Activation of Phosphatidylinositol-Specific Phospholipase C in Response to HDL3 and LDL Is Markedly Reduced in Cultured Fibroblasts From Tangier Patients
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 α and ε isoforms) was markedly reduced. Thus, the data indicate an impaired PI-PLC activation in response to lipoproteins in Tangier fibroblasts.
- Received April 27, 1995.
- Accepted June 9, 1995.
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 membrane14 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.
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).
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. [γ-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 α (from Amersham) all other PKC antibodies were obtained from Dr Doriano Fabbro, Ciba-Geigy.
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.
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 isotachophoresis26 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-β-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 α (monoclonal) and δ, ε, and ζ (polyclonal) by using standard procedures. The PKC/antibody complex was visualized by autoradiography by using 125I-labeled goat anti-mouse (α) and goat anti-rabbit (ε and ζ) 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 α-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 1×8 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.
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.
Protein determinations were performed according to the method described by Smith et al.30 Cell viability was tested by Trypan Blue exclusion.
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.
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⇓).
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.
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⇓).
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).
Western blot analysis was used to identify various isoforms of PKC. Pilot studies revealed that fibroblasts (both normal and Tangier) expressed PKC α, ε, ζ, and trace amounts of PKC δ, while PKC β1, β2, γ, and η were undetectable. The amount of PKC ε 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 ε was 171±15 aU (n=2, performed in quadruplicate) with 62±1% (n=2) basal-membrane association. PKC α (normal, 65±15 aU; Tangier, 59±9 aU) and PKC ζ (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 α 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 α by 26±2% and 31±5%, respectively (n=2). After stimulation with HDL3 and LDL, membrane association of PKC ε was increased by 13±2% and 11±1%, respectively (n=2). In Tangier fibroblasts, however, neither PKC α nor PKC ε 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⇓.
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 α and PKC ε in both normal and Tangier fibroblasts (data not shown). Membrane association of PKC ζ 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 ζ 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).
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.
Disturbed cellular lipid and lipoprotein metabolism in Tangier disease21 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 α and ε 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 α37 and PKC ε38 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
|[Ca2+]i||=||intracellular calcium concentration|
|PI-PLC||=||phosphatidylinositol-specific phospholipase C|
|PKC||=||protein kinase C|
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.
Voyno-Yasenetskaya TA, Dobbs LG, Erickson SK, Hamilton RL. Low density lipoprotein- and high density lipoprotein-mediated signal transduction and exocytosis in alveolar type II cells. Proc Natl Acad Sci U S A. 1993;90:4256-4260.
Mendez AJ, Oram JF, Bierman EL. Protein kinase C as a mediator of high density lipoprotein receptor-dependent efflux of intracellular cholesterol. J Biol Chem. 1991;266:10104-10111.
Daniels RJ, Guertler LS, Parker TS , Steinberg D. Studies on the rate of efflux of cholesterol from cultured human skin fibroblasts. J Biol Chem. 1981;256:4978-4983.
Fielding CJ, Fielding PE. Evidence for a lipoprotein carrier in human plasma catalyzing sterol efflux from cultured fibroblasts and its relationship to lecithin:cholesterol acyltransferase. Proc Natl Acad Sci U S A. 1981;78:3911-3914.
Miller NE. Mechanisms and approaches to therapy. In: Miller NE, ed. Atherosclerosis. New York, NY: Raven Press; 1984:156-168.
Phillips MC, Johnson WJ, Rothblatt GH. Mechanisms and consequences of cellular cholesterol exchange and transfer. Biochim Biophys Acta. 1987;777:209-276.
Fielding CJ, Fielding PE. Cholesterol transport between cells and body fluids: role of plasma lipoproteins and the plasma cholesterol esterification system. Med Clin North Am. 1982;6:363-373.
Slotte JP, Oram JF, Bierman EL. Binding of high density lipoproteins to cell receptors promotes translocation of cholesterol from intracellular membranes to the cell surface. J Biol Chem. 1987;262:12904-12907.
Fredrickson DS. The inheritance of high density lipoprotein deficiency (Tangier disease). J Clin Invest. 1964;43:228-236.
Assmann G, Schmitz G, Brewer HB Jr. Familial HDL deficiency: Tangier disease. In: Scriver CR, Beaudet AS, Sly WS, Valle D, eds. The Metabolic Basis of Inherited Disease. 6th ed. New York, NY: McGraw-Hill Book Co; 1989:1267-1282.
Law SW, Brewer HB Jr. Tangier disease: the complete amino acid sequence for proapo AI. J Biol Chem. 1985;260:12810-12814.
Schmitz G, Assmann G, Robenek H, Brennhausen B. Tangier disease: a disorder of intracellular membrane traffic. Proc Natl Acad Sci U S A. 1985;82:6305-6309.
Schmitz G, Robenek H, Lohmann U, Assmann G. Interaction of high density lipoproteins with cholesteryl ester laden macrophages: biochemical and morphological characterization of cell surface binding, endocytosis and resecretion of high density lipoproteins by macrophages. EMBO J. 1985;4:613-622.
Schmitz G, Fischer H, Beuck M, Hoecker K-P, Robenek H. Dysregulation of lipid metabolism in Tangier-monocyte derived macrophages. Arteriosclerosis. 1990;10:1010-1019.
Robenek H, Schmitz G. Abnormal processing of Golgi elements and lysosomes in Tangier disease. Arterioscler Thromb. 1991;11:1007-1020.
Rogler G, Trümbach B, Klima B, Lackner KJ, Schmitz G. High density lipoprotein–mediated efflux of newly synthesized cholesterol is impaired in fibroblasts from Tangier patients while membrane desorption and efflux of lysosomal cholesterol are normal. Arterioscler Thromb Vasc Biol. 1995;15:683-690.
Nowicka G, Brüning T, Böttcher A, Kahl G, Schmitz G. Macrophage interaction of HDL subclasses separated by free flow isotachophoresis. J Lipid Res. 1990;31:1947-1963.
Nowicka G, Brüning Th, Grothaus B, Kahl H-G, Schmitz G. Characterization of apolipoprotein B-containing lipoprotein subpopulations by free flow isotachophoresis. J Lipid Res. 1990;31:1173-1186.
Grynkiewicz G, Ponie M, Tsien RY. A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem. 1985;260:3440-3450.
Shears SB. Metabolism of the inositol phosphates produced upon receptor activation. Biochem J. 1989;260:313-324.
Martinson EA, Goldstein D, Brown JH. Muscarinic receptor activation of phosphatidylcholine hydrolysis: relationship to phosphoinositide hydrolysis and diacylglycerol metabolism. J Biol Chem. 1989;264:14748-14754.
Exton JH. Signaling through phosphatidylcholine breakdown. J Biol Chem. 1990;265:1-4.
Bjorkerud S, Bjorkerud B. Lipoproteins are major and primary mitogens and growth promoters for human arterial smooth muscle cells and lung fibroblasts in vitro. Arterioscler Thromb. 1994;14:288-298.
Van Blitterswijk WJ, Hilkmann H, de Widt J, van der Bernd RL. Phospholipid metabolism in bradykinin-stimulated human fibroblasts, I: biphasic formation of diacylglycerol from phosphatidylinositol and phosphatidylcholine, controlled by protein kinase C. J Biol Chem. 1991;226:10337-10343.
Schmitz G, Brüning T, Williamson E, Nowicka G. The role of HDL in reverse cholesterol transport and its disturbances in Tangier disease and HDL deficiency with xanthomas. Eur Heart J. 1990;11:197-211.