© 1999 American Heart Association, Inc.
Original Contributions |
Growth and Cell Cycle Abnormalities of Fibroblasts From Tangier Disease Patients
From the Institut für Klinische Chemie und Laboratoriumsmedizin, Universität Regensburg, Regensburg, Federal Republic of Germany.
Correspondence to Prof Dr G. Schmitz, Institut für Klinische Chemie und Laboratoriumsmedizin, Universität Regensburg, Franz-Josef-Strauß-Allee 11, D-93042 Regensburg, Germany. E-mail gerd.schmitz{at}klinik.uni-regensburg.de
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Abstract |
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Abstract—We have investigated the abnormal proliferation and morphology of fibroblasts from patients with Tangier disease (TD), a high density lipoprotein (HDL) deficiency syndrome that is characterized by impairment of HDL3-mediated lipid efflux and Gi-protein–mediated signaling via phosphatidylinositol-specific phospholipase C (PI-PLC) and phospholipase D (PLD). TD fibroblasts displayed a 30% to 50% reduced in vitro growth rate and a 1.6-fold increased cell surface area. The response to different mitogens was diminished, and asynchronously growing TD fibroblasts showed 4.4±0.3% S-phase and 19.1±0.5% G2/M-phase cells compared with 9.7±0.6% and 7.8±0.5%, respectively, in controls. Monensin, but not brefeldin A, induced an S- and G2/M-phase distribution in control cells similar to that found in TD fibroblasts. This effect of monensin was accompanied by an increase of ceramide levels in controls, whereas TD fibroblasts already had a 2.5-fold increased basal ceramide concentration. Incubation of control cells with C2 ceramide and threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP) mimicked the effect of monensin on the cell cycle. The inhibition of neither Gi protein function by pertussis toxin nor PLD by butanol resulted in a G2/M-phase arrest. Propranolol, known to increase phosphatidic acid levels, was ineffective in reversing the G2/M-phase arrest in TD fibroblasts. In addition, cDNA sequences and mRNA expression of the participants of PI-PLC or PLD signaling, ie, G-protein subunits
i1, i2, and i3;
phosphatidylinositol transfer proteins- and -ß; and ADP
ribosylation factors 1 and 3 were found to be normal. Thus, growth and
cell cycle abnormalities in TD fibroblasts are likely to be related to
impaired Golgi function and sphingolipid signaling rather than
inoperative G-protein signal transduction. Because PDMP was also found
to decrease HDL3-mediated lipid efflux in control but not
TD fibroblasts, similar pathways seem to be involved in the
disturbances of lipid transport and growth retardation.
Key Words: Tangier disease • ceramide • Golgi apparatus • cell cycle • cholesterol efflux
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Introduction |
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Tangier disease (TD) is a rare, autosomal recessive disorder of cellular lipid and lipoprotein metabolism. Concomitant with a severe reduction of HDL levels in plasma, there is cholesteryl ester deposition in various tissues.1 Metabolic studies in TD patients have shown that the reduced levels of HDL are most likely due to an increased catabolism of HDL or its precursors.2 3 The underlying genetic defect affects intracellular lipid transfer processes, as indicated by the following observations: (1) In contrast to normal mononuclear phagocytes (MNPs), TD MNPs degrade internalized HDL completely in lysosomes.4 5 (2) On cholesterol loading with acetylated LDL, TD MNPs accumulate 2 types of unusual lysosome.6 (3) HDL3-mediated efflux of newly synthesized cholesterol and phospholipids is markedly reduced in TD fibroblasts.7 8 9 This defect in intracellular lipid transport can be normalized by 1,2-dioctanoylglycerol, a short-chain analogue of the second messenger 1,2-diacylglycerol, and by propranolol, a drug known to increase intracellular phosphatidic acid levels.10 Thus, the previously demonstrated impaired HDL3-mediated signal transduction in TD fibroblasts (ie, reduced activation of phosphatidylinositol-specific phospholipase C [PI-PLC]11 12 and a phosphatidylcholine-specific phospholipase D [PLD]13) seems to be causally linked to defective HDL3-dependent lipid transport. Because both PI-PLC and PLD were found to be coupled to a pertussis toxin (PTX)–sensitive G protein, it is possible that the genetic defect in TD is associated with a member of this G-protein family.
However, a characteristic finding in different cell types of TD
patients is an abnormal morphology of the Golgi apparatus.
In MNPs and fibroblasts of TD patients, the cisternae of the
trans-Golgi region are markedly dilated and the more dense form of the
unusual lysosomes seems to degrade Golgi
material.6 Thus, a dysfunction of the Golgi
apparatus is very likely involved in the disturbed
intracellular transport process. Moreover, recent progress in the
understanding of Golgi function clearly indicates a close relationship
between vesicular Golgi transport and signal transduction
mechanisms.14 The mammalian phosphatidylinositol
transfer protein (PITP) isoforms- and -ß have been shown to be
required for the formation of secretory vesicles from the
trans-Golgi-network.15 The underlying mechanism
probably involves the promotion of phosphatidylinositol-4,5-biphosphate
synthesis, which seems to be critical for vesicular budding and is also
essential for PI-PLC– and PLD-mediated signal
transduction.16 17 18 In addition, ADP ribosylation
factor (ARF), a small, monomeric G protein that is needed for the
recruitment of coat proteins to the Golgi, has been shown to be an
activator of PLD, and the need for ARF in vesicle budding
can be bypassed by providing PLD.19 Thus, a
disturbance in the regulation of phospholipid
metabolism may link impaired Golgi function and signal
transduction in TD. This hypothesis is supported by enhanced turnover
of phospholipids in TD cells that results in a net increase of the
cellular concentration of sphingomyelin6 20 and
may also relate to the impaired signal transduction and Golgi traffic
in TD cells. Sphingolipid-derived molecules are important second
messengers with cross-talk to other signaling pathways, including
PI-PLC21 and PLD.22 23
Furthermore, ceramide, the initial breakdown product of
sphingomyelin hydrolysis, has been reported to inhibit Golgi
function24 and exhibit specific antiproliferative
effects by causing cell arrest at the G1/S and
G2/M transitions of the cell
cycle.25 26 To further elucidate the complex
regulatory disturbances in TD fibroblasts, we investigated the
relationship of sphingomyelin metabolism, Golgi
function, and Gi-protein–coupled signal
transduction to the previously observed abnormal morphology and growth
behavior as well as the impaired HDL3-mediated
lipid efflux of cultured TD fibroblasts. Additionally, cDNA sequences
and mRNA expression of Gi1 through 3 as well
as of PITP-, PITP-ß, ARF1, and ARF3 were compared in TD and
control cells.
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Methods |
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Subjects
Cutaneous fibroblasts were obtained from 4 patients homozygous for TD4–7,11: patient 1, female, aged 64 (triglycerides, 2.94 to 4.89 mmol/L; cholesterol, 2.02 to 2.67 mmol/L); patient 2, male, aged 57, brother of patient 1 (triglycerides, 1.58 to 2.24 mmol/L; cholesterol, 1.16 to 1.50 mmol/L); patient 3, a 42-year-old Pakistani male (triglycerides, 3.42 to 3.88 mmol/L; cholesterol, 1.5 to 2.02 mmol/L); and patient 4, male, aged 50 (triglycerides, 2.41 to 2.78 mmol/L; cholesterol, 1.6 to 2.22 mmol/L). Patients 3 and 4 are related to neither patients 1 and 2 nor to each other. Four lines of control fibroblasts were cultured from the skin of normolipemic individuals who underwent abdominal surgery. Cells were used between passages 8 and 15. Cutaneous fibroblasts and peripheral monocytes were only obtained after the subjects had given informed consent.
Materials
Cell culture media and FCS were obtained from
Gibco-BRL. All other biochemicals and solvents, including the
antibodies,
threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol
(PDMP), N-acetyl-sphingosine (C2 ceramide), brefeldin A
(BFA), monensin, epidermal growth factor (EGF), platelet-derived
growth factor (PDGF) -AB, hydroxyurea, ceramide type III, and
Ficoll/Hypaque, were from Sigma. Laboratory-Tek chamber slides were
obtained from Nunc Inc; 6-well plates and polypropylene tubes were from
Falcon. The Cycle Test PLUS DNA reagent kit was obtained from
Becton-Dickinson. High-performance thin-layer
chromatography (HPTLC) plates were from Merck. The
Expand high-fidelity polymerase chain reaction (PCR) system was from
Boehringer Mannheim. Reverse transcription (RT) kits were
obtained from Promega, and QIAEXII gel extraction kit was from Qiagen.
X-OMAT x-ray film was obtained from Kodak. Hybond-N,
125I–PDGF-AB, 125I-EGF,
[32P]ATP, [3H]choline,
and [14C]cholesterol were from
Amersham.
Cell Culture and Growth Kinetics
Fibroblasts were routinely cultured in Dulbecco's modified
Eagle's medium (DMEM) supplemented with L-glutamine,
nonessential amino acids, and 10% FCS under 5%
CO2 in a humidified incubator at 37°C. The
experiments described herein utilized cells at passages 8 to 15.
Contamination of cells with Mycoplasma was routinely tested
for, and only Mycoplasma-free cells were used for
experiments. For analysis of growth kinetics, cells were seeded
into 6-well plates at a density of 10 000 cells per well and incubated
with DMEM supplemented with 10% FCS or 0.5% BSA (negative control)
for the times indicated in the figure legends. Cell numbers were
determined by enzymatic dissociation with 0.04% trypsin in PBS and
counted in a Neubauer chamber. Cell numbers on day 0 were determined 12
hours after seeding of 104 cells per well.
Viability of cells was determined by trypan blue exclusion.
Morphometric Analysis of Cultured Fibroblasts
Fibroblasts were grown to confluence on
Laboratory-Tek chamber slides. They were fixed by
immersion in 70% ethanol at -20°C and stained by incubation with a
monoclonal antibody against -tubulin and a
fluorescence-labeled anti-mouse IgG. This procedure permitted
very accurate identification of cell borders. The cell surface was
measured by integration of the maximal surface area in a confocal laser
scan microscope CSLM (Leitz).
Scanning Electron Microscopy
Control and TD fibroblasts were fixed in phosphate-buffered
3.5% glutaraldehyde solution, pH 7.4. Subsequent to
fixation, the cells were rinsed 3 times with PBS; sequentially
dehydrated in 30%, 50%, and 70% alcohol and 3 times in 99% alcohol;
and dried by the critical-point method on Balzer equipment. Immediately
after critical-point drying, the cells were sputter-coated with a 20-Å
gold layer and visualized with a scanning electron microscope (Zeiss,
Oberkochen).
PDGF and EGF Binding Assays
Fibroblasts (106 cells) were seeded on
10-cm dishes and incubated for 24 hours with 10% FCS. Cells were then
carefully rinsed 3 times with PBS and incubated for 2 hours with
serum-free medium. Cells were subsequently incubated at 4°C with 0.2
to 10 nmol/L 125I-EGF or with 0.02 to 1.0 nmol/L
125I–PDGF-AB in HEPES buffer supplemented with
1% BSA. Nonspecific binding was determined by inclusion of a 200-fold
excess of unlabeled human recombinant EGF or PDGF-AB. Thereafter cells
were washed extensively with buffer and lysed with 0.2% SDS. Aliquots
were taken for gamma counting in a Pharmacia LKB gamma counter and for
protein determination. Scatchard plots were constructed by using
GraphPad software.
Isolation of Peripheral, Human MNCs
Peripheral, human MNCs from a healthy volunteer and
a TD patient (patient 1) were isolated by leukapheresis. Subsequent to
density gradient centrifugation over Ficoll/Hypaque,
monocytes were separated by counterflow centrifugal
elutriation.27 Elutriated monocytes that were
>90% pure, as determined by immunophenotyping in an FACScan
(Becton-Dickinson) analysis, were used for further
analysis.
Cell Cycle Analysis
Determination of cell cycle distribution was performed by a
modification of the method according to Vindelov et
al.28 After treatment of cells (indicated in the
text or the figure legends), they were washed twice with 2 mL PBS,
detached with 0.04% trypsin in PBS, transferred to a Falcon tube, and
centrifuged at 250g for 10 minutes. The resulting
cell pellet was gently resuspended in 100 µL of a solution containing
trypsin in a spermine tetrahydrochloride detergent buffer for the
enzymatic digestion of cell membranes and cytoskeletons (solution A;
Cycle Test PLUS DNA reagent kit) and incubated for 30 minutes at
37°C. Subsequently, 100 µL of a citrate buffer with spermine
tetrahydrochloride containing a trypsin inhibitor and
ribonuclease A was added (solution B; Cycle Test PLUS DNA reagent kit),
and the mixture was incubated for 15 minutes at room temperature to
inhibit trypsin activity and digest the RNA. Finally, 100 µL of
propidium iodide solution was added (solution C; Cycle Test PLUS DNA
reagent kit) and incubated for at least 15 minutes on ice. The
propidium iodide stoichiometrically binds to DNA at a final
concentration of 
125 µg/mL. The isolated and stained nuclei were
directly analyzed and kept at 4°C in the dark. Propidium
iodide fluorescence was stable for 4 hours under these
conditions. Flow-cytometric cell-cycle analysis was performed
on an FACScan (Becton-Dickinson) equipped with a doublet discrimination
module and 15-mW argon laser using an excitation wavelength at 488 nm
and emission at 580 nm. The flow cytometer was calibrated with
ethanol-fixed chicken erythrocyte nuclei included in the DNA quality
control particle kit (Becton-Dickinson) according to the instructions
of the manufacturer. The cell cycle distribution
(G1 phase, S phase, and
G2/M phase) of isolated nuclei was determined by
Cellfit software (Becton-Dickinson). Histogram analysis was
performed using the R-Fit model.29 The
coefficient of variation of the G1 phase peak was
<8%. Cell doublets were excluded by gating by using the doublet
discrimination module.
Quantification of Ceramide Levels
For the analysis of basal ceramide concentration,
fibroblasts were grown to confluence. To investigate the influence of
different drugs on intracellular ceramide levels, confluent fibroblasts
were treated with the indicated agonist for 24 hours. Cells were then
sonicated in ice-cold PBS, and an aliquot was set aside for protein
determination. Subsequently, the lipids were extracted according to the
method of Bligh and Dyer.30 Cellular lipids were
separated by HPTLC. Samples were dissolved in chloroform/methanol and
applied, together with external standards, to 10x20-cm silica gel
HPTLC plates (Merck) by using a capillary dispenser (Camag).
Separation of ceramide was performed twice with
chloroform/methanol/acetic acid, 19:9:1,
vol/vol/vol.31 A standard curve was prepared by
using ceramide type III (the mean molecular weight was estimated
according to the manufacturer's specifications). Ceramide type IV was
used as an internal standard. For staining, HPTLC plates were dipped in
10% CuSO4 (in 8%
H3PO4) for 12 seconds and
heated to 170°C for 15 minutes. Quantification of ceramide was
carried out by scanning the plates with a TLC scanner II from Camag in
the absorption mode. For the standards used, a mean molecular
weight was estimated according to the manufacturers'
specifications.
Determination of HDL3-Mediated Cholesterol
and Phospholipid Efflux
Metabolic Labeling of Fibroblasts
On reaching confluence, fibroblasts were rinsed and incubated
for 48 hours in DMEM containing 10% FCS supplemented with 3 µCi/mL
[3H]choline chloride to label
choline-containing phospholipids and 1.5 µCi/mL
[14C]cholesterol. For the last 12
hours of pulse incubation, 40 µmol/L PDMP was supplemented to
the medium when indicated. After incubation with the radioactive
tracer, the medium was removed, and cells were rinsed twice with PBS
supplemented with 0.5% BSA and once with PBS alone.
Determination of Lipid Efflux From Fibroblasts
Cells were incubated for 12 hours at 37°C in DMEM containing
0.5% BSA supplemented with 100 µg/mL HDL3 with
or without 40 µmol/L PDMP. After incubation, the media were
removed and centrifuged at 800g to precipitate any
detached cells. Cells were rinsed 3 times with PBS and lysed with 0.2%
SDS. Aliquots of cell lysates were taken for protein determination.
Lipids were extracted from supernatant media and cell lysates according
to the method of Bligh and Dyer.30
3H and 14C radioactivities
were measured by liquid scintillation counting in total lipid extracts
from cell lysates and in the incubation media for distinguishing
14C-labeled cholesterol from
3H-labeled phospholipids. Lipid efflux in
response to HDL3 is expressed as the percentage
of "total radioactivity" that appeared in the medium after the
chase period: [disintegrations per minute (dpm) in medium/(dpm in
medium+dpm in cells)]. Specific HDL3-mediated
efflux was calculated as the difference between the efflux to
"HDL3+0.5% BSA" minus the nonspecific efflux
to "0.5% BSA alone."
RNA Isolation and RT-PCR Conditions
Fibroblasts between passages 8 and 15 were grown to
confluence, and total RNA was isolated by using previously described
procedures. Gi1-, Gi2-,
Gi3-, ARF1-, ARF3-, PITP-–, and
PITP-ß–specific DNA probes were generated using corresponding primer
pairs in a PCR reaction (Expand high-fidelity PCR system). cDNA was
generated using a commercially available RT kit (Promega). The primers
were derived from the 5'- and 3'-untranslated regions where sequences
are less conserved. The following primer pairs were used for
amplification: for Gi1,
Gai1-3,
5'-AACAACTT-TGGTCAAGTCCCAGAT-3';
Gai1-5,
5'-CTAGGAGAGAGAAAGG-ATTCCCCT-3'; for
Gi2, Gai2-3,
5'-CATACCCGCCAGGCCCCGCT-GCCC-3';
Gai2-5, 5'-GGCGGCCGGCGGACGGCGGGATG-3'; for
Gi3, Gai3-3,
5'-GTAACTTTCACTAACATCCATGCT-3';
Gai3-5, 5'-GAGTCCGGGCCCGTGTCCCCTCTC-3'; for
ARF1, ARF1-3, 5'-TTGGATCCGTGTCCTTCCACCTGTCCACA-3'; ARF1-5,
5'-GTGAATTCGAGGGCAAGAGGAGTGAGAG-3'; for ARF3,
ARF3-3, 5'-TTGGATCCAGCAGCTGCTGTGATCATGGGCAAT-3'; ARF3-5,
5'-CCGAATTCCCTGGCCACACTTGCATGGAGAGGAA-GG-3'; for
PITP-, PITP--3,
5'-TTGGATCCTGACCATGGACGACA-TTCGAAGGATG-3'; PITP--5,
5'-GCGAATTCAATGGGCACAGA-ACGGCTGCGTCCG-3'; for PITP-ß,
PITP-ß-3,
5'-GTGGATCCATCT-CACGATGGAAGACATTAGGAG-3';
PITP-ß-5,
5'-CTGAATTCCGTCAACACTGCAAGATACTGGTCA-3'.
The cycling conditions were as follows: 2 minutes at 94°C; followed
by 10 cycles of 10 seconds at 93°C; 30 seconds at 62.5°C, 62.0°C,
and 61.0°C for Gi1-,
Gi2-, and Gi3-specific
primers, respectively, and 30 seconds at 59°C, 62.0°C, and 61.0°C
for ARF1-, ARF3-, and PITP-– and PITP-ß–specific primers;
55 seconds at 72°C; and an additional 30 cycles with an incremental
increase of the elongation time by 20 seconds in each cycle. After a
final extension for 10 minutes at 72°C, the PCR products were
analyzed on agarose gels. The fragments of interest were
gel-purified (QIAEXII gel extraction kit), cloned into pUC18 plasmids,
and sequenced using an automated fluorescence-based
sequencer.
Northern Blot Analysis
For Northern blot analysis, 15 µg
(Gi1 to 3) and 10 µg (PITP-/ß and ARF1/3)
of total RNA were separated on 1.2% formaldehyde agarose gels, and RNA
was transferred to a nylon membrane and immobilized by UV
cross-linking. 32P-labeled, random-primed DNA
probes were prepared using the cloned cDNA fragments for
Gi1, Gi2,
Gi3, PITP, PITPß, ARF1, and ARF3. Northern
blots were hybridized overnight at 65°C (G proteins) or 60°C (PITPs
and ARFs) in 50 mmol/L PIPES (pH 6.5), 100 mmol/L NaCl,
50 mmol/L Na3PO4 (pH
7.0), 1 mmol/L EDTA, and 5% SDS in the presence of the probe.
Blots were washed in 5% SDS and 1x SSC for 10 minutes at room
temperature and subsequently in 5% SDS and 1x SSC for 20 minutes at
65°C (G proteins) or 60°C (PITPs and ARFs). The blots were exposed
to x-ray film at -80°C for 6 to 47 hours with intensifier
screens.
Protein Determination
Protein quantification was performed according to the method of
Smith et al.32
Statistical Analysis
Results are expressed as mean±SEM. The overall mean was
calculated from the mean values of multiple determinations performed on
the indicated number of TD patients and control subjects. Statistical
significance was assessed by Student's t test for paired or
unpaired values and set at P<0.05.
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Morphological Characterization of TD Fibroblasts
Because previous studies had indicated that fibroblasts from TD patients appeared to be larger than those of control subjects, the cells' morphologies were investigated by morphometry. At least 2 days after cells had reached confluence, the average surface area of TD fibroblasts (5015±215 µm2, n=2) was significantly greater (P<0.01) than that of control fibroblasts (3090±110 µm2, n=2).
Figure 1
shows a
representative scanning electron microscopy picture of
fibroblasts from control subjects and TD patients. Control fibroblasts
were homogeneous in size and orientation (Figure 1A). In
contrast, TD fibroblasts grew heterogeneously in culture.
They were more variable in size and showed irregular orientation on
the culture dish (Figure 1B). These differences were independent of the
passage number of the cells.
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Analysis of In Vitro Growth of Fibroblasts From TD Patients
in Response to FCS
On the basis of the abnormal morphology and the empirical
observation of a reduced in vitro growth of TD fibroblasts, we
performed detailed analysis of cell proliferation. Growth
kinetics of fibroblasts of 4 different control subjects and 3 TD
patients cultured in DMEM supplemented with 10% FCS were determined
over a period of 10 days (Figure 2). All
cells used in the experiments had comparable passage numbers. As of day
3 in culture, the cell numbers of control fibroblasts (25 981±3162
cells per well) were significantly (P<0.002) higher than
those of TD fibroblasts (14 729±1879 cells per well; Figure 2). After
8 days in culture the number of control fibroblasts per well reached a
maximum of 103 026±25 429, whereas the average cell number of TD
fibroblasts was only 29 967±5948 (Figure 2). In culture medium
supplemented with 0.5% BSA instead of 10% FCS, neither control nor TD
fibroblasts showed any significant variations in cell number over the
entire 8-day culture (Figure 2). The cell numbers on day 0, determined
12 hours after seeding of 10 000 cells per well, were 8870±195 for TD
fibroblasts and 9475±180 for control fibroblasts.
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, 
) and TD (
, 
) fibroblasts in response to 10% FCS. Cells
(10 000) were seeded in 6-well dishes and incubated with DMEM and
0.5% BSA with (
The growth retardation of TD fibroblasts could not be overcome by the
addition of conditioned medium derived from cultures of control
fibroblasts. TD fibroblasts were incubated with conditioned medium
either with or without the addition of fresh FCS for 7 days, and under
both conditions the numbers of TD fibroblasts were 
50% of the
control cell counts (data not shown).
Mitogenic Effects of EGF and PDGF on TD
Fibroblasts
The mitogenic effects of 2 growth factors were tested
by cell cycle analysis. As shown in Figure 3, the incubation of asynchronously
growing control fibroblasts with 30 ng/mL EGF or PDGF-AB for 24 hours
further increased the proliferation of these cells. This effect is
indicated by a marked increase in the percentage of cells in the S and
G2/M phases (Figure 3). In contrast, TD
fibroblasts did not respond to either EGF or PDGF-AB with an increase
of cells in the S or G2/M phase (Figure 3).
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Binding of EGF and PDGF to TD Fibroblasts
To investigate whether a reduced expression of growth factor
receptors might be responsible for the impaired mitogenic
response of TD fibroblasts to EGF and PDGF-AB, the binding of these
mitogens to their cellular receptors was determined. No significant
differences were found between the cell lines for
Bmax and Kd. In a
representative experiment, Bmax
values for PDGF-AB and EGF were 8.13 and 1.15 pmol/mg cell protein in
control fibroblasts and 8.01 and 1.35 pmol/mg cell protein in TD
fibroblasts, respectively.
Cell Cycle Distribution in TD Fibroblasts
Because the cell cycle studies with PDGF-AB and EGF
indicated a substantially increased number of
G2/M-phase cells in TD cultures, the cell cycle
distribution of synchronized as well as asynchronously growing
fibroblasts of different passages and from an extended number of TD
patients (n=3) and control subjects (n=4) was determined in multiple
experiments (Figure 4). The results
confirmed the findings shown in Figure 3
. Compared with control
fibroblasts, TD fibroblasts showed a significantly higher number of
total cells in the G2/M phase. This result was
independent of whether the cells were synchronously (6.6±0.4% versus
19.5±2.8%, P<0.003; Figure 4A) or asynchronously
(7.8±0.5% versus 19.1±0.5%, P<0.001; Figure 4B)
growing. In addition, the percentage of asynchronously growing cells in
the S phase was significantly reduced in TD fibroblasts (9.7±0.6%
versus 4.4±0.3%, P<0.003; Figure 4B). After 24-hour
incubation of asynchronously growing control fibroblasts with
hydroxyurea (2.5 µmol/L), a drug known to halt the cell cycle in
the S phase, nearly all cells disappeared from the
G2/M phase (0.9±0.7%) and accumulated in the
G1 (86.6±1%) or S (12.5±1.7%) phase of the
cell cycle (n=2). In contrast, the same treatment of TD fibroblasts
reduced the percentage of cells in the G2/M phase
only gradually (17.6±1.5% versus 21.8±2.7%; n=2). Prolonged
incubation with hydroxyurea for up to 5 days did not result in a
further decline of G2/M-phase cells in TD
fibroblasts.
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Influence of Inhibitors of Golgi Function on Cell
Cycle Distribution
To investigate an involvement of Golgi function in the growth and
cell cycle abnormalities, we analyzed the influence of monensin
and BFA on cell cycle distribution. In control fibroblasts, monensin
but not BFA significantly decreased the percentage of S-phase cells,
reaching 11.8±0.9% at 100 µmol/L monensin compared with
16.6±0.6% without monensin (Figure 5A).
In TD fibroblasts, however, the already low percentage of cells in the
S phase was not affected (Figure 5A). Concomitantly, in control
fibroblasts monensin markedly increased the percentage of cells in the
G2/M phase in a concentration-dependent manner
(Figure 5B). The percentage of cells in the G2/M
phase increased from a basal level of 8.4±0.2% without monensin to
23.9±1.2% with 100 µmol/L monensin, which was very similar to
those values found in TD fibroblasts under basal and
monensin-containing conditions (Figure 5B). Furthermore, similar to TD
fibroblasts, the number of G2/M-phase cells in
monensin-treated control fibroblasts could not be decreased by
treatment with hydroxyurea (2.5 µmol/L) for 24 hours. In
contrast to monensin, BFA at the tested concentrations did not
significantly affect the cell cycle distribution in either control or
TD fibroblasts (Figure 5C and 5D).
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Determination of Basal Ceramide Concentration in TD
Fibroblasts
Because TD cells are known to have disturbed sphingolipid
metabolism and ceramide is known to inhibit cell
proliferation as well as Golgi function, the intracellular
concentration of ceramide was determined in confluent cultures.
Ceramide levels were found to be significantly increased in TD compared
with control fibroblasts, with 4.5±0.7 compared with 1.8±0.2 nmol/mg
cell protein, respectively (P<0.005 for control versus TD).
These results represent mean±SEM values from 3 TD patients and
5 control subjects, respectively.
Influence of Monensin and BFA on Intracellular Ceramide
Concentration
To evaluate the effect of monensin on intracellular ceramide
concentrations, asynchronously growing (72 hours, 10% FCS) control and
TD fibroblasts were incubated with different concentrations of monensin
for 24 hours. In control fibroblasts monensin significantly
(P<0.05, n=2) increased ceramide levels by 35±18%,
42±18%, and 45±17% at 20, 40, and 60 µmol/L, respectively.
The already 2.5-fold increased basal ceramide concentrations in TD
fibroblasts were not significantly affected. Incubation with 10
µmol/L BFA had no effect on intracellular ceramide concentrations in
control cells (n=2).
Influence of Modulators of Intracellular Ceramide Concentration on
Cell Cycle Distribution
The relationship between the increased ceramide concentrations and
the cell cycle abnormalities in TD fibroblasts was investigated by
analyzing the influence of PDMP, an inhibitor of
glucosylceramide synthase,25 and of C2 ceramide
on cell cycle distribution. PDMP, which elevates intracellular ceramide
concentrations, significantly decreased the percentage of S-phase cells
in control fibroblasts at 40 µmol/L (Figure 6A) and increased
G2/M-phase cells from 7.0±0.8% without PDMP to
11.9±0.9% at 10 µmol/L and to 8.1±0.7% at 40 µmol/L
(Figure 6B). In contrast to control fibroblasts, cell cycle
distribution in TD fibroblasts was not significantly affected (Figure 6A and 6B). To exclude the possibility that these effects of PDMP were
mediated by depletion of higher glycosphingolipids and not due to
increased ceramide concentrations, a short-chain analogue of natural
ceramide, C2 ceramide, was tested for its influence on the cell cycle.
C2 ceramide at 40 µmol/L significantly decreased the number of
S-phase cells in control fibroblasts (Figure 6C) and increased the
percentage of cells in the G2/M phase at 10 as
well as at 40 µmol/L (Figure 6D). Like PDMP, C2 ceramide did not
affect cell cycle distribution in TD fibroblasts (Figure 6C and 6D).
The influence of PDMP and C2 ceramide on the number of control
fibroblasts in the G2/M phase was confirmed by
experiments in which control cells were synchronized with 0.5% BSA in
the presence of either substance. Similar to the effect on
asynchronously growing fibroblasts, the percentage of
G2/M-phase cells was significantly increased in
synchronized cells after 48-hour treatment (data not shown).
|
Influence of PTX and Modulators of the PLD Signaling Pathway on
Cell Cycle Distribution and Intracellular Ceramide
Concentration
To determine a potential role of impaired
Gi-protein–mediated signal transduction and PLD
signaling, the influence of PTX, an inhibitor of
Gi proteins33; butanol, a
competitive substrate of PLD that reduces PLD-catalyzed
production of phosphatidic acid34; and
propranolol,10 an
inhibitor of phosphatidic acid hydrolase, on cell cycle
distribution was investigated (the
Table). Butanol at a concentration of
0.3% did not significantly affect cell cycle distribution of control
and TD fibroblasts (the Table). Additional experiments showed that even
1.0% butanol did not induce a G2/M-phase arrest
(data not shown). Propranolol at 100 µmol/L and PTX
at 100 ng/mL reduced the percentage of cells in the S phase in both
control and TD fibroblasts, while the number of
G2/M-phase cells was unaffected (the Table).
Furthermore, neither butanol nor PTX increased intracellular ceramide
concentrations (data not shown).
|
Influence of PDMP on HDL3-Mediated Lipid
Efflux
Because TD fibroblasts are characterized by a marked impairment of
HDL3-mediated cholesterol and
phospholipid efflux, the influence of PDMP (40 µmol/L) on these
lipid transport processes was analyzed. As shown in Figure 7, PDMP significantly decreased specific
HDL3-mediated efflux of
[14C]cholesterol and
[3H]phospholipids in control cells, whereas the
already reduced efflux in TD fibroblasts was not further
diminished.
|
Cloning and Sequencing of Gi1, Gi2,
and Gi3 and Determination of the mRNA Expression Levels
of These G-Protein Subunits in TD Fibroblasts
To determine whether mutations in Gi1,
Gi2, or Gi3 accounted
for the impaired signal transduction or the cell cycle and growth
abnormalities in TD cells, we cloned and sequenced the corresponding
regions. cDNAs were reverse-transcribed from RNA isolated from cultured
fibroblasts from 4 TD patients. DNA sequencing revealed no alterations
at the cDNA level with an impact on the amino acid sequence.
Furthermore, analysis of mRNA expression from fibroblasts of 4
healthy controls and 4 TD patients demonstrated similar levels and
normal sizes for all 3 transcripts in TD fibroblasts when compared with
controls.
Cloning, Sequencing, and Determination of mRNA Expression of ARF1
and 3 as Well as PITP- and -ß in TD Fibroblasts
Because the monomeric G proteins of the ARF family and the PITPs
are related to PLD and PI-PLC signaling as well as vesicular Golgi
transport, the cDNA sequence as well as the mRNA expression of ARF1,
ARF3, PITP-, and PITP-ß were analyzed in fibroblasts and
monocytes from 1 TD patient and 1 control subject, respectively.
Similar to the G-protein subunits, no alterations at the cDNA level
with an impact on the amino acid sequence were detected. Likewise, the
mRNA levels and transcript sizes of all 4 genes were not different
between TD and control cells.
|
Discussion |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
The current study shows that cultured fibroblasts from patients with TD are characterized by an abnormal morphology and a markedly reduced in vitro growth compared with fibroblasts from control subjects. The reduced proliferation of TD fibroblasts is due to an impaired cellular response that cannot be explained by a lack of either well-known autocrine or paracrine growth factors or a reduced attachment of the initially seeded fibroblasts. Furthermore, by stimulating cells with EGF as well as PDGF-AB, it was demonstrated that the impaired cellular response was not restricted to a specific mitogen, and a defect at the level of ligand-receptor interaction was also excluded. Thus, the observed growth retardation in TD fibroblasts is due to either an inhibition of mitogenic signaling or an intracellular defect that critically interferes with the processes of cell replication. It is unlikely that a less efficient recovery with a higher number of cell divisions in primary culture might explain the observed growth retardation, because TD fibroblasts can be cultured to the same number of passages as control fibroblasts until they discontinue in vitro growth.
Independent of cell passage number, a markedly higher percentage of TD fibroblasts were found in the G2/M phase of the cell cycle, which was characteristic for fibroblasts from different TD patients, independent of whether the former were in a growing or resting state. The failure to synchronize TD fibroblasts with hydroxyurea, a drug known to block cells in the S phase of the cell cycle, indicates that the increased percentage of G2/M-phase cells in TD fibroblasts may be due to a real arrest rather than a delay in G2/M. The G2/M arrest may be linked to the higher average cell surface area in cultures of TD fibroblasts, because enlarged size is a typical feature of G2/M-phase cells.
An arrest in the G2/M phase of the cell cycle has so far been described after treatment of cells with DNA-damaging drugs and is found in mammalian cells after irradiation or treatment with certain cytostatics. The present study indicates that monensin treatment of cultured fibroblasts of healthy controls can also induce an accumulation of cells in the G2/M phase of the cell cycle. Monensin, an Na+ ionophore capable of causing collapse of Na+ and H+ gradients, has gained widespread acceptance as an inhibitor of Golgi function, with its most profound effects on the transcisternae of the Golgi apparatus stacks.35 The concentration-dependent ability of monensin to convert control fibroblasts to a TD phenotype (ie, increased G2/M-phase cells and reduced S-phase cells) suggests that an impaired Golgi function in TD fibroblasts may be associated with the cell cycle and growth abnormalities. This concept is supported by both the absent effect of monensin in TD fibroblasts as well as the previously demonstrated appearance of hyperplastic and dilated trans-Golgi elements in fibroblasts and MNPs of TD patients.6 Similarly, swelling of trans-Golgi cisternae is the most consistent visual in vitro demonstration of a monensin-induced effect on a membranous cell compartment.35 In contrast, inhibition of Golgi function by BFA, which is mainly characterized by the redistribution of Golgi components back to the endoplasmic reticulum,36 had no effect on the cell cycle. Other cellular processes that can be inhibited by monensin and that are known to involve the Golgi apparatus include retroendocytosis of HDL particles,37 the processing of acetylated LDL,38 and the transport of newly synthesized sphingomyelin and glucosylceramide to the plasma membrane39 are also impaired in cells of TD patients46 (impaired transport of sphingomyelin and glucosylceramide in TD fibroblasts are from D.M. et al, unpublished data, 1997). Recently, a reduced HDL-mediated cholesterol efflux, which is a characteristic finding in TD fibroblasts, was found after disruption of Golgi function in fibroblasts of healthy subjects.40 However, monensin also affects compartments of the endocytotic pathway by alteration of the vesicular pH,35 and although the lysosomotropic amine chloroquine showed no G2/M-arresting effect and the pHi of TD cells was normal (W.D. et al, unpublished data, 1998), other monensin effects besides those on the Golgi apparatus cannot be excluded.
Ceramide has been described as a negative regulator of the cell cycle25 26 41 and was recently shown to inhibit Golgi transport processes and to induce dilation of the trans-Golgi region similar to that seen in TD fibroblasts.24 Furthermore, ceramide is known to inhibit PLD, which has been suggested to critically participate in vesicular traffic.42 Thus, the 2-fold increased ceramide level in TD fibroblasts can potentially link both the impaired Golgi function and the cell cycle abnormalities in TD cells. In accordance with this hypothesis, the effect of monensin on cell cycle distribution in control cells was accompanied by an increase of ceramide concentration. However, independent of whether ceramide is involved in abnormal Golgi function of TD fibroblasts or not, our data argue for ceramide's role in mediating the G2/M-phase arrest and the reduced growth in these cells as well as in the monensin-treated control cells. This conclusion is supported by the following observations: (1) PDMP, an inhibitor of glucosylceramide synthase,25 which is known to increase intracellular ceramide levels, induced a significant increase in G2/M-phase cells in fibroblasts from control subjects but not from TD patients. (2) This PDMP effect was reproduced by C2 ceramide, and it is thus unlikely that reduced synthesis of glycosphingolipids is responsible for the effect of PDMP. Recently, similar findings were reported in 3T3 fibroblasts, wherein a short-chain analogue of ceramide as well as PDMP was able to induce a G2/M-phase arrest.25 (3) Furthermore, our data support previous reports demonstrating ceramide as a negative regulator of cell growth,41 since ceramide and PDMP at 40 µmol/L markedly reduced the number of growing control fibroblasts in the S phase to a level comparable to that observed in TD cells. However, it cannot be excluded that the observed effects of C2 ceramide and PDMP are mediated by some downstream metabolites of ceramide. Thus, the present data suggest that either the increased ceramide by itself or some downstream sphingolipid mediators in TD fibroblasts contribute to the altered cell cycle distribution and the reduced mitogenic response of these cells. Although a general growth retardation is not observed in TD patients, impairment of cell cycle regulation may nonetheless occur under certain conditions in vivo. Thus, one can speculate whether a reduced mitogenic response of certain cells may be related to the lack of excessively increased atherogenic risk despite the nearly total absence of HDL. A functional role for sphingolipids in the pathomechanism of altered cellular function in TD is further supported by the reduction of the HDL3-mediated lipid efflux in control cells by PDMP, whereas the already reduced efflux in TD fibroblasts was not affected.
Impairment of Gi-protein–mediated signal
transduction (eg, activation of PI-PLC and PLD) was demonstrated to be
relevant for the reduced HDL3-mediated lipid
efflux of TD fibroblasts.7 11 13 Because PLD is
also thought to participate in mitogenic
signaling,43 the question arises as to whether
defective Gi-protein–mediated signaling is
involved in the growth and cell cycle abnormalities of TD fibroblasts.
We have demonstrated that neither the blockade of
Gi protein function with
PTX33 nor the inhibition of phosphatidic acid
generation by butanol34 was able to induce a
G2/M-phase arrest or to elevate cellular ceramide
levels in control cells. Also propranolol, a substance
known to increase intracellular phosphatidic acid
concentrations,10 was ineffective in normalizing
the cell cycle distribution in TD cells. These findings support the
following interpretations: (1) Together with a normal expression of
transcripts for -subunits of the Gi proteins,
ARF1,44 ARF3,45
PITP-,46 and PITPß,47
all of which are known to be involved in PI-PLC– or PLD-mediated
signal transduction, the inoperative Gi-protein
signaling in TD fibroblasts is likely due to functional downregulation
rather than a structural defect. (2) The effect of ceramide on the cell
cycle is unlikely to be mediated via inhibition of PLD. Downregulation
of PLD- and PI-PLC–mediated signal transduction in TD fibroblasts may
involve ceramide and other sphingolipids48 but
can also be related to caveolae, because these special membrane domains
are considered to be signal transduction centers and their formation
depends on a functional Golgi
apparatus.49 50 The involvement of
caveolae in the pathophysiology of TD is further supported by the
recent observation that HDL-mediated cholesterol efflux
occurs via these membrane domains.51
Owing to the small pedigrees, molecular biology approaches that were successfully used to identify the genetic defects of other diseases will not be promising in the currently available TD families. Therefore, we tried to reduce the number of potential candidate genes by elucidating the functional interrelation of the different cellular abnormalities. The presented data contribute to this approach by linking Golgi function and sphingolipid signaling to a characteristic growth and cell cycle abnormality as well as the reduced HDL3-mediated cholesterol efflux observed in TD fibroblasts.
|
Acknowledgments |
|---|
This work was supported by Deutsche Forschungsgemeinschaft (DFG DR 348/2-1) (to Wolfgang Drobnik). The expert technical assistance of Stella Potra is greatly appreciated. This study was possible only with the continuing cooperation of the patients.
Received October 24, 1997; accepted April 9, 1998.
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