluorescence followed by 10 mmol/L EGTA for minimum fluorescence according to the procedures of Grynkiewicz et al.²⁸

Cell Fractionation for PKC Assay
Confluent fibroblasts (six-well plates) were incubated for 1 hour in serum-free medium and then exposed to specific stimuli for various times (three wells for each measurement). After incubation the cells were chilled on ice, washed twice with ice-cold PBS, and scraped from the flasks into ice-cold PBS. All subsequent procedures were conducted at 4°C. After collection of cells by centrifugation at 1000g for 3 minutes, cell pellets were resuspended in 1 mL sonication buffer (20 mmol/L Tris-HCl, pH 7.4, 0.5 mmol/L Na₂-EDTA, 0.5 mmol/L EGTA, 0.25 mol sucrose, 50 µg/mL leupeptin, 5 µg/mL antipain, 5 µg/mL aprotinin, 50 µg/mL phenylmethylsulfonyl fluoride, and 10 mmol/L 2-ß-mercaptoethanol) and sonicated two times for 20 seconds each, and the cell lysate was centrifuged at 128 000g for 30 minutes by using a Beckmann TLX ultracentrifuge and a 120.2 TLA rotor. The supernatant (representing the cytosolic fraction) was withdrawn, and the pellet (representing the membrane fraction) was suspended in sonication buffer and sonicated for two times for 30 seconds each. After determination of protein concentrations, aliquots of the samples were either stored at -20°C in electrophoresis sample buffer²⁹ for Western blot analysis or immediately analyzed for PKC activity.

Western Blot Analysis of PKC
Equal amounts (50 µg) of protein from the cytosolic and membrane fractions from fibroblasts were resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (8% gels).²⁸ Proteins were transferred to nitrocellulose membranes and probed with specific antibodies for PKC (monoclonal) and , , and (polyclonal) by using standard procedures. The PKC/antibody complex was visualized by autoradiography by using ¹²⁵I-labeled goat anti-mouse () and goat anti-rabbit ( and ) IgG. Membranes were exposed to x-ray films overnight at -70°C. Quantification was achieved by densitometry.

PKC Activity Assay
Assays were performed by employing the Amersham PKC enzyme assay kit in accordance with the manufacturer's protocols. Briefly, equal amounts of protein dissolved in 25 µL sonication buffer (as described above) were added to 25 µL of component mixture containing equal volumes of the following components, each dissolved in 50 mmol/L Tris-HCl buffer, pH 7.5: calcium buffer (12 mmol/L calcium acetate); lipid (8 mol/L -phosphatidyl-L-serine and 24 µg/mL phorbol 12-myristate 13-acetate); peptide (900 µmol/L PKC-specific substrate); and 30 mmol/L dithiothreitol. Reactions were started by adding [³²P]ATP (3000 Ci/mmol) dissolved in a Tris-HCl buffer (pH 7.5) containing 150 µmol/L ATP and 45 mmol/L magnesium acetate, and the mixture was incubated at 25°C for 25 minutes. After the reactions were terminated with dilute acid, phosphorylated peptides were collected by adsorption to Whatman P81 paper. After the papers were washed, radioactivity was determined by scintillation counting.

Treatment of Cells for Determination of Inositol Phosphates and Phosphoinositides
Fibroblasts were cultured in six-well plates in the presence of 6 µCi/mL myo-[³H]inositol during the last 48 hours of growth, after which unincorporated radiolabel was removed by washing twice with Hanks' balanced salt solution. Cultures were preincubated for 1 hour with serum-free medium prior to addition of stimuli.

Assay of Inositol Phosphates
Stimulation was terminated by adding 0.2 vol ice-cold 20% perchloric acid, after which 25 µg phytic acid hydrolysate dissolved in 4 µL H₂O was added to each well. After 20 minutes at 4°C proteins were removed by centrifugation at 2000g. The supernatants were titrated with cold 10N KOH to pH 7.5 and kept on ice. The precipitated KClO₄ was removed by centrifugation, and supernatants were diluted with H₂O to a volume of 10 mL. Samples were loaded on 1-mL Dowex AG 1x8 columns, and inositol phosphates were resolved by elution with the following solutions (in mol/L): free inositol with distilled water, InsP₁ with ammonium formate 0.2 and formic acid 0.1, InsP₂ with ammonium formate 0.4 and formic acid 0.1, and InsP₃ with ammonium formate 0.8 and formic acid 0.1. An aliquot of each fraction was taken for scintillation counting. This method allowed the analysis of percent changes of different inositol phosphate species but no quantification.

Assay for Phosphoinositides
Stimulation was terminated by adding 3 mL chloroform/methanol (1:2, vol/vol) containing 20 µL of 6 mol/L HCl. After 30 minutes at 4°C phase separation was achieved by adding 1 vol chloroform and 1 vol of 2 mol/L KCl. After centrifugation the lower phase was dried under nitrogen and redissolved in chloroform/methanol (9:1, vol/vol) prior to resolution by thin-layer chromatography with a one-dimensional system of chloroform/methanol/88% ammonia/water (90:90:7:20, vol/vol/vol/vol). Silica gel 60 plates that had been oxalated (1% potassium oxalate impregnation) were generally used. Standards of phosphoinositides were used to characterize spots on the chromatography plates.

1,2-DAG Determination
In order to measure generation of 1,2-DAG, fibroblasts were cultured in the presence of 2 µCi/mL [³H]palmitic acid during the last 48 hours of growth. Unincorporated radiolabel was removed by washing with Hanks' balanced salt solution, and cells were preincubated for 1 hour with serum-free medium prior to stimulation. Stimulation was terminated by adding 2 vol ice-cold methanol containing 0.25% HCl. Cells were scraped from the dish, and the suspensions were mixed with 1 vol ice-cold chloroform and maintained for 20 minutes at 4°C before sonication and subsequent pelleting of proteins by centrifugation. The supernatants were mixed with 1 vol ice-cold chloroform and 1 vol of 1 mol/L NaCl to yield two phases that were separated by centrifugation at 2000g for 10 minutes at 4°C. The lower chloroform phase was evaporated to dryness under a stream of nitrogen, and samples were redissolved in chloroform/methanol (95:5). The diacylglycerols were separated from the other lipid species by thin-layer chromatography. Samples were chromatographed on Whatman LK5D thin-layer chromatography plates. The chromatogram was developed in the solvent system benzene/chloroform/methanol (80:15:5, vol/vol/vol). The position of the diacylglycerols was located by autoradiography, and radiolabel was quantified by scraping of the relevant areas and subsequent scintillation counting. Relevant areas were defined by including standards of 1,2-DAG.

Other Methods
Protein determinations were performed according to the method described by Smith et al.³⁰ Cell viability was tested by Trypan Blue exclusion.

Statistical Analysis
The results are expressed as mean±SD; n refers to the number of different control and Tangier fibroblasts used in the experiments. Statistical significance was assessed by Student's t test for paired or unpaired values.

	Results

Top Abstract Introduction Methods Results Discussion References

 
HDL₃- and LDL-Induced [Ca²⁺]_i Transients in Tangier Compared With Normal Fibroblasts
Calcium responses of normal and Tangier fibroblasts to various concentrations of HDL₃ and LDL were examined. The maximum response to HDL₃ in both normal and Tangier cells was obtained at 100 µg/mL (Fig 1A). The maximum [Ca²⁺]_i increase in normal fibroblasts (315±69 nmol/L) was significantly higher (P<.001) than that in Tangier fibroblasts (136±35 nmol/L). Concentration response profiles show that saturation levels were reached at different LDL concentrations in normal (50 µg/mL) and Tan- gier (10 µg/mL) fibroblasts (Fig 1B). In addition, at saturating concentrations of LDL, the maximum [Ca²⁺]_i increase in normal fibroblasts was significantly higher than that in Tangier fibroblasts (P<.001). Pretreatment of cells with 100 ng/mL pertussis toxin for 4 hours completely abolished the intracellular calcium responses to both lipoproteins in normal as well as Tangier fibroblasts (Fig 2).

View larger version (16K): [in this window] [in a new window]   Figure 1. Line graphs showing dose response of (A) HDL3- and (B) LDL-induced calcium signals in normal () and Tangier () fibroblasts. Fura 2–loaded fibroblasts were stimulated with the indicated concentrations of HDL3 or LDL. Each point shows the maximal [Ca2+]i obtained after stimulation (reached within 30 seconds) and represents mean±SD of three experiments, each performed in duplicate, from fibroblasts of three Tangier patients and three control subjects, respectively.

View larger version (50K): [in this window] [in a new window]   Figure 2. Kinetic graphs of (A) HDL3- and (B) LDL-induced calcium signals in normal and Tangier fibroblasts. Fura 2–loaded fibroblasts were stimulated by HDL3 or LDL (50 µg/mL each) with or without 100 ng/mL pertussis toxin for 4 hours. Each kinetic is representative of three experiments from fibroblasts of three Tangier patients and three control subjects, respectively.

In normal fibroblasts, depletion of intracellular Ca²⁺ stores by the addition of 1 µmol/L thapsigargin, an inhibitor of the Ca²⁺-ATPase of the endoplasmic reticulum,³¹ significantly reduced the maximum HDL₃- and LDL-induced [Ca²⁺]_i increase by 187±62 and 189±43 nmol/L, respectively (P<.001 versus [Ca²⁺]_i increases in the absence of thapsigargin; n=3). In Tangier fibroblasts thapsigargin treatment had no effect on the maximum [Ca²⁺]_i responses to either HDL₃ or LDL. In accordance with this, removal of extracellular calcium completely abolished the minor lipoprotein-induced [Ca²⁺]_i increase in Tangier fibroblasts (n=3).

HDL₃- and LDL-Induced Inositol Phosphate Formation in Normal and Tangier Fibroblasts
Since the calcium experiments indicated a defective release of calcium from internal stores in Tangier fibroblasts, the formation of inositol phosphates in response to lipoproteins was examined in normal and Tangier fibroblasts. The experiments were performed in the absence of LiCl. Since the method used for the measurement of inositol phosphates did not allow absolute quantification, results are given as percent changes compared with unstimulated cells. Incubation of normal fibroblasts with 100 µg/mL HDL₃ for 30 seconds increased InsP₁ content by 57±4%, whereas Tangier fibroblasts responded with only a slight increase of 13±6% at 30 seconds (P<.01 above control values; Fig 3A). Stimulation of normal and Tangier fibroblasts with 100 µg/mL LDL yielded similar results to those obtained with HDL₃ (Fig 3B). Both HDL₃- and LDL-induced increases of InsP₁ content were significantly higher in normal compared with Tangier fibroblasts (P<.001). In both normal and Tangier fibroblasts only minor changes in InsP₂ and InsP₃ contents were found after stimulation with HDL₃ and LDL, most likely due to the rapid conversion of InsP₃ and InsP₂ into InsP₁.^{3 30} To ensure that the increase of InsP₁ reflected the activation of PI-PLC, the degradation of PIP₂ in response to 100 µg/mL HDL₃ was investigated. PIP₂ content was significantly decreased by 20±3% (P<.01, n=3) after a 30-second stimulation in normal fibroblasts, while in Tangier fibroblasts only a very slight decrease (4±3%; n=3) was observed.

View larger version (12K): [in this window] [in a new window]   Figure 3. Line graphs showing (A) HDL3- and (B) LDL-stimulated InsP1 formation in normal () and Tangier () fibroblasts. myo-[3H]Inositol–prelabeled fibroblasts were stimulated without (zero time; control) or with 100 µg/mL HDL3 or LDL for the indicated times, and the content (as disintegrations per minute) of [3H]InsP1 was measured. Data are expressed as the percentage increase in [3H]InsP1 content per milligram cell protein relative to [3H]InsP1 content per milligram cell protein in unstimulated controls. Results represent mean±SD of three experiments, each performed in duplicate, from fibroblasts of three Tangier patients and three control subjects, respectively.

HDL₃- and LDL-Induced Formation of 1,2-DAG in Tangier and Normal Fibroblasts
Since 1,2-DAG is the second product of PI-PLC activity, the influence of HDL₃ and LDL on cellular 1,2-DAG content was examined in normal and Tangier fibroblasts. Incubation of normal fibroblasts with 100 µg/mL HDL₃ induced a sustained increase of cellular 1,2-DAG content, which reached 75±12% above control at 5 minutes (Fig 4A). In Tangier fibroblasts the HDL₃-induced increase in 1,2-DAG was significantly diminished (P<.001 versus normal fibroblasts at 5 minutes). LDL-induced 1,2-DAG accumulation in normal fibroblasts reached a maximum of 35±3% above control after 2 minutes of stimulation (Fig 4B). The maximum increase of 21±3% in LDL-stimulated Tangier fibroblasts was significantly lower (P<.001 versus normal fibroblasts at 2 minutes). In normal and Tangier fibroblasts the LDL-induced increase in 1,2-DAG was transient, and levels of 1,2-DAG declined to control values within 5 minutes (Fig 4B).

View larger version (11K): [in this window] [in a new window]   Figure 4. Line graphs showing (A) HDL3- and (B) LDL-stimulated generation of 1,2-DAG formation in normal () and Tangier () fibroblasts. Fibroblasts were prelabeled with [3H]palmitic acid and incubated for the indicated times without (zero time; control) or with 100 µg/mL HDL3 or LDL. Data express the percentage increase in [3H]DAG content per milligram cell protein relative to [3H]DAG content per milligram cell protein under control conditions. Results represent mean±SD of three experiments, each performed in duplicate, from fibroblasts of three Tangier patients and three control subjects, respectively.

HDL₃- and LDL-Induced Redistribution of PKC in Tangier Compared With Normal Fibroblasts
Since elevated levels of [Ca²⁺]_i and 1,2-DAG result in the translocation of PKC from the cytosol to the cell membrane fraction, the redistribution of PKC in response to lipoproteins was investigated by using the PKC activity assay. Total PKC activity in normal (97±25 pmol · mg cell protein^-1 · min^-1) did not differ significantly from that in Tangier (69±19 pmol · mg cell protein^-1 · min^-1) fibroblasts. Membrane-associated activity in unstimulated fibroblasts was 33±1% in normal and 34±1% in Tangier cells. Total PKC activity and basal-membrane association were determined by the analysis of fibroblasts from three different Tangier patients and three control subjects, respectively (two performed in triplicate and one performed in quadruplicate).

Membrane PKC activity in normal fibroblasts peaked (21±2% above control) at 2.5 minutes after addition of HDL₃ and thereafter declined toward control values (Fig 5A). In Tangier fibroblasts membrane-bound PKC activity transiently increased by 8±2% at 1 minute after addition of HDL₃ and thereafter also returned to control values (Fig 5A). This maximum increase was significantly lower than that in normal fibroblasts (P<.01). After exposure to 100 µg/mL LDL, membrane-associated PKC activity in normal fibroblasts increased by 24±3% at 1 minute and remained at this level for at least the next 4 minutes (Fig 5B). In Tangier fibroblasts LDL induced only a transient increase of 10±1% at 1 minute, with a subsequent decline to basal values. At all times tested the PKC activity response to LDL in Tangier fibroblasts was significantly lower than that in normal fibroblasts (P<.01).

View larger version (12K): [in this window] [in a new window]   Figure 5. Line graphs showing effect of (A) HDL3 and (B) LDL on redistribution of total PKC activity in normal () and Tangier () fibroblasts. Cells were incubated for the indicated times without (zero time; control) or with 100 µg/mL HDL3 or LDL, after which the distribution of PKC activity (as picomoles [32P]ATP transferred to substrate per milligram per minute) between the membrane and cytosolic fractions was analyzed by PKC activity assay. Data express the percentage increase of membrane-associated PKC activity relative to membrane-bound PKC activity in unstimulated controls. Results represent mean±SD of three experiments, each performed in duplicate, from fibroblasts of three Tangier patients and three control subjects, respectively.

Western blot analysis was used to identify various isoforms of PKC. Pilot studies revealed that fibroblasts (both normal and Tangier) expressed PKC , , , and trace amounts of PKC , while PKC ß₁, ß₂, , and were undetectable. The amount of PKC expressed in normal fibroblasts was 116±14 aU (n=2, performed in quadruplicate) with a basal-membrane association of 62±2% (n=2). In Tangier fibroblasts the amount of PKC was 171±15 aU (n=2, performed in quadruplicate) with 62±1% (n=2) basal-membrane association. PKC (normal, 65±15 aU; Tangier, 59±9 aU) and PKC (normal, 76±18 aU; Tangier, 97±10 aU) were also expressed in substantial amounts (for each value n=2, performed in quadruplicate). Membrane association of PKC in unstimulated normal fibroblasts (34±4%; n=2) was markedly lower than that in unstimulated Tangier fibroblasts (72±1%; n=2).

Stimulation of normal fibroblasts with 100 µg/mL HDL₃ or LDL for 2.5 minutes increased membrane association of PKC by 26±2% and 31±5%, respectively (n=2). After stimulation with HDL₃ and LDL, membrane association of PKC was increased by 13±2% and 11±1%, respectively (n=2). In Tangier fibroblasts, however, neither PKC nor PKC was translocated from the cytosol to the membrane fractions in response to HDL₃ or LDL. Representative blots demonstrating the different PKC redistribution responses to HDL₃ in normal and Tangier fibroblasts are presented in Fig 6.

View larger version (38K): [in this window] [in a new window]   Figure 6. Autoradiographs showing effect of HDL3 on redistribution of PKC isoforms in normal (top) and Tangier (bottom) fibroblasts. Cells were incubated without (con) or with 100 µg/mL HDL3 for 2.5 minutes, after which the distribution of PKC and PKC between the membrane (memb.) and cytosolic (cyt.) fractions was analyzed by Western blotting. Blots are representative for each isoform. Arrows indicate electrophoretic mobility of 80-kD PKC and 90-kD PKC .

The validity of the translocation assay was tested by stimulation for 5 minutes with 100 nmol/L phorbol 12-myristate 13-acetate, which markedly increased the membrane-bound fraction of PKC and PKC in both normal and Tangier fibroblasts (data not shown). Membrane association of PKC in normal and Tangier fibroblasts was not influenced by HDL₃, LDL, or phorbol 12-myristate 13-acetate (data not shown). The absence of an effect on PKC is consistent with the fact that the C1 phorbol ester–binding domain of this isoform contains only one zinc finger motif and thus does not bind phorbol ester or 1,2-DAG.³¹

Bradykinin-Induced [Ca²⁺]_i Transient and Inositol Phosphate Formation in Tangier Compared With Normal Fibroblasts
Bradykinin-induced signaling was also investigated in normal and Tangier fibroblasts. The peak [Ca²⁺]_i response to 1 µmol/L bradykinin in normal fibroblasts (1140±242 nmol/L; n=3 performed in duplicate) was not significantly different from that in Tangier fibroblasts (993±219 nmol/L; n=3 performed in duplicate). In normal and Tangier fibroblasts bradykinin-induced calcium transients were insensitive to pertussis toxin pretreatment (data not shown).

Bradykinin-induced changes in InsP₁, InsP₂, and InsP₃ did not differ significantly between normal and Tangier fibroblasts. After stimulation with 1 µmol/L bradykinin, InsP₁ content increased steadily over the entire incubation period, reaching 151±13% after 5 minutes in both normal and Tangier fibroblasts (Fig 7A). InsP₃ content was markedly elevated above control values within 30 seconds of the addition of bradykinin (normal, 61±15%; Tangier, 80±15%) and returned to basal levels after 5 minutes. Levels of InsP₂ were similarly altered (data not shown).

View larger version (10K): [in this window] [in a new window]   Figure 7. Line graphs showing bradykinin-stimulated (A) InsP1 and (B) 1,2-DAG formation and (C) bradykinin-induced redistribution of PKC activity in normal () and Tangier () fibroblasts. Fibroblasts were stimulated without (zero time; control) or with 1 µmol/L bradykinin for the indicated times. InsP1 and 1,2-DAG determinations were enabled by prelabeling cells with myo-[3H]inositol and [3H]palmitic acid, respectively. Contents (as disintegrations per minute) of [3H]InsP1 and [3H]1,2-DAG were measured. A and B, Data are expressed as the percentage increase in contents per milligram cell protein relative to corresponding contents in unstimulated controls. C, Data for PKC redistribution are expressed as the percentage increase of membrane-associated PKC activity relative to membrane-bound activity in unstimulated controls. All results represent mean±SD of three experiments, each performed in duplicate, from fibroblasts of three Tangier patients and three control subjects, respectively.

Bradykinin-Induced 1,2-DAG Formation and PKC Redistribution in Normal Compared With Tangier Fibroblasts
Bradykinin (1 µmol/L) induced a transient increase of 1,2-DAG that reached maximum levels of 120±12% and 143±15% above control at 2 minutes in normal and Tangier fibroblasts, respectively (P=NS; Fig 7B). Bradykinin-induced redistribution of PKC activity also did not differ between normal and Tangier fibroblasts (Fig 7C). Maximum increases in membrane-associated PKC activity in normal and Tangier fibroblasts (30±3% and 29±3%, respectively) were obtained within 1 minute and thereafter declined toward control values.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
Disturbed cellular lipid and lipoprotein metabolism in Tangier disease^{21 23 24} and especially observation of reduced HDL₃-mediated cholesterol efflux that could be normalized by 1,2-DAG²⁵ led us to the present study. We aimed to elucidate whether HDL₃- and LDL-induced signal transduction is impaired in Tangier fibroblasts and thereby contributes to disturbed cellular lipid metabolism in Tangier disease.

We have demonstrated that compared with normal fibroblasts, intracellular Ca²⁺ signaling in response to HDL₃ and LDL in Tangier fibroblasts is greatly diminished. Thapsigargin, an inhibitor of the Ca²⁺-ATPase of the endoplasmic reticulum,³¹ had no effect on the [Ca²⁺]_i responses to either HDL₃ or LDL in Tangier fibroblasts. Since thapsigargin is known to deplete intracellular Ca²⁺ stores,³¹ we conclude that the impaired Ca²⁺ signaling is due to an inability of HDL₃ and LDL to release Ca²⁺ from internal stores. Accordingly, the minor lipoprotein-induced increases of [Ca²⁺]_i in Tangier fibroblasts result from an influx of extracellular Ca²⁺, since removal of extracellular calcium abolished the minor calcium signals.

Lipoprotein-induced increases of InsP₁ content were markedly lower in Tangier compared with normal fibroblasts. It is well established that InsP₃, a product of PI-PLC activity, promotes Ca²⁺ release from intracellular pools³ and that InsP₃ is rapidly converted to InsP₁.^{3 32} Thus, the minor increase of InsP₁ content, the small decrease in PIP₂, and the significantly reduced increase of 1,2-DAG content in response to HDL₃ and LDL in Tangier fibroblasts indicate an impaired activation of PI-PLC as the cause for the lack of Ca²⁺ release from internal pools. Currently, it is unknown which isoforms of PI-PLC are activated by HDL₃ or LDL. However, 1,2-DAG generation could also result via activation of phospholipases other than PI-PLC, eg, phosphatidylcholine-specific phospholipase C³³ and the sequential action of PLD and phosphatidate hydrolase.³⁴ Since the present data do not allow discrimination between these different routes of 1,2-DAG generation, we cannot rule out the possibility that HDL₃- and LDL-induced activation of other phospholipases is also impaired in Tangier fibroblasts. Since both 1,2-DAG and Ca²⁺ are major activators of PKC,³⁵ the diminished ability of both lipoproteins to generate these second messengers in Tangier fibroblasts is probably responsible for the reduced PKC activation, which is reflected by decreased cytosol-to-membrane translocation of PKC activity.³⁵ Impaired PKC activation was further demonstrated by the lack of lipoprotein-stimulated translocation of the PKC and isoforms.

Although the calcium signaling and formation of inositol phosphates in response to both lipoproteins were similar, the kinetics of 1,2-DAG generation and PKC activation differed between HDL₃ and LDL in control fibroblasts, indicating specificity of HDL₃- and LDL-induced signal transduction. HDL₃ induced a prolonged formation of 1,2-DAG, which might indicate the activation of phosphatidylcholine-specific phospholipase C in addition to the activation of PI-PLC. In contrast, LDL-induced 1,2-DAG generation was typical for a sole activation of PI-PLC. Specificity of lipoprotein-induced signaling is further supported by similar calcium signals in response to apoA-I liposomes and whole HDL particles and the greatly reduced calcium signal in response to tetranitromethane-HDL₃ (data not shown). Signal transduction that is specific for each lipoprotein in turn supports the idea of a functional relation between HDL₃- and LDL-induced signal transduction and their respective effects on cellular lipid and lipoprotein metabolism. Thus, HDL₃ but not tetranitromethane-modifed HDL activates PKC; this activation induces HDL₃-mediated efflux of newly synthesized cholesterol,^{6 7} a process that enables removal of excess cholesterol from peripheral cells. Mendez et al⁶ have shown that inhibitors of PKC diminish this HDL₃-induced efflux in human fibroblasts, but a stable analogue of 1,2-DAG is able to enhance it. We have observed reduced efflux of newly synthesized cholesterol in response to HDL₃ in Tangier fibroblasts that could be normalized by the addition of 1,2-DAG, a potent PKC activator.²⁵ These observations and the currently presented data, which show a reduced ability of HDL₃ to activate PKC in Tangier fibroblasts, strongly argue for a physiological role of HDL-induced signaling in the regulation of HDL-mediated cholesterol efflux. Furthermore, the current work indicates an impaired activation of PI-PLC as being the most likely reason for reduced PKC activation in Tangier fibroblasts. Defective transport of cholesterol from tissues to HDL might contribute to hypoalphalipoproteinemia in Tangier disease, and thus impaired signal transduction could be an important factor in the pathophysiology of this disease.

Because we do not yet know which cellular processes are influenced by LDL-induced signal transduction, the functional consequences of impaired PKC activation in response to LDL in Tangier fibroblasts are unclear. However, it is reasonable to consider the possibility that impaired signaling by LDL in Tangier fibroblasts might also underlie some disturbances of cellular lipid metabolism in these cells. Besides its effects on lipoprotein metabolism, an impaired activation of PKC isoforms in response to lipoproteins might also influence other cellular functions. Lipoproteins³⁶ as well as PKC ³⁷ and PKC ³⁸ are implicated in the regulation of fibroblast proliferation; impaired lipoprotein-induced signaling might, besides its effects on lipid and lipoprotein metabolism, lead to growth abnormalities. This issue is under investigation in our laboratory.

Stimulation of fibroblasts with bradykinin, an acknowledged activator of PI-PLC in fibroblasts,³⁹ yielded no differences between Tangier and normal fibroblasts. This proves the validity of the assays used and excludes nonspecific impairment of signal transduction in Tangier fibroblasts.

Presently, the underlying cause for impaired PI-PLC activation in response to HDL₃ and LDL is unknown. Reduced binding to fibroblasts is not responsible for impaired signaling with HDL₃, since HDL₃ binding is impaired in neither fibroblasts²⁵ nor monocytes⁴⁰ from Tangier patients. In accordance with previous data,⁴¹ our data suggest involvement of pertussis toxin–sensitive G-proteins in lipoprotein-induced signaling, which might reveal a particular level at which the defect in lipoprotein-induced signal transduction could be localized, although a defect in a particular isoform of PI-PLC must also be considered. In addition, PI-PLC is regulated by intracellular messengers such as PKC, which can result in the inhibition of enzyme activity.⁴²

In summary, the reduced HDL₃- and LDL-induced activation of PI-PLC in fibroblasts of Tangier patients is likely to be responsible for some of the abnormalities of lipid and lipoprotein metabolism observed in this disease.

	Selected Abbreviations and Acronyms

 

aU = arbitrary units [Ca2+]i = intracellular calcium concentration 1,2-DAG = 1,2-diacylglycerol InsP1 = inositol monophosphate InsP2 = inositol biphosphate InsP3 = inositol trisphosphate PBS = phosphate-buffered saline PIP2 = phosphatidylinositol 4,5-biphosphate PI-PLC = phosphatidylinositol-specific phospholipase C PKC = protein kinase C

	Acknowledgments

 
The expert technical assistance of Renate Glätzl, Stella Potra, Thomas Woodtli, and Sybille Bertschin-Woodtli is greatly appreciated. This study was only possible with the continuing cooperation of the patients.

Received April 27, 1995; accepted June 9, 1995.

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