From the
National Hellenic Research Foundation,
Institute of Biological Research and Biotechnology, 48 Vas. Constantinou Ave.,
Athens 11635, Greece, the ¶Department of
Biochemistry, University of Bristol, School of Medical Sciences, Bristol, BS8
1TD, United Kingdom, and the ||Université
Denis Diderot-Paris 7, Laboratoire de Biologie et Biochimie Cellulaire du
Vieillissement, 2 Place Jussieu, Paris 75005, France
Received for publication, January 30, 2003 , and in revised form, April 30, 2003.
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ABSTRACT |
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INTRODUCTION |
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Although often neglected, protein degradation is a major intracellular
function, which is not only responsible for housekeeping but also for the
regulation of important cellular functions, such as homeostasis and survival.
The proteasome is a non-lysosomal threonine protease. It is responsible for
the degradation of many intracellular proteins, including abnormal, misfolded,
denatured, or otherwise damaged proteins (reviewed in Ref.
4) as well as perfectly healthy
proteins, which have to be removed for normal cellular functioning (reviewed
in Ref. 5). The 20 S proteasome
is a 700-kDa protease composed of 28 subunits arranged as a barrel-shaped
structure of four heptameric rings. The two outside rings contain one copy
each of seven different but related -type subunits. Likewise, both
inside rings contain one copy each of seven different but related
-subunits where the catalytic sites are localized. The proteasome
possesses multiple endopeptidase activities including chymotrypsin-like
(CT-L),1 PGPH, and T-L
(reviewed in Refs.
68).
In mammalian cells, the 20 S core complex can be flanked at both ends by 19 S
regulatory complexes or by 11 S (PA28) complexes, giving rise to the 26 S
proteasome and the PA28 20 S proteasome complexes, respectively
(reviewed in Refs. 9 and
10). The 26 S proteasome is in
charge of the ATP-dependent degradation of ubiquitinated proteins (reviewed in
Ref. 11).
Aging is accompanied by abnormalities in intracellular proteolysis (reviewed in Refs. 5, 12, and 13). We and others have shown that the proteolytic activities of the 20 S proteasome are remarkably reduced upon aging of several human tissues as well as in senescent primary cultures (1421). These findings indicate that during aging there is an impaired function of the proteasome, which in turn may result in the accumulation of oxidized proteins (reviewed in Refs. 22 and 23). Limited data exist with regard to the understanding of the molecular basis of such events. Using oligonucleotide array techniques, the expression of several proteasome subunits has been found to decline with age in: human dermal fibroblasts (24), the skeletal muscle of the C57BL/6 mouse (25), and the epididymis of the Brown Norway rat (26). Similarly, decreased proteasome quantity has been observed in senescence MRC5 human embryonic fibroblasts (17) as well as in skin fibroblasts and keratinocytes derived from aged donors (14, 16, 18); in contrast, unchangeable proteasome content has been reported in few other studies (1921). However, in all these preliminary studies only one or two representative subunits of the 20 S complex were analyzed. These fragmented data suggest that aging has profound effects on the proteasome, which in turn may influence its proteolytic activities.
In this study, we have taken a detailed molecular approach regarding the
role of the proteasome in replicative senescence and survival of human
fibroblasts. We first demonstrate that senescent cells exhibit reduced levels
of all examined proteasomal activities that are accompanied by lower
proteasome content and protein expression levels of some but not all
proteasome subunits. We demonstrate that when the proteasome is inhibited
partially a senescence-like phenotype is triggered. Finally, we investigate
the effects of stable overexpression of 1 or
5 subunits in cellular survival to proteasome inhibitors and
oxidative stress. This is the first report of enhanced cellular survival that
is attributed to a better function of the proteasome because of the
overexpression of one of its subunits. Accordingly, these results strongly
suggest a central role of the proteasome during cellular senescence and
survival in response to stress in human fibroblasts.
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EXPERIMENTAL PROCEDURES |
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Cell Lines and Culture ConditionsHuman embryonic
fibroblasts WI38, WI38/T, IMR90, and MRC5 were obtained from the European
Collection of Cell Cultures and were maintained in Dulbecco's modified Eagle's
medium (Invitrogen) supplemented with 10% fetal bovine serum (v/v), 100
units/ml penicillin, 100 µg/ml streptomycin, 2 mM glutamine, and
1% non-essential amino acids (complete medium). WI38 fibroblasts were seeded
at a density of 2 x 105 cells/75-cm2 flask (unless
otherwise noted), were subcultured at a split ratio 1:2 when cells reached
confluence, until they entered senescence at about 40 cumulative population
doublings. In all experimental procedures described below early passage
(young; cumulative population doublings <25) and late passage (senescent;
cumulative population doublings >39) WI38 cultures were used. Cells were
fed 16 h prior to the assay and cell number was determined in duplicates
using a Coulter Z2 counter (Coulter Corp.).
-Galactosidase StainingStaining for
-galactosidase activity was performed as previously described
(29). Briefly, 1.5 x
105 cells were seeded in 6-well plates. After 24 h cells were
washed with PBS, fixed in 0.2% glutaraldehyde and 2% formaldehyde for 5 min,
washed again with PBS, and finally stained for 24 h at 37 °C in the
absence of CO2, in staining solution (150 mM NaCl, 2
mM MgCl2, 5 mM
K3Fe(CN)6, 40 mM citric acid, and 12
mM sodium phosphate, pH 6.0) containing 1 mg/ml
5-bromo-4-chloro-3-indolyl-
-D-galactoside.
Immunofluorescence Antigen Staining and Confocal Laser Scanning
Microscope AnalysisFor immunofluorescence labeling, cells grown on
coverslips were washed with cold PBS and were subsequently fixed with 4%
freshly prepared paraformaldehyde in PBS followed by cell permeabilization
with 0.2% Triton X-100 in PBS; fixation with 20 °C cold MetOH
yielded similar results. Immunolabeling of proteasomes was carried out using
antibodies against 6 and
7 subunits. The
antibodies were diluted in PBS containing 0.1% Tween 20 and 3% bovine serum
albumin (blocking buffer); the secondary anti-mouse IgG/fluorescein
isothiocyanate-conjugated antibody was diluted 1:300 in blocking buffer.
Images of the mounted coverslips were taken using a Leica TCS SP-1 confocal
laser scanning microscope. Routine procedures, applied as controls to
demonstrate the specificity of the antibody used, were: (a) the usage
of normal serum instead of the reactive antibody and (b) omission of
the first antibody. All controls appeared free of any immunofluorescence
background.
Stable TransfectionsExpression vectors encoding for the
full-length 1 subunit and
5 subunit cDNAs
(plasmids pREP7-hygro.
1 and pBJ1-neo.
5,
respectively) were the generous gifts of Drs. K. Tanaka and K. Rock
(30,
31). The WI38/T cells were
transfected with either plasmids or empty vectors by using the electroporation
method. Briefly, 107 cells were mixed with 50 µg of DNA and were
electroporated at 260 V, 960 microfarads (Gene PulserTM, Bio-Rad).
Transfected cells were split 48 h later and were maintained in complete medium
containing 400 µg/ml G418 (for pBJ1-neo and pBJ1-neo.
5
plasmids) or 100 µg/ml hygromycin-B (for pREP7-hygro and
pREP7-hygro.
1 plasmids). Colonies of stable transfectants
were isolated after 34 weeks of selection and propagated into cell
lines. In all cases, transfectants were tested for the appropriate protein
expression levels by immunoblot analysis.
Clonogenic Assays104 cells (WI38/T cell lines
stably transfected with empty vectors, 1 and
5) were seeded in 6-well plates in duplicates. After 24 h,
cells were treated with 100 nM epoxomicin, 10 µM
MG132, or 300 µM H2O2 for 2.5 h in fresh
medium. Cultures were then washed thoroughly and maintained in complete medium
until they formed colonies (about 10 days), which were scored by Crystal
Violet staining. In parallel, the number of treated cells after the same
recovery period was estimated in duplicate experiments. Empty vectors
(pREP7-hygro and pBJ1-neo) gave similar results in all assays (shown as mean
values in Figures and Table I).
Each experiment was performed at least 4 times. For immunoblot detection of
carbonyl groups into proteins, 105 cells were seeded in 6-well
plates in normal medium and 24 h later they were treated with 300
µM H2O2 for 30 min. Proteins were
extracted 24 h after H2O2 treatment.
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Proteasome Inhibitors (Epoxomicin and MG132) Treatment2 x 105 cells were seeded in normal medium and 24 h later they were treated in triplicates with epoxomicin or MG132 (dissolved in Me2SO), or an equal volume of the solvent (control cultures) in the presence of normal medium. Constant treatment with epoxomicin, a more potent and specific inhibitor of the proteasome, was performed as follows: fresh medium containing 20 nM epoxomicin was added in cell cultures every 24 h continuously for 4 days (epoxomicin was replaced every day to exclude the possibility of its inactivation at 37 °C). Repetitive treatment with MG132 was performed as follows: 10 µM MG132 was added in cell cultures for 2 h. Cells were washed with PBS, left to recover for 22 h, and treatment was repeated 3 additional times. Cultures at this stage were named as 4 + 0 days (i.e. 4 days of treatment, constantly with epoxomicin or repetitively with MG132). These cultures were re-seeded, washed thoroughly with PBS, and then maintained in normal medium for an additional period of 1 or 2 weeks. Assays were performed immediately after treatment (4 + 0 days) as well as 1 (referred to as 4 + 7 days) and 2 weeks (referred to as 4 + 14 days) later.
Quantitative Analysis of Cellular ApoptosisFor the quantitative analysis and determination of the cytoplasmic histone-associated DNA fragments, that are indicative of on-going apoptosis, the Cell Death Detection ELISAPLUS photometric enzyme immunoassay method (Roche Diagnostics) was used. Briefly, 105 cells were seeded in triplicates in 6-well plates for 24 h and were then treated with 100 nM epoxomicin, 10 µM MG132, or 300 µM H2O2 for 2.5 h, followed by a recovery period in complete medium for an additional 24 h. The extent of the induced apoptosis was then quantitatively measured by the ELISAPLUS death assay, according to the manufacturer's instructions. For the determination of the induced apoptosis in WI38 cells continuously treated with epoxomicin, 105 cells were seeded in triplicates in 6-well plates for 24 h and were then treated with 20 nM epoxomicin or the solvent (Me2SO, control cultures) for 4 days as described in the previous section. Triplicate sets of samples were analyzed right after treatment (4 + 0 days), 1 (4 + 7 days), and 2 weeks (4 + 14 days) after treatment.
Cellular Proliferation-DNA Synthesis AnalysisFor the determination of DNA synthesis the Cell Proliferation ELISA 5-bromo-2-deoxyuridine colorimetric immunoassay method (Roche Diagnostics) was used. Briefly, 2 x 103 cells were seeded in 96-well plates for 24 h and were then treated with 20 nM epoxomicin or the solvent (Me2SO, control cultures) for 4 days as described previously. Sextuplicate sets of samples were labeled for 3 h with 5-bromo-2-deoxyuridine and DNA synthesis was analyzed right after treatment (4 + 0 days), 1 (4 + 7 days), and 2 weeks (4 + 14 days) after treatment, according to the manufacturer's instructions.
Proteasome Peptidase Assays and Protein DeterminationCT-L, PGPH, and T-L activities of the proteasome in crude extracts were assayed with the hydrolysis of fluorogenic peptides, LLVY-AMC, LLE-NA, and LSTR-MCA, respectively, for 30 min at 37 °C, as described previously (16). Proteasome activity was determined as the difference between the total activity of crude extracts or fractions and the remaining activity in the presence of 20 µM MG132. Assays of 26 S proteasomes were carried out in 25 mM Tris/HCl buffer, pH 7.5, containing 5 mM ATP and assays of 20 S proteasomes were performed in 50 mM Hepes buffer, pH 7.5, containing 0.02% SDS as described previously (32). Fluorescence was measured using a PerkinElmer Life Sciences 650-40 fluorescence spectrophotometer. Protein concentrations were determined using the Bradford method with bovine serum albumin as a standard.
Immunoblot AnalysisCells were harvested at the indicated
time points, lysed in non-reducing Laemmli buffer, and fractionated by
SDS-PAGE (12% separating gel) according to standard procedures
(33). After electrophoresis,
proteins were transferred to nitrocellulose membranes for blotting with
appropriate antibodies. Secondary antibodies conjugated with horseradish
peroxidase and enhanced chemiluminescence were used to detect the bound
primary antibodies. Immunoblot detection of carbonyl groups into proteins was
performed with the OxyBlotTM protein oxidation detection kit (Intergen)
according to the manufacturer's instructions. Protein loading was tested by
stripping each membrane and reprobing it with -actin antibody.
Preparation of Cell Extracts and Separation of Proteasome Complexes by Gel FiltrationCells were lysed in 20 mM Tris/HCl buffer, pH 7.5, containing 5 mM ATP and 0.2% Nonidet P-40. Extracts were centrifuged at 11,500 x g for 10 min at 4 °C and then an equal amount of protein from early/late passage cells (0.51.5 mg) was fractionated by gel filtration using a Pharmacia Superose 6 FPLC column equilibrated in 20 mM Tris/HCl buffer, pH 7.5, containing 10% glycerol, 5 mM ATP, and 100 mM NaCl (34). Fractions were collected and samples were analyzed by SDS-PAGE and immunoblot analysis. The peak fractions containing 26 S and 20 S proteasomes were determined by activity assays (CT-L activity), which were carried out in duplicate.
Immunoprecipitation of Proteasomes and Two-dimensional Gel
ElectrophoresisImmunoprecipitated proteasomes were prepared as
follows. Cell monolayers were washed and scraped into cold PBS containing 10
mM phenylmethylsulfonyl fluoride and 10 µg/ml aprotinin. For
radioactive-labeled immunoprecipitation, prior to washing and scraping, cells
were starved in methionine-deficient medium for 1 h, followed by pulse
labeling for 4 h with 100 µCi/ml [35S]methionine in
methionine-deficient medium. Scraped cells were diluted directly in 20
mM Tris/HCl buffer, pH 7.5, containing 5 mM ATP, 10%
glycerol, and 0.2% Nonidet P-40 containing 10 mM
phenylmethylsulfonyl fluoride and 10 µg/ml aprotinin (lysis buffer). Cell
extracts (all equilibrated in lysis buffer) were then cleared by adding normal
mouse serum and protein-A agarose beads for 3 h at 4 °C. Meanwhile,
protein A-agarose beads equilibrated in lysis buffer were coupled with
12 µg of antibody against 6 subunit for 3 h at 4
°C with constant rocking. The target antigen was then immunoprecipitated
by adding in the pre-coupled antibody against
6
subunit-protein A the pre-cleared extracts. Binding reactions were performed
overnight at 4 °C with constant rocking. Immunoprecipitated protein
complexes were collected, washed 4 times in 50 mM Tris/HCl buffer,
pH 7.5, containing 5 mM ATP, 75 mM NaCl, 10% glycerol,
and 0.2% Triton X-100 (washing buffer), and eluted from the agarose beads by
boiling for 5 min in the washing buffer without NaCl. Controls used to
demonstrate the specificity of the observed immunoprecipitations included the
use of normal serum instead of the specific antibody. Following
immunoprecipitation of proteasomes, samples were processed for one-dimensional
SDS-PAGE as described above.
Two-dimensional gel electrophoresis was carried out as follows. Immunoprecipitated proteasomes (starting from 650 µg of crude extracts) were diluted in sample buffer (9 M urea, 2% CHAPS, 2% Pharmalytes pH 310, 20 mM dithiothreitol and bromphenol blue). The first dimension was performed with Immobilines Drystrips (nonlinear, pH 310, length 13 cm) using the Multiphor II system (Amersham Biosciences). The Drystrip was rehydrated with the sample in a reswelling tray (Amersham Biosciences) overnight at room temperature and then focused for 50,000 volt hours (23 h). After focusing, the strips were equilibrated in 50 mM Tris/HCl buffer, pH 6.8, 6 M urea, 30% (v/v) glycerol, 1% (w/v) SDS (equilibration buffer) supplemented with 1% dithiothreitol for 15 min and in equilibration buffer containing 2.5% (w/v) iodoacetamide and traces of bromphenol blue for 15 min. The second dimension was performed on a 12% SDS-PAGE using the Protean II system (Bio-Rad). Gels were fixed, amplified, dried, and directly exposed to low energy screen of STORM 860 phosphorimager. Gels were analyzed by the "STORM 860" and "Image Master 2D Elite analysis" softwares (Amersham Biosciences).
Statistical AnalysisStatistical calculations and graphs were performed with the Microsoft Excel software. Data were analyzed by analysis of variance single factor and p value was used to determine the level of significance (p < 0.05). All values were reported as mean (the average of three independent experiments) ± S.E., unless otherwise indicated.
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RESULTS |
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It is well documented that oxidized proteins are degraded by the proteasome. Therefore a decrease of proteasomal activities would be expected to lead to increased levels of oxidized proteins. As shown in Fig. 1B (left panel) we observed accumulation of oxidized proteins in senescent cells consistent with the reduction in proteasome activities observed in these cells. We have also investigated the ability of senescent cells to ubiquitinate proteins for degradation by the proteasome. As shown in Fig. 1B (right panel), we observed increased amounts of ubiquitinated proteins in senescent cells.
We next investigated the loss of proteasome function in senescent
fibroblasts. Therefore we examined whether there is a quantitative difference
of the proteasome content between early and late passage WI38 cells. A
detailed immunoblot analysis of several representative proteasomal subunits,
of both the 20 S proteasome and the 19 S complex, was performed. We started by
examining the expression levels of the catalytic -subunits
1,
2, and
5 that contain
the catalytic centers of PGPH, T-L, and CT-L activities of the proteasome,
respectively. All three subunits were found to have decreased levels in late
passage WI38 cells (Fig.
1C) compared with the early passage cells. As
1,
2, and
5 subunits can be
replaced by
1i,
2i, and
5i,
respectively, in immunoproteasomes
(35) we explored the
possibility of subunit substitution in senescent cells.
5i
and
2i were found equally expressed in early and late passage
WI38 cells, whereas
1i was not detected in either cultures
(data not shown). Therefore these data suggest that the decreased levels of
1,
2, and
5 subunits are
not compensated by
1i,
2i, and
5i subunits in senescent WI38 fibroblasts. To clarify whether
there was a general decrease of the proteasomal subunits with senescence we
continued the analysis by examining several other
- and
-subunits
of the 20 S core. A representative analysis for
7,
4,
6, and
7 is shown in
Fig. 1C. No
significant differences in the expression levels were observed between early
and late passage WI38 cells for any of these subunits.
Because the 20 S core is flanked with the 19 S regulatory complexes to form the 26 S proteasome, representative subunits of this complex were also examined. All the subunits that were analyzed (S4, S5a, S6a, S6b, S8, and S14; see Fig. 1C) were found to be down-regulated in late passage cells. In conclusion, taking together all of the data, in late passage WI38 cells there is a decrease of all three proteasome activities that is accompanied by accumulation of oxidized and ubiquitinated proteins. Moreover, senescent cells exhibit reduced expression levels of the catalytic centers of the 20 S proteasome and subunits of the 19 S regulatory complex.
To investigate further the down-regulation of specific proteasome subunits
in senescent cells, next we examined the overall amount of newly synthesized
proteasome in both early and late passage WI38 cells. For these experiments,
immunoprecipitated radiolabeled proteasomes were analyzed by two-dimensional
gel electrophoresis. Such an analysis is shown in
Fig. 2, A and
B. It is first worth mentioning that with the same amount
of total protein, the radiolabeled starting and immunoprecipitated material in
late passage cells accounted for 50 and 20% of those obtained from early
passage cells, respectively. This reveals a different rate of protein
synthesis between early and late passage WI38 cells, a known feature of
replicative senescence (reviewed in Ref.
1), but also an even higher
decrease of newly synthesized proteasome in late passage cells. Specifically,
it was estimated by "Image Master 2DElite analysis" of
two-dimensional gels that both 20 S and 26 S newly synthesized proteasomes
were reduced approximately to less than 15% in senescent cells as compared
with the young ones. Proteasome content in both early and late passage cells
was further investigated by "cold" immunoprecipitation followed by
immunoblot analysis. As shown in Fig.
2C, for
7 and
1
subunits, the detected amount of both subunits was decreased in the senescent
cell extracts, thus indicating an overall lower amount of assembled
proteasomes in late passage cells. Taking together these data show both a
decreased rate of proteasome subunit synthesis as well as a decrease of
assembled proteasome content in late passage cells. These observations are in
agreement with the decreased proteasome activities reported in
Fig. 1A.
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We investigated further the proteasome complexes in early and late passage
cells after separation by gel filtration. In accordance with previously
reported data a decrease in both 26 S and 20 S proteasome complexes was
observed in senescent cells (data not shown). Some significant differences
were found in the immunoblots for the late passage fractionated cell extracts.
These showed an accumulation of some low molecular weight cross-reacting
material (free subunits?) for -type proteasome subunits
6 and
7 in the late passage cells
(Fig. 2E) that was not
present in early passage cells (Fig.
2D). Although further analysis is needed, this intriguing
observation could indicate that these subunits may not integrate efficiently
in assembled proteasomes in senescent cells.
Partial Proteasomal Inhibition by Specific Proteasome Inhibitors
Elicits a Senescence-like Phenotype in Early Passage WI38
FibroblastsThe previous results have revealed that the proteasome
is highly affected during replicative senescence. Therefore, we investigated
the effect of proteasomal inhibition in early passage WI38 cells. Two
proteasomal inhibitors were used, namely epoxomicin (an irreversible
inhibitor) and MG132 (a reversible inhibitor), in independent experiments. It
is known that total inhibition of the proteasome with specific inhibitors for
long periods triggers apoptosis
(36). However, there are no
reports regarding the effect of these reagents in primary human fibroblasts.
Therefore, in pilot experiments, we examined the time- and
concentration-effect of the referred inhibitors in early passage WI38
fibroblasts. We tested different ranges of concentration of epoxomicin (1
nM to 1 µM) and MG132 (100 nM to 50
µM) for various periods of treatment (1 h to 4 days). In
accordance with earlier reports, apoptosis was evident in high epoxomicin (1
µM) and MG132 (>10 µM) concentrations added
constantly in medium for 24 h (data not shown). However, when cells were
treated with lower concentrations we observed mainly cytostatic and to a
lesser extent cytotoxic effects. Treatment with a low dose of epoxomicin or
high dose of MG132 once for a short period of time (i.e. up to 2 h)
resulted in a reversible growth arrest as cells recovered fully and started
proliferating again soon after the withdrawal of the inhibitor from the tissue
culture medium. However, when cells were left in the presence of the same
concentration of the inhibitor for longer periods or repetitively
(i.e. more than one treatment), an irreversible growth arrest was
observed. An example of the effect of a low dose of epoxomicin (20
nM for 4 days; for conditions see "Experimental
Procedures") in WI38 cells is shown in
Fig. 3A (left
panel). Specifically, whereas control cells (Me2SO treated)
were increased from 2.37 x 106 ± 0.09 x
106 (cells) right after treatment (CON/4 + 0 days) to 56.65 x
106 ± 0.19 x 106 (cells) after 2 weeks of
recovery (CON/4 + 14 days; 24-fold increase), epoxomicin-treated cells
were about half as much as the control cells right after treatment (1.27
x 106 ± 0.02 x 106 cells at INH/4 + 0
days). This difference accounts both for some cells that died by apoptosis but
mainly for cells that ceased to proliferate (see below). Importantly, no
further cell decrease was observed as assayed at INH/4 + 7 and INH/4 + 14 days
(Fig. 3A, left
panel, gray bars). Instead, these cells were viable and they were unable
to divide even when supplemented with fresh, inhibitor-free medium.
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First we examined the characteristics of epoxomicin-treated WI38 cells. As shown in Fig. 3B (left panel), limited apoptosis is triggered during the 4 days of treatment (INH/4 + 0 days); however, there is no further ongoing apoptosis 1 (INH/4 + 7) and 2 (INH/4 + 14) weeks after treatment, in accordance with the steady cell numbers counted in these conditions (see Fig. 3A, left panel). It is worth mentioning that WI38 cells are relatively resistant to apoptosis (enrichment factor of apoptosis was arbitrary set to 1; see comparative apoptosis analysis between WI38 and WI38/T cells in Fig. 5) and, therefore, the exhibited 1.8 times of induction of apoptosis in epoximicin-treated cells (INH/4 + 0 days) as compared with control cells (CON/4 + 0 days) represents a small fraction of treated cells.
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Because epoxomicin-treated cells were unable to divide, next we examined
whether these cells show the characteristic features of senescence. As shown
in Fig. 3B (right
panel), treated cells right after treatment (INH/4 + 0 days) exhibited
significantly reduced DNA synthesis levels as compared with the control cells
(CON/4 + 0 days). This rate could not be restored 1 (INH/4 + 7 days) and 2
weeks (INH/4 + 14 days) after treatment (gray bars), in contrast to
the control cells that exhibited the expected levels of DNA synthesis
throughout the experiment (black bars). In addition,
epoxomicin-treated cells exhibited a morphology typical of senescent cells,
thus, they were enlarged, they did not line up in parallel arrays, they had
larger nuclei (an example can be seen in
Fig. 4C), and they
were positive to the senescence biomarker -galactosidase
(Fig. 3C). At protein
level, treated cells were also found to overexpress several biomarkers of
senescence. As it is shown in Fig.
3D the epoxomicin-treated cells exhibit elevated protein
levels of both forms of ApoJ
(37) as well as the
cyclin-dependent kinase inhibitors p21
(38) and p16
(39). Finally, the status of
oxidized proteins was analyzed and as expected, the treated cells had a high
amount of oxidized proteins as opposed to the control cultures
(Fig. 3D).
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Next, we have addressed the issue whether induction of a senescence-like phenotype by epoxomicin is a feature common to other primary human fibroblasts cell lines. Therefore we have repeated epoxomicin treatment in IMR90 and MRC5 cells. As shown in Fig. 3A epoxomicin treatment resulted in the induction of irreversible growth arrest in both IMR90 (right upper panel) and MRC5 (right lower panel) cells in a manner similar to WI38 cells (left panel). Moreover, the earlier mentioned biomarkers of senescence were also tested in MRC5 and IMR90 cells and showed expression patterns similar to the ones found in WI38 cells (data not shown). Finally, a similar induction of a senescence-like phenotype was observed after delivering 10 µM MG132 for 2 h for 4 continuous days in WI38 cells (data not shown), thus excluding the possibility of a specific effect of epoxomicin itself. Taking together all these data (i.e. same effect of (a) epoxomicin treatment in three different primary human fibroblast cell lines and (b) delivery of two different proteasome inhibitors in WI38 cells) we exclude the possibilities either of a cell line-specific induction of a senescence-like phenotype or of a selection of a cellular subpopulations with possible unknown phenotypic traits that could account for a different survivability. Thus partial proteasomal inhibition by specific inhibitors elicits a senescence-like phenotype in early passage fibroblasts.
We have also examined the proteasome function per se in WI38-treated cells. First, we determined the levels of the three proteolytic activities, CT-L, PGPH, and T-L, in these cells. As shown in Fig. 4A for epoxomicin-treated cells all three activities were found to be altered. As expected, both CT-L and T-L activity were immediately reduced after treatment (INH/4 + 0 days) because this inhibitor targets the catalytic center of these activities. Similar low levels of CT-L activity were recorded even 2 weeks after treatment (INH/4 + 14 days). Regarding T-L activity, we observed a slight increase in cultures being left to recover for 2 weeks after treatment (INH/4 + 14 days). As expected, PGPH activity was not immediately affected, because epoxomicin does not primarily block this activity, but as cultures progressed into an irreversible growth arrest state it was eventually reduced to low levels. Neither the expression of biochemical markers of senescence nor the three major proteasomal activities showed any significant changes between CON/4 + 0 days and CON/4 + 14 days.
We next addressed the issue whether the inhibition of the proteasome in
epoxomicin-treated cells could result in changes in the expression levels of
proteasome subunits, by triggering a possible autoregulatory mechanism.
Therefore, we examined the protein expression levels of representative
subunits of the 20 S proteasome (2 and
5
catalytic subunits) as well as of the 19 S regulatory complex (S6b), as these
subunits were found to be down-regulated during replicative senescence
(Fig. 1C). No
significant differences in the expression levels of these subunits between the
control cultures (CON/4 + 0 days) and the epoxomicin-treated cells (INH/4 + 0,
4 + 7, 4 + 14 days) were observed (Fig.
4B).
Finally, to further investigate the morphology of early, late passage, and
epoxomicin-treated early passage cells (INH/4 + 14 days) as well as the
distribution of the proteasome in these cells, we immunolocalized
representative proteasome subunits. Examples for 6 and
7 subunits are shown in
Fig. 4C. In accordance
with previously reported data, epoxomicin-treated early passage cells
exhibited identical morphology to the late passage cells. In addition, in all
cases antigens were mainly found to be distributed in the nucleoplasm, whereas
the nucleolus appeared free of these proteasome subunits. A dispersed
punctuate pattern was also evident in the cytoplasm. However, as the earlier
presented immunoblot analysis of gel filtration fractions (see
Fig. 2, D and
E) indicates the possible presence of free
6 and
7 subunits in late passage cells, it
is not feasible to draw additional conclusions regarding either quantitative
changes or differences in intracellular distribution of assembled proteasome
in these cells.
Overexpression of 1 and
5 Catalytic Subunits Leads to Increased
Proteasomal Activities and to Increased Capacity of the Clones to Cope Better
with StressAs
1 and
5 subunits
were found to be down-regulated in late passage cells and, in addition, they
constitute the catalytic centers of PGPH and CT-L activities, respectively, we
decided to overexpress these two subunits in mammalian cells to check whether
the proteasome can be "activated." We have decided not to employ
primary WI38 fibroblasts for these experiments for several reasons. Retroviral
infections in primary cells is the regular practice to test whether the
studied gene immortalizes, extends lifespan, or inhibits cellular
proliferation. Thus far, only infections of few nuclear oncogenes
(immortalize), telomerase (extends lifespan), and some onco-suppressors
(inhibit proliferation) have given these phenotypes
(40). We have no reason to
believe that overexpression of a single proteasome subunit will immortalize,
extend lifespan, or inhibit cellular proliferation in a similar manner to
oncogenes, telomerase, or onco-suppressors, respectively. Instead, we aimed at
addressing the question whether overexpression of a proteasome subunit results
in proteasome activation and, in turn, in increased capacity of transfected
cells to cope with stress. This particular issue cannot be addressed either,
by using primary cells because by the time that is needed to select stable
clones, infectants have reduced proliferative capacity as they are near
senescence and therefore any follow up survival assay is not feasible. In
contrast established cell lines, by not suffering from limited proliferative
capacity, offer a considerable advantage for performing survival assays.
Therefore we have used such an established WI38 cell line, namely WI38/T
cells. First we have compared the sensitivity of the two cell lines. As shown
in Fig. 5 (top panel,
CON) WI38/T cells are
5 times more apoptosis prone than WI38 cells
(because of the introduction of SV40 T Ag, reviewed in Ref.
41), as it was assayed in
normal growing conditions by an ELISA cell death detection method that
measures apoptosis. Treatment of both cell lines with 100 nM
epoxomicin, 10 µM MG132, or 300 µM
H2O2 for 2.5 h resulted in massive cell death in WI38/T
cells in contrast to WI38 cells that were, in these conditions, relatively
resistant (Fig. 5, lower
panel). Furthermore, the number of WI38/T cells that survived treatment
and proliferated as assayed by clonogenic assays (for conditions see
"Experimental Procedures") were significantly lower than the
corresponding number of WI38-treated cells (data not shown).
Having established that WI38/T cells are more responsive to a variety of
cytotoxic agents than the corresponding primary cells we have transfected
constructs overexpressing the 1 and
5
proteasome subunits in these cells. Several stable clones were isolated and
were propagated to cell lines. Those cell lines exhibited similar growth rates
and morphological characteristics with the parental cell lines even after
several months of cultivation. Four representative WI38/T clones,
1.8,
1.13,
5.8, and
5.19, were chosen for further analysis. These clones
exhibited a moderate overexpression of
1 and
5 subunits (see below), thus resembling the differences of
expression levels observed between early and late passage cells (see
Fig. 1C). As shown in
Fig. 6A, for
1.8 and
5.19 these stable clones were found
to overexpress
1 subunit 35-fold and
5 subunit 23-fold. Moreover, and in agreement with
previously published similar studies, the overexpression of the
5 subunit resulted to the overexpression of
1 subunit
(31); however, the inverse
phenomenon was also observed, i.e.
1 transfectants
exhibited elevated levels of
5 subunit.
|
All of the 1 and
5 stable clones tested
exhibited enhanced levels of CT-L and PGPH activities as compared with
vector-transfected cell lines (2.02.3 fold for CT-L and
1.42.3-fold for PGPH; see Fig.
6B). Transfection with either vector alone (pBJ1-neo and
pREP7-hygro) had no effect on proteasome activity. These data demonstrate that
increased levels of
1 and
5 subunits cause
an increase in proteasome activity. Therefore we tested the survival ability
of our clones when subjected to different stresses. First, WI38/T cells
transfected with
1,
5, and each vector were
exposed to high concentrations of epoxomicin (100 nM) and MG132 (10
µM) for 2.5 h. Their survival capacity was then measured (by
determining colony formation and number of treated cells) after a recovery
period of 10 days (Fig. 7, A and
B, and Table
I). All of the clones exhibited higher survival rates as compared
with the appropriate vector-transfected cell line. Specifically,
1.8 and
1.13 overexpressing cell lines
exhibit 2.8- and 1.5-fold (for epoxomicin) and 6.7- and 2.8-fold (for MG132)
higher proliferating ability as compared with control cells. Similarly,
5.8 and
5.19 overexpressing cell lines
exhibited 3- (for epoxomicin) and 5-fold (for MG132) higher proliferating
ability compared with control cells, respectively. When the survival rates of
the stable transfectants were calculated depending on their ability to form
clones (clonogenic assays) similar results were obtained: cell lines that
overexpress
1 subunit exhibit 1.31.7 (for epoxomicin)
and 1.82.3-fold (for MG132) higher survival rates than control cells
and cell lines that overexpress
5 subunit exhibit
1.82.0-fold (for epoxomicin) and 2.22.4-fold (for MG132) higher
survival rates than control cells (Table
I).
|
As the proteasome is in charge of proteolysis of oxidized proteins we
treated our cultures once with sublethal doses of H2O2
(300 µM) for 2.5 h and their survival capacity was then measured
as above (Fig. 7C).
Again, all 1- and
5-transfected cell lines
exhibited higher proliferating ability compared with vector-transfected cell
lines ranging from 2.3-fold for
1.8 and
1.13
to 4.5-fold for
5.8 and
5.19. When
clonogenic assays were performed, cell lines that overexpress
1 subunit exhibit 2.13.5-fold higher survival rates
compared with the vector-transfected cells. Similarly, cell lines that
overexpress
5 subunit exhibit 3.13.5-fold higher
survival rates as compared with the control cells
(Table I). When our cultures
were treated once with a lower sublethal dose of H2O2
(100 µM), the results were similar (data not shown). Finally the
status of oxidized proteins was determined in
1 and
5 clones following treatment with 300 µM
H2O2. As shown in
Fig. 7D all tested
transfectants exhibited lower amounts of oxidized proteins than vector
transfectants after a recovery period from stress of 24 h. Thus we conclude
that the differences in the proteolytic activities we observed in
1 and
5 overexpressing cell lines can be
translated to functional differences of the proteasome because transfectants
exhibit an increased capacity to cope better with various stresses.
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DISCUSSION |
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A number of studies have demonstrated that impairment of proteasome function is associated with cellular senescence; however, the available data are fragmented and contradictory (1521, 42). To understand the involvement of the proteasome, we have taken a detailed molecular and biochemical approach of WI38 fibroblasts undergoing replicative senescence. It is clear that all three examined activities of the proteasome are reduced in senescent cells. Accordingly, we have observed increased amounts of both oxidized and ubiquitinated proteins in these cells. Therefore we believe that the observed accumulation of oxidized proteins reflects both their increased rate of production as cells progress into senescence as well as the decreased ability of the proteasome to degrade them efficiently. Similarly, as the 26 S complex of the proteasome has impaired proteolytic activity in senescence, the increased levels of ubiquitinated proteins may simply reflect queuing of "labeled for degradation proteins" derived from a functional ubiquitination system. In support of this, Taylor's group (43) has shown that upon oxidative stress and aging in the rat lens, the levels of ubiquitin mRNA, high molecular weight ubiquitin aggregates, and activity of the ubiquitination enzymes, E1 and E2, increase. Another possibility might be of a functional system of ubiquitination in senescent cells, but an impaired system of protein recognition before (i.e. no ubiquitination of the appropriate proteins) or after the ubiquitination (i.e. no entrance of the targeted proteins in the proteasomal cavity). Given the importance of this cellular system, further studies are needed to elucidate the function of the ubiquitination machinery during replicative senescence.
Can the reduced proteasomal activities of senescent fibroblasts be related
to decreased proteasome content in these cells? Several lines of evidence
support this hypothesis. The radiolabeled two-dimensional gel electrophoresis
analysis indicates a decreased rate of synthesis of proteasome subunits. The
cold immunoprecipitation/immunoblot analysis proposes the presence of less
assembled proteasome in senescent cells as compared with young ones. The gel
filtration data indicate the existence of reduced 26 S and 20 S proteasome
complexes in these cells. In agreement with these findings, the immunoblot
analysis has shown that the protein expression levels of the catalytic
subunits of the 20 S complex and ATPases of the 19 S regulatory complex to be
down-regulated in senescence. Interestingly, we did not observe a change in
the expression levels of other subunits, for example, some -subunits of
the 20 S complex that can accumulate in lower molecular weight forms in late
passage cells. The work of Keller et al.
(42) provides strong
supporting evidence as it reports differences of both
- and
-20 S
proteasome subunits during the aging process of the spinal cord. In addition,
Lee et al. (25),
using oligonucleotide array techniques for gene expression profiling, have
found that the expression of some but not all proteasome subunits decline with
aging in the skeletal muscle of mice, including
2 and S6a
subunits. Ly et al.
(24), using a similar
experimental approach, found age-related alterations in expression of several
proteasome subunits in human dermal fibroblasts. This intriguing observation
may suggest the possibility of a rate-limiting expression of proteasome
subunit(s) in senescence.
A major finding of this study is that partial inhibition of the proteasome
induces a senescence-like phenotype in early passage human fibroblasts. The
treated young proliferating cells adapted these senescence-like features in a
short period of time as a consequence of perturbation of their proteasomal
pathway. To our knowledge this is the first report that demonstrates that
cells treated constantly with low doses of epoxomicin or high but chronically
limited doses of MG132 induce an irreversible growth arrest state, in contrast
to the known initiation of apoptosis at higher doses
(36). Epoxomicin is a highly
specific and irreversible inhibitor of the proteasome. It covalently modifies
four catalytic subunits of the 20 S complex, namely 5,
5i,
2, and
2i, resulting in
an inhibition of primarily the CT-L and T-L activities and to a lesser extent
the PGPH activity (44). The
observed slight increase of T-L in cultures being left to recover for 2 weeks
after treatment (INH/4 + 14 days; Fig.
4A) could be attributed to a possible activation of other
protease(s) possessing a T-L activity, in accordance with findings by Glas
et al. (45), which
they demonstrate that when proteasome function is lost, another group of
proteases compensate for this loss. MG132 (Z-Leu-Leu-Leu-H) is a leupeptin
analogue. It is a reversible inhibitor of the proteasome and it inhibits
primarily the CT-L activity and to a lesser extent the PGPH activity
(46). Few other reagents are
known to induce a senescence-like phenotype when delivered into primary
proliferating mammalian cells. These include introduction by retroviral
infection of the ras oncogene
(47), treatment with
H2O2
(48), or inhibition of the
phosphatidylinositol 3-kinase pathway
(49). However, irreversibly
growth-arrested cells because of the effect of these reagents do not exhibit
all features of "normal replicative senescence." For instance,
H2O2-treated cells have long telomeres
(48) and ras-induced
senescent cells overexpress a set of genes that do not associate with normal
in vitro aging (50).
Similarly, our preliminary analysis indicates that proteasome
inhibitor-treated cells exhibit similar levels of expression of
2,
5, and S6b subunits as compared with
non-treated cells. We were not surprised with this finding as: (a)
there is no evidence that these inhibitors affect the transcription or the
translation of these proteasomal subunits and (b) not every
biochemical feature of replicative senescence should also be a feature of a
senescence-like phenotype being induced by various non-related reagents or
experimental conditions.
We have shown enhanced function of the proteasome after stable
transfections of either 1 or
5 catalytic
subunits. It is worth noting that overexpression of either two subunits
resulted in the overexpression of the other subunit. In accordance with our
data, Gaczynska et al.
(31) have also shown that
5-transfected HeLa cells exhibit
1 elevated
levels. Preliminary results from both two-dimensional gel electrophoresis
analysis and subunit expression levels of
1 and
5 stable cell lines suggest activation of several other
proteasomal subunits.2
These data support the argument of a possible common regulation of these
subunits and, in addition, may also imply the existence of an autoregulatory
mechanism. A common transcriptional regulation of the proteasomal subunits has
been reported in yeast
(5153).
26 of the 32 proteasomal subunit genes have been found to be preceded in their
promoters by proteasome-associated control element. RPN4 has been found to be
the factor that binds on this element and consequently is a transcriptional
activator of these genes (51).
However, no human homologue of RPN4 has been identified thus far. Wojcik and
DeMartino (54) have recently
applied the use of RNAi technology in S2 cells derived from
Drosophila. Different double stranded RNAs were used for several
proteasomal subunits. Each double stranded RNA greatly reduced the mRNA level
of its respective targeted subunit and, moreover, most of them also
significantly increased the mRNA levels of several others, but not all,
non-targeted subunits. However, limited data exist regarding experimentation
in human cells. Davies' work (reviewed in Ref.
4) indirectly implies that few
proteasomal subunits may be regulated in the same way. Seven days of daily
treatment with a
6 antisense oligonucleotide severely
depressed the intracellular levels of several, but not all, proteasome
subunits in both cultured liver epithelial cells and in K562 human
hemetopoitic cells. In conclusion, although one could not rule out the
possibility of a transcriptional co-regulation of proteasome subunits in human
cells, by analogy to RPN4 in yeast, further studies are needed to unravel
fully this intriguing issue.
Our stable clones exhibit elevated levels of proteasome activities that are
accompanied by increased capacity to cope with various stresses. To our
knowledge this is the first report that demonstrates that proteasome
activation by overexpression of one of its catalytic subunits results in
increased cell survival to both proteasome inhibitors and to oxidative stress.
Gaczynska et al. (31)
have also transfected 1 and
5 subunits in
HeLa cells. Their clones have elevated levels of some but not all proteasomal
activities (
1 subunit stimulated PGPH activity without
altering other peptidase activities, whereas
5 subunit
reduced CT-L and T-L activities). The discrepancy regarding the proteasomal
activities in
1- and
5-transfected HeLa and
WI38/T cells is possibly because of the different cellular type used. There
are several cases in the literature where stable overexpression of the same
gene construct has different phenotypes when introduced into different cell
lines. For instance, introduction of the catalytic subunit of telomerase
efficiently extends the lifespan in WI38 cells but it does very poorly in MRC5
cells (55).
Accumulation of abnormal proteins is determined by their rates of
formation, but of equal importance are their rates of hydrolysis and
elimination. Therefore it is reasonable to expect that cells possessing
elevated proteasome activity, like our WI38/T 1 and
5 clones, to confer enhanced protection to oxidants like
H2O2. We have previously studied proteasome activity in
fibroblasts derived from "control" donors of different ages (18 to
80 years old), as well as from healthy centenarians, because these individuals
represent the best model of successful aging (reviewed in Ref.
56). Although we observed a
decreased activity of the proteasome with increasing age, importantly,
analysis of RNA and protein levels of several proteasome subunits,
determination of CT-L and PGPH activities, and levels of oxidized proteins,
has revealed that healthy centenarians have an active proteasome as compared
with control donors (16). Thus
we have concluded that cells possessing a functional proteasome may be
equipped with an extra anti-aging arsenal. In support to our belief, it has
been suggested by Davies and colleagues
(4) that the ability of the
proteasome to degrade oxidized proteins serves as a secondary cellular
antioxidant defense system.
Primary human diploid fibroblasts reach senescence because of both genetic
and stochastic factors. Telomere shortening and changes in expression of genes
involved in cell cycle check-points (e.g. p21 and p16) represent some
examples of the genetic background that underlies senescence. Agents that
induce failure of cellular homeostasis (like oxidants) belong to the even
bigger category of those hundred stochastic factors that are capable of
inducing senescence on top of the genetic background. We believe that the
impaired function of the proteasome during replicative senescence is linked
with both genetic and stochastic factors. The observed down-regulation of the
-catalytic subunits of the 20 S proteasome and subunits of the 19 S
regulatory complex should be related to the genetic background that underlies
senescence, at least in the case of WI38 fibroblasts. Identifying the pathways
of regulation of these subunits in mammalian cells, similarly to the RPN4
pathway in yeast, will be the subject of future studies toward the genetic
manipulation of the function of this multicatalytic enzyme. The decreased
proteasome activity in senescent cells is likely to be due not only to the
down-regulation of specific subunits but also mainly to the accumulation of
oxidized and other damaged forms of proteins. Anti-aging strategies should be
aimed based on the activation of the proteasomal proteolytic capacity or its
maintenance. In any case it is expected that the regulation and the function
of the proteasome will become a key issue in future aging research.
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FOOTNOTES |
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Recipient of a Ph.D. fellowship from the Bodosaki Foundation.
** To whom correspondence should be addressed. Tel.: 30-210-7273756; Fax: 30-210-7273677; E-mail: sgonos{at}eie.gr.
1 The abbreviations used are: CT-L, chymotrypsin-like; ApoJ, apolipoprotein
J/clusterin; E1, ubiquitin activating enzyme; E2, ubiquitin-conjugating
enzyme; LLE-NA, N-Cbz-Leu-Leu-Glu--naphthylamine; LLVY-AMC,
Suc-Leu-Leu-Val-Tyr-7-amido-4-methylcoumarin; LSTR-MCA,
N-tBoc-Leu-Ser-Thr-Arg-7-amido-4-methylcoumarin; MG132,
Z-Leu-Leu-Leu-H; PGPH, peptidylglutamylpeptide hydrolyzing; T-L, trypsin-like;
WI38/T, SV40 T Ag WI38 VA 13 cell line; PBS, phosphate-buffered saline; ELISA,
enzyme-linked immunosorbent assay; CHAPS,
3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; CON, control;
INH, inhibitor.
2 N. Chondrogianni and E. S. Gonos, unpublished data.
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ACKNOWLEDGMENTS |
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REFERENCES |
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