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* Institute of Pathology,
Clinics of Physical Medicine and Rehabilitation, and
Institute of Medical Immunology, Charité, Humboldt University, Berlin, Germany; and
§ Ethel Percy Andrus Gerontology Center and Division of Molecular Biology, the University of Southern California, Los Angeles, California 90089-0191, USA
1Correspondence: Ethel Percy Andrus Gerontology Center, University of Southern California, 3715 McClintock Ave., Room 306, Los Angeles, CA 90089-0191, USA. E-mail: kelvin{at}usc.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: ceroid pigment • protein turnover • lysosome • oxidative stress • free radicals
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and
that cell cycle checkpoint control may be effected by the tumor
suppressors p53 and pRB (2)
. The finding that telomere
shortening can be induced by oxidative stress (3
4
5)
recently connected this area of study with the free radical theory of
aging.
Our understanding of the aging of individual postmitotic cells is much
less advanced, although many theories have been proposed. One such
postulate is that the accumulation of heavily damaged, oxidized, and
cross-linked proteins may contribute to the aging process in
postmitotic cells (6
7
8)
. Numerous studies have identified
a major role for the 20S proteasome in the removal of oxidatively
modified proteins in mammalian cells (9
10
11
12)
, and
proteasome depletion prevents cells from degrading oxidized proteins
(13
, 14)
. Oxidized protein aggregates can inhibit the
proteolytic activity of the proteasome in vitro (15
, 16)
. Therefore, the accumulation of heavily damaged, oxidized,
and aggregated proteins during postmitotic aging may diminish the
effectiveness of proteolytic enzymes.
Lipofuscin and ceroid are fluorescent pigments of aggregated polymers
derived from oxidation products of proteins and lipids, which are
cross-linked by covalent and hydrophobic bonds (17)
.
Lipofuscin and ceroid accumulate during aging, most obviously in
postmitotic cells. A close correlation has been reported between
lipofuscin/ceroid accumulation and aging rate in several mammalian
species despite widely differing maximum life spans (18
, 19)
. Lipofuscin/ceroid accumulation within aging cells might be
due to increased production of reactive oxygen species (7
, 20)
or a decline in the efficiency of protein repair and/or
degradation systems (21
, 22)
. Experimentally,
lipofuscin/ceroid accumulation can be accelerated by increased
oxidative stress (19
, 23)
and by inhibition of lysosomal
proteases and lipases (24)
, conditions that accelerate the
aging process in general (24
25
26)
. Accordingly,
lipofuscin/ceroid accumulation is regarded as one of the best-known
biomarkers of aging (27)
. Since lipofuscin/ceroid is a
biological marker of aging and cross-linked proteins are able to
inhibit proteases, we decided to study the influence of oxidative
stress and artificial lipofuscin/ceroid (4
, 28)
on protein
turnover and proteolytic enzymes in aging postmitotic fibroblasts.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 4)
. This treatment results in an irreversible
senescence-like growth arrest after ~4 wk and shortens the normal
postmitotic life span of 1–2 years down to only about 3 months
(4)
.
Parallel cultures were grown under normoxia and treated with synthetic
lipofuscin/ceroid twice a week for up to 4 wk. Artificial
lipofuscin/ceroid was prepared from liver mitochondria as described
(4
, 28)
. Briefly, a crude liver mitochondrial preparation
(3.0 mg protein/ml) was UV-peroxidized to completion as judged by
return to basal levels of the production of thiobarbituric
acid-reactive materials within the preparation. The preparation was
extensively homogenized to obtain particles of mitochondrial size and
the equivalent of 0.3 mg protein was added to 106
cells. Additional controls were performed with WI-38 cells held under
normoxia for up to 2 months as density-inhibited cultures. Some of
these control cultures were treated with nonfluorescent latex spheres
(1 µm in diameter, PLANO, Marburg, Germany) instead of
lipofuscin/ceroid.
Measurement of lipofuscin/ceroid content
For electron microscopy, 1 x 106
cells were centrifuged at 900 rpm and the pellets were fixed in
glutaraldehyde/osmium tetroxide. Between 25 and 35 cells were
photographed at a final magnification of 5000x using an EM 10 electron
microscope (Zeiss, Oberkochen, Germany) in a systematic random manner.
The fraction of secondary lysosomes within the fibroblast cytoplasm
(excluding the nuclei) was estimated by point counting using a grid
with a 10 mm mesh size. Cellular autofluorescence in the yellow-green
range of the spectrum (563–607 nm), which stems mainly from
lipofuscin/ceroid, was also separately measured by flow cytometry using
a Becton-Dickinson FACScan as described previously (4)
.
Measurement of protein turnover
For measurements of overall protein synthesis and proteolytic
activity, cells were washed twice with phosphate-buffered saline and
incubated for 16 h with 35S-labeled
methionine (35S-Met) in methionine-free medium
(Sigma). After labeling, cells were again washed twice and cultured
further in (methionine-rich) standard medium. Protein synthesis was
determined by calculating the difference between the radioactivity in
the medium before and after 16 h of incubation. Radioactivity was
measured by scintillation counting. For proteolysis measurements,
trichloroacetic acid (TCA) soluble counts were determined as described
previously (13
, 14)
.
Protease activity determinations
The maximal activity of proteolytic systems was analyzed
according to Inubushi et al. (29)
and Grune et al.
(13
, 14)
. Between 0.3 and 1 x
106 cells were lysed in 150 µl of 1 mM
dithiothreitol during vigorous shaking for 1 h at 4°C. The
lysates were immediately used for determination of proteolytic
activities.
Proteasome activity
The lysates were centrifuged for 30 min at 14,000 g.
Supernatants were incubated in 50 mM Tris-HCl buffer (pH 7.8)
containing 20 mM KCl, 0.5 mM MgOAc, and 1 mM dithiothreitol. The
fluorogenic peptide suc-LLVY-MCA was used as a substrate at a final
concentration of 200 µM. After a 1 h incubation at 37°C,
proteolysis was terminated by addition of an equal volume of ice-cold
ethanol. Measurements of proteolysis (release of the MCA fluor) were
performed at 380 nm excitation and 440 emission after addition of 0.125
M sodium borate (pH 9.0) using free MCA (a fluorogenic peptide used to
measure proteasome activity) as standard for quantification.
Activity of lysosomal proteases
Lysates were sonicated for 2 min on ice in a SONOPLUS GM70. The
activity assay was performed by incubation of lysates at 37°C for 30
min in the presence of 200 µM z-FR-MCA (a fluorogenic peptide used to
measure lysosomal cathepsin activity) as substrate. The incubation
buffer consisted of 50 mM sodium acetate (pH 5.5), 8 mM cysteine
hydrochloride, and 1 mM EDTA. The reaction was stopped and measurements
of MCA release were performed, as described for the determination of
proteasome activity.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Under these conditions, proliferation
ceases completely within 4 wk; this phenomenon is referred to as
proliferative senescence (3)
. Most of the postmitotic life
span is completed within the next 2 to 3 months, and extensive cell
death occurs after 12 to 14 wk of hyperoxia (4)
. Protein
turnover was measured in intact WI-38 fibroblasts. Incorporation of
[35S] methionine into newly synthesized
proteins was taken as a measure of protein synthesis. Incorporation of
[35S] into proteins did not change
significantly during 12 wk of fibroblast incubation under normobaric
hyperoxia (Fig. 1A
). Under the same conditions, however, protein degradation
as measured by the liberation of TCA-soluble counts after removal of
unincorporated label underwent a series of important changes (Fig. 1B
). Total protein degradation increased by as much as 30%
after 5 wk of hyperoxia, but thereafter declined to a level 25% lower
than original after 12 wk of incubation under hyperoxic conditions
(Fig. 1B
, inset).
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From the results of Fig. 1
, we concluded that the main changes in
protein turnover during postmitotic aging might be those in protein
catabolism. Therefore, we decided to test the overall capacity of the
main proteolytic systems in WI-38 cells using various fluorogenic
peptide substrates. Degradation of z-FR-MCA under acidic conditions in
the cell lysate gives a good approximation for the activity of
lysosomal cathepsins (29)
, whereas in the centrifuged
(membrane-free) lysate of cells, proteasome represents the main
suc-LLVY-MCA-degrading activity (13
, 14)
. As reported in
Fig. 2
, a decline in the activity of both lysosomal proteases and cytosolic
proteasome occurs during postmitotic aging. Both proteolytic systems
lose more than 70% of their initial activity, indicating a dramatic
loss of the overall capacity of the cell to degrade proteins.
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Hyperoxia causes increased oxygen radical production (30)
and an increased rate of formation of radical-damaged proteins.
Accumulation of lipofuscin/ceroid pigment is a major consequence of
oxidative damage to proteins, which can be followed by the measurement
of cellular yellow-green autofluorescence. As shown in Fig. 3A
, the autofluorescence of WI-38 cells increases continuously
during ‘aging’ under hyperoxic conditions, becoming especially marked
after permanent inhibition of proliferation at around wk 4 of the
treatment. Increased production of damaged proteins, due to increased
formation of reactive oxygen species, may overwhelm the capacity of
proteolytic systems. In addition, the accumulated oxidized proteins may
cause a further decrease in the activity of proteolytic systems
themselves, as discovered in earlier in vitro experiments
(15
, 16)
. Therefore, we decided to directly test whether
exogenous lipofuscin/ceroid-like material, if taken up by cells, can
change protein turnover in WI-38 fibroblasts.
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Confluent WI-38 fibroblasts were cultivated under normoxic conditions
and synthetic lipofuscin/ceroid was added to the cells twice a week.
Uptake and incorporation of exogenous lipofuscin/ceroid was carefully
monitored both by electron microscopic morphometry and flow cytometry
with 563–607 nm autofluorescence. The amount of lipofuscin/ceroid
phagocytosed over time is shown as autofluorescence in Fig. 3B
. A continuous increase in cellular autofluorescence was
found during incubation with lipofuscin/ceroid. After 4 wk of
lipofuscin/ceroid treatment, the level of autofluorescence was
comparable to that of WI-38 cells after 7–10 wk of normobaric
hyperoxia (compare Fig. 3
, panels A and B).
Electron microscopic morphometry of secondary lysosomes confirmed this
result (data not shown). Increases in secondary lysosomes after 2 wk of
lipofuscin/ceroid treatment were similar to those seen after 7 wk of
hyperoxia as measured by morphometry and autofluorescence (Fig. 3C
). Control experiments using latex particles of a size
similar to the lipofuscin/ceroid-like material showed only a slight
increase in autofluorescence (Fig. 3B
, insert), which was
actually equal to the accumulation of lipofuscin/ceroid we observed in
untreated confluent fibroblasts under normoxia (data not shown).
To test the influence of lipofuscin/ceroid accumulation on protein
turnover in WI-38 fibroblasts, we performed
[35S] incorporation experiments with
lipofuscin/ceroid-treated cells. As shown in Fig. 4
, overall protein degradation declined in a time-dependent manner in
cells treated with lipofuscin/ceroid, so that after 4 wk of
lipofuscin/ceroid treatment protein degradation had decreased by
~20%. As measured by [35S] incorporation,
protein synthesis remained unchanged (Fig. 4
, insert). Control
incubations using inert latex particles revealed no influence on
protein turnover. There was, however, a significant influence of
lipofuscin/ceroid on the proteolytic activities of both the lysosomal
and proteasomal systems. As demonstrated in Fig. 5
, the activity of lysosomal cathepsins increased by ~30%, whereas the
cytosolic activity of proteasome declined by 25%. Therefore, despite
the overall decrease in proteolytic specific activities observed in
‘aging’ WI-38 fibroblasts (Fig. 2)
, an increase in the total activity
of lysosomal cathepsins occurs. It will also be noted that both
proteasome and lysosomal cathepsin-specific activities decreased in the
normobaric hyperoxia studies of Fig. 2
, whereas lysosomal
cathepsins actually increased after external treatment with
lipofuscin/ceroid. This apparent contradiction is, in fact, explained
by an increase in secondary lysosomes induced by artificial
lipofuscin/ceroid in WI-38 cells (see Fig. 6
, and Fig. 3C
).
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To determine whether the changes in proteasome activity, overall
proteolysis, and lipofuscin/ceroid accumulation reported in Figs. 1
2
3
4
5
6
were temporally related, we performed studies of hyperoxia-treatment
and lipofuscin/ceroid treatment in WI-38 cells in which all data was
collected on the same time scale. As shown in Fig. 7A
, hyperoxia caused an initial increase in overall protein
degradation until the point (5 wk) when lipofuscin/ceroid began to
accumulate. From wk 5 through wk 12, lipofuscin/ceroid accumulated
whereas proteasome activity and overall proteolysis declined.
Lipofuscin/ceroid-treatment of WI-38 fibroblasts caused a gradual
lipofuscin/ceroid accumulation and a similarly gradual decline in both
proteasome activity and overall protein degradation over a 4 wk period
(Fig. 7B
). The data of Fig. 7
reveal a rather tight negative
correlation between cellular accumulation of lipofuscin/ceroid (whether
added directly or induced by hyperoxia), proteasome inactivation, and
loss of intracellular proteolysis.
|
To test whether or not the inverse relationship between
lipofuscin/ceroid accumulation and proteasomeactivity/overall
proteolysis seen in Fig. 7
and the divergence of lysosomal and
proteasomal activities during lipofuscin/ceroid treatment seen in Fig. 5
are limited to the WI-38 cell line, we next examined three other
nontransformed fibroblast cell lines. In all these cell lines,
lipofuscin/ceroid treatment caused lipofuscin/ceroid accumulation
(Fig. 8D
) and gradually and significantly decreased overall protein degradation
over a 4 wk treatment period (Fig. 8A
). This was accompanied
by a decline in the activity of proteasome (Fig. 8B
),
whereas lysosomal cathepsin activity actually increased significantly
in all cell lines tested (Fig. 8C
).
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Since our results indicated that lipofuscin/ceroid might inhibit the
proteasome during aging, we decided to directly test the ability of
lipofuscin/ceroid to inhibit proteasome in vitro. Using
purified proteasome and our artificial lipofuscin/ceroid preparation,
we indeed found a clear, dose-dependent inhibition of proteasome
activity (Fig. 9
). These data clearly demonstrate the strong, direct inhibitory effect
of lipofuscin/ceroid on the proteasome.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, considerable evidence has been presented to support
the involvement of free radicals in the aging process. One example is
the demonstration of an accumulation of oxidized proteins during aging
(32)
. The original free radical theory of aging
concentrated on increased oxidant production and decreased primary
antioxidant enzyme protection, but the discovery of damage removal and
repair systems (or secondary antioxidant systems) has led to a
reevaluation of the theory and new proposals for declining damage
removal capacity (33)
. Indeed, age-associated accumulation
of oxidized proteins may well be the result of a decline in the
proteolytic activity of damage removal systems (16)
.
Decreased proteolytic activities have been reported in aging brains
(32)
. Additional studies using protease inhibitors like
leupeptin or E-64 (24
, 25)
have indicated a key role for
proteases in the aging process. Previous studies focused on the role of
lysosomal proteases in aging, but it is known that the proteasome is
mostly responsible for the degradation of oxidized cytosolic proteins
(9
10
11
12)
. Several groups have demonstrated a role of the
proteasome in the selective recognition (34
, 35)
and
degradation of oxidized proteins (9
10
11
12
13
14
15
16
, 21
, 22)
. The
proteasomal capacity of cells is high, and the removal of oxidized
proteins depends largely on substrate supply (13
, 14)
. The
present data indicate an initial increase in overall protein
degradation in cells exposed to hyperoxia, i.e., to an increased oxygen
radical flux. Such an increase has previously been demonstrated using
other radical-generating systems (13
, 14)
. After 5 wk of
hyperoxia, however, a progressive decline in protein degradation
occurred in WI-38 cells, reflecting the decreased activity of
proteolytic enzymes.
In this study, lipofuscin/ceroid accumulation during hyperoxia-induced
cellular ‘aging’ was correlated with decreased overall protein
degradation as well as with decreased proteasome activity and lysosomal
cathepsin activity. In cells forced to accumulate artificial
lipofuscin/ceroid under normoxic conditions, overall protein
degradation again decreased, as did the activity of proteasome. An
apparent increase in lysosomal cathepsin activity in cells treated with
artificial lipofuscin/ceroid was actually caused by an increase in the
number of secondary lysosomes. These results are in accord with a major
role for the proteasome in normal protein turnover, as proposed
previously (36)
. Inhibition of proteasome by cross-linked
proteins was previously demonstrated only in in vitro
systems, and now we present evidence that this process also occurs in
living cells. Accumulation of lipofuscin/ceroid and cross-linked
proteins during aging may be a self-accelerating process, leading to a
gradual decline in cellular proteolytic activities. In retinal
pigmented epithelial cells, it has been reported that lipofuscin/ceroid
might contain lysosome-disrupting cationic detergents, which can
inhibit proteolysis (37
, 38)
.
One should consider that the activity of proteolytic systems measured
using only artificial substrates as performed by others (39
, 40)
provides only limited insight into the real protein turnover
of living cells. From the present results, combining both artificial
substrates and overall intracellular proteolysis, we conclude that
oxidized cross-linked proteins and lipofuscin/ceroid are possible
inhibitors of the proteasome during postmitotic aging. The proteasome
seems to be an important proteolytic system underlying age-dependent
changes.
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ACKNOWLEDGMENTS |
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Received for publication September 17, 1999. Accepted for publication December 7, 1999.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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