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1,
* Clinics of Physical Medicine and Rehabilitation and
Institute of Pathology, Medical Faculty (Charité), Humboldt University Berlin, D-10098 Berlin, Germany; and
Ethel Percy Andrus Gerontology Center, and Division of Molecular Biology, 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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, however, the present
study of hyperoxic senescence in nondividing cells actually revealed
marked increases in lysosomal cathepsin activity in all but the very
‘oldest’ postmitotic cells. Our comparative studies of proliferating
(1)
and nonproliferating (this paper) human BJ fibroblasts
reveal a good correlation between the accumulation of
oxidized/cross-linked proteins and the decline in proteasome activity
and overall cellular protein turnover during in vitro
senescence, which may predict a causal relationship during actual
cellular aging.—Sitte, N., Merker, K., von Zglinicki, T., Davies,
K. J. A., Grune, T. Protein oxidation and degradation during
cellular senescence of human BJ fibroblasts: part II—aging of
nondividing cells.
Key Words: aging • cross-linked proteins • lysosomes • proteasome • protein oxidation • proteolysis • protein turnover • senescence
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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3
4
5
6
7
8
9
10)
. Fluorescent age pigments are also
called lipofuscin, ceroid, or ceroid-like material
(2
3
4
5
6
7
8
9
10)
. Although accumulation seems to occur
preferentially in postmitotic cells (2
3
4
5
6
7
8
9
10)
, these
pigments are also found in hepatocytes, a cell type that undergoes
active mitosis (11)
. Two principal explanations have been
proposed for the increase in lipofuscin accumulation with age. The
first focuses on the theory that increased lipofuscin is caused by an
age-related enhancement of autophagocytosis, a decrease in
intralysosomal degradation, and/or a decline in exocytosis
(2)
. The second explanation is based on ineffective
degradation of oxidized/cross-linked proteins, which then aggregate and
promote autophagocytosis in postmitotic cells (12)
.
Over the past few years we and others have published a series of papers
outlining the relationship between protein oxidation and proteolysis by
the proteasome in postmitotic cells (13
14
15
16
17
18
19
20
21
22
23)
. These
studies were performed in erythrocytes and reticulocytes and in both
cardiac muscle and skeletal muscles in vitro. More detailed
studies on the importance of the proteasome for the intracellular
degradation of oxidized proteins were performed in dividing cell lines
(21
22
23
24
25
26)
. All of these studies concluded that proteins are
inherently susceptible to oxidative damage, which alters their
proteolytic susceptibility. It was further established that low-level
oxidative damage greatly increases proteolytic susceptibility, whereas
extensive oxidative damage causes decreased proteolysis due to
aggregation and cross-linking of the substrate proteins (12
, 27
28
29)
. Extensive oxidation and cross-linking seem to be key
steps in the formation of fluorescent age pigments, and it is clear
that oxidized proteins are located within these pigments (5
, 10)
. Further modification and oxidation processes are also
involved in the formation of these age-linked fluorophores, such as
modification by lipid peroxidation products and by glycation (2
, 8)
.
In the companion paper (1)
, we report that human BJ
fibroblasts undergo a clear decline in proteasome activity during
proliferative senescence and exhibit a diminishing responsiveness of
the proteasome to acute oxidative stress. These processes are
accompanied by an increased accumulation of oxidized proteins. We
proposed that these processes are interactive and mutually propagating,
so that a constant minor accumulation of a small number of
oxidized/cross-linked protein molecules occurs throughout proliferative
life, because some oxidized proteins will always ‘escape’ the
proteasome. Eventually the cellular concentrations of these
accumulating protein oxidation products may reach a level that causes a
generalized inhibition of the proteasome, because they bind but cannot
be degraded. The consequent decrease in effective cellular proteasome
activity may then cause a more rapidly diminishing ability to degrade
oxidized proteins and, therefore, accumulation of protein oxidation
products occurs more rapidly during the latter stages of proliferative
senescence. The accompanying studies (1)
may provide a
good initial model for the accumulation of oxidized proteins in
proliferative tissues such as the lung and liver, and mitotic cells
such as glia and astrocytes. In the present paper we have tested these
ideas in postmitotic and confluent quiescent or nondividing BJ
fibroblasts, which should better model the senescence phenotype in
postmitotic tissues such as brain, cardiac muscle, and skeletal muscle.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 33)
. After 0, 4, 8, 12, 16,
and 20 wk of hyperoxia, various parameters of protein turnover and
protein oxidation were investigated. Cell numbers were counted at each
of these times in each experiment to determine the loss of living cells
under hyperoxic conditions.
Protease activity determinations
The maximal activities of lysosomal cathepsins and the
proteasome were analyzed, respectively, according Inubushi et al.
(30)
and Grune et al. (35)
. Between 0.3 and
1.6 x 106 cells were washed twice with
phosphate-buffered saline and then subjected to hypotonic lysis 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
Nonlysed cells, membranes, and nuclei were removed by
centrifugation at 14,000 g for 30 min. Supernatants were
incubated in a buffer consisting of 50 mM Tris-HCl (pH 7.8), 20 mM KCl,
0.5 mM Mg-acetate, and 1 mM dithiothreitol. After 1 h incubation
with 200 µM of one of the fluorogenic peptides (suc-LLVY-MCA for the
chymotrypsin-like activity, z-PFR-MCA for the trypsin-like activity,
and z-LLE-ßNA for the peptidyl-glutamyl-hydrolyzing activity),
hydrolysis was stopped by addition of an equal volume of ice-cold
ethanol and by further dilution with 0.125 M sodium borate (pH 9.0).
Fluorescence products were monitored at 380 nm excitation and 440 nm
emission for MCA or 335 nm excitation and 410 nm emission for ßNA,
using free MCA or ßNA, respectively, as standards.
Activity of lysosomal cathepsins
Lysates were sonicated for 2 min on ice in a SONOPLUS GM70. The
proteolytic activity assay was performed by incubation of lysates at
37°C for 30 min in a buffer containing 50 mM sodium acetate (pH 5.5),
8 mM cysteine hydrochloride, and 1 mM EDTA in the presence of 200 µM
z-FR-MCA as fluorogenic peptide substrate. The reaction was terminated
by addition of an equal volume of ice-cold ethanol, and measurements of
MCA release were performed as described above to determine proteasome
activity.
Protein carbonyl measurement
Protein carbonyl content was determined in cell lysates (4
mg/ml) by the ELISA of Buss et al. (31)
with modifications
by Sitte et al. (34)
. The detection system used was an
anti-dinitrophenyl rabbit IgG-antiserum (Sigma, Deisenhofen, Germany)
as primary antibody and a monoclonal anti-rabbit IgG antibody
peroxidase conjugate (Sigma) as secondary antibody. Development was
performed with o-phenylenediamine.
Oxidized and cross-linked proteins
Oxidized/cross-linked proteins (lipofuscin-like or
ceroid-like material) in samples of 
3 x
105 cells were determined by measuring the
cellular autofluorescence in the yellow-green range of the spectrum
(563–607 nm) by flow cytometry using a BECTON-DICKINSON FACScan as
described previously (33)
.
Immunoblots
After equalizing the protein content of cell lysates or
centrifuged cell lysates (14,000 g for 30 min), proteasome
subunits were analyzed by sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE) under reducing conditions, performed
according to Laemmli et al. (34)
. For analysis of
protein-bound carbonyls, samples were modified with
2,4-dinitrophenylhydrazine prior to SDS-PAGE, as described by Levine et
al. (35)
. Proteins were transferred from SDS-PAGE gels to
nitrocellulose membranes using a Bio-Rad Trans-Blot apparatus and
incubated with either an anti-rabbit proteasome antibody (Affinity,
Exeter, U.K.) or an anti-rabbit DNP antibody (Sigma). The secondary
antibody was an anti-rabbit IgG peroxidase conjugate, which was
detected by chemiluminescence using the ECL assay (Amersham, Little
Chalfont, U.K.).
Measurement of overall proteolysis
The degradation of metabolically radiolabeled proteins in
confluent fibroblasts was measured after a 20 h labeling procedure
(25)
. During labeling, cells were incubated with
[35S]-methionine in methionine-free minimal
essential medium. After 20 h of incubation at 37°C, all
nonincorporated radiolabel was removed and the cells were washed twice
with phosphate-buffered saline. Afterward, complete tissue culture
medium (containing ‘cold’ methionine) was added and the liberation of
acid-soluble radioactivity was followed. The degradation of
metabolically radiolabeled proteins was quantified, after addition of
an equal volume of 20% trichloroacetic acid, by liquid scintillation
counting of the acid-soluble counts in the supernatant after
centrifugation at 14,000 g for 10 min.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) with aging of nondividing cells, we
cultivated confluent BJ fibroblasts of different proliferation stages
under hyperoxic conditions for 20 wk (Fig. 1A
). Under the experimental conditions of hyperoxia, cell
cultures of different proliferation stages lose living cells (Fig. 1B
). As expected in cell cultures of the latest
proliferation stage, cells die off continuously from the beginning of
the experiment whereas cell cultures of younger proliferation stages do
not lose cells before 12 or 16 wk of hyperoxia (Fig. 1B
).
Since all our biochemical investigations were performed only with the
remaining (attached) cells, we determined the viability of this cell
fraction. The determination of viability using trypan blue exclusion
demonstrated that in all harvested cell suspensions, 98% of cells were
fully viable. Figure 1
also reveals an important difference between
cells of PD 46 or 62 and those of PD 74. At PD 74 the cells could just
be said to have become truly postmitotic (Fig. 1A
), and
these were the only cell populations to show no lag phase in their
response to hyperoxia (Fig. 1B
). In contrast, the confluent
and quiescent (but still capable of mitosis) PD 46 and PD 62 cultures
exhibited much greater resistance to the senescence-accelerating
effects of hyperoxia (Fig. 1B
).
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Protein turnover
Overall intracellular proteolysis declined significantly
after 4 wk of hyperoxia in all cells tested (Fig. 2
). Overall intracellular protein degradation decreased to about the same
level after 20 wk of hyperoxia in cells of all proliferation stages
(Fig. 2)
. We next tested whether the declining overall proteolysis seen
in Fig. 2
could be explained by diminishing proteasome activities or by
decreasing lysosomal proteolysis. As in our studies of proliferative
senescence (please see the accompanying paper, ref 1
), we
used different fluorogenic peptide substrates to determine the maximal
activities of these proteolytic systems. All three proteasome peptidase
activities as well as lysosomal cathepsin activity declined
significantly as a function of proliferation stage (Fig. 3A
, B
).
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Similar to our results on proliferative senescence (1)
, we
again found that all three of the proteasome peptidase activities
declined during the aging of nondividing BJ fibroblasts (Fig. 3A
). In stark contrast, the activity of lysosomal cathepsins
either increased or remained essentially unchanged (Fig. 3B
). Quantitatively, the cells lost 50–80% of their
chymotrypsin-like and trypsin-like proteasome activities, and as much
as 100% of their peptidyl-glutamyl-hydrolyzing activity during 20 wk
of hyperoxia-accelerated senescence (Fig. 3A
). In every
case, proteasome activity losses were greater in the postmitotic cells
(PD 74) than in the confluent quiescent cells of PD 46 or 62 (Fig. 3A
). One possible explanation for these results could be a
loss of actual proteasome protein during senescence. To test this
possibility, we used a polyclonal anti-proteasome antibody to test for
relative levels of proteasome in cells of different PD. As shown in
Fig. 4
; however, there were no significant losses of proteasome subunits due
to either proliferation stage or hyperoxic senescence.
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Accumulation of oxidized and cross-linked proteins during aging of
nondividing fibroblasts
Since it we have proposed that loss of proteasome activity should
cause an accumulation of oxidized proteins during the cellular aging
process (1)
, we wanted to test whether declining
proteasome activity was also accompanied by an increased accumulation
of oxidized proteins during aging under hyperoxic conditions. As
demonstrated in Fig. 5A
, cellular protein carbonyl content increased significantly
during hyperoxic aging. This increase in protein carbonyls seems to
involve a large number of proteins of various molecular size (Fig. 5B
). An even more dramatic increase in oxidized/cross-linked
protein accumulation was revealed by autofluorescence studies. As shown
in Fig. 6
, oxidized/cross-linked proteins increased during hyperoxic aging of
nondividing cells, particularly during the final 5 wk of hyperoxia.
This increase is much more dramatic than that seen during proliferative
senescence (1)
.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Several authors have reported decreased proteasome activities during
aging of various animals (36
37
38
39)
, although not all
authors have obtained the same results (40
41
42)
. Of
particular interest, Conconi et al. reported an age-related decline of
the peptidyl-glutamyl hydrolyzing activity in rat liver
(37
38
39)
. These data seem to be consistent with our
finding that the peptidyl-glutamyl-hydrolyzing activity exhibits the
most dramatic changes of all proteolytic activities investigated.
Since the proposal of the free radical theory of aging by Harman
(43)
, much evidence has accumulated to support the
involvement of oxidative stress in the aging process. An increased
accumulation of oxidized proteins has been reported in flies and in
mammalian brain tissue, liver, and lenses (36
, 44
45
46
47
48)
.
However, when using these models it is impossible to judge the effects
of proliferative aging vs. the senescence of quiescent or postmitotic,
nondividing, cells. This limitation may be more important than is
immediately apparent, since even cells that are capable of mitosis may
spend significant periods of time in a quiescent state before
continuing to divide actively again and finally becoming postmitotic.
Although a mammalian brain contains postmitotic neurons, it also has
astrocytes and glia that can actively divide. Similarly, the liver
contains numerous subpopulations of hepatocytes, some of which are
undergoing active mitosis, some are quiescent, and some are postmitotic
but still alive. In Table 1
we have made a comparison of the relative changes that occur during
proliferative senescence (from the accompanying paper, ref
1
) and the hyperoxic aging of nondividing cells examined
in this paper. are summarized. Table 1
reveals the most interesting
aspects of these studies, which has only been made possible by
simultaneously studying different modes of in vitro
senescence in the same cell line, under, otherwise, identical
conditions.
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The chymotrypsin-like and the trypsin-like activities of the proteasome
were essentially equally affected during senescence (Table 1)
. The loss
of 60–70% of the initial activity during proliferative senescence
is comparable to the 50–60% loss during aging under hyperoxic
conditions. The peptidyl-glutamyl-hydrolyzing activity was much more
affected during proliferative senescence (80% loss), and under
hyperoxic conditions it actually declined to zero. From Table 1
it can
readily be seen that aging under both proliferative conditions and in
nondividing cells involves a loss of proteasomal activity.
As shown in Table 1
, protein carbonyl concentration as a measure for
oxidized proteins increased by 50% during proliferative senescence,
and was somewhat further increased during the senescence of nondividing
cells. Only a moderate increase of cellular autofluorescence occurred
during proliferative senescence (50% increase), whereas a large
accumulation of fluorescent material was evident under nondividing
conditions. These data are certainly consistent with the idea that
proteins become permanently oxidized and tend to aggregate and form
insoluble particles, which accumulate in aging cells.
Besides the activity of the proteasome system, we also used fluorogenic
peptides to measure lysosomal cathepsin activity, resulting in some of
the most interesting results of our studies. As shown in Table 1
,
lysosomal cathepsin activity declined by 60% during proliferative
senescence, whereas the changes during the aging of nondividing cells
were much more complex. During the hyperoxic aging of nearly
postmitotic cells (PD 74), lysosomal cathepsin activity initially
increased and then declined (see Fig. 3B
and Table 1
).
During the hyperoxic aging of younger (PD 46 and PD 62.1) but still
quiescent (nondividing) cells, lysosomal cathepsin activity in
proteases increased markedly (Fig. 3B
and Table 1
). This
might be an important protective mechanism, since fluorescent
oxidized/cross-linked materials formed in the cytoplasm actually seem
to be accumulated within lysosomes. Although cells of lower PD seem to
be able to produce functionally intact lysosomes with functioning
cathepsins in response to hyperpoxic senescence, postmitotic cells seem
less able to maintain appropriate lysosomal function. Perhaps most
important for this study, it is quite clear from the results of Table 1
that the senescence-associated intracellular accumulation of oxidized
and cross-linked proteins correlates quite well with declining
proteasome activity in each of our senescence models, whereas there is
no apparent consistent relationship with lysosomal proteolytic
activity.
Declining proteasome activity was also accompanied by a decreased
overall protein turnover, which is in good agreement with the data of
Rock et al. (49)
, which indicate that proteasome
responsible for the degradation of short-lived cellular proteins. The
role of proteasome in the degradation of oxidized proteins has been
demonstrated by many studies from our laboratory and others
(12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29)
. Recently we demonstrated that the proteasome is
responsible for the degradation of oxidized proteins in mammalian cells
(12
, 21
, 24
25
26)
. Since the proteasome exists in an
ATP-stimulated and an ATP-independent form, it is important to note
that, in in vitro systems, the ATP-independent form is able
to degrade oxidized proteins preferentially (12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29)
.
Whether the removal of oxidized proteins in living cells is due to the
activity of the ATP-stimulated or the ATP-independent form is still
under discussion (12
, 50
51
52)
. Since oxidation causes some
rearrangement of protein structures, combined with an exposure of
hydrophobic domains to the surface, it was suggested that
hydrophobicity is the ‘marker’ for degradation by the proteasome
(23
, 53)
. On the other hand, it was demonstrated that
oxidized proteins tend to aggregate (12)
if they are not
degraded. Aggregated and cross-linked proteins are able to inhibit the
proteasome (27
28
29)
. Since these aggregation products
accumulate during the aging of fibroblasts, as shown by accumulation of
oxidized/cross-linked material in this study, it is possible that the
proteasome is inhibited by such aggregates. That proteasome
inactivation is a posttranslational process is supported by a constant
proteasome content as determined by Western blotting. One possible
mechanism may be the inhibition of the proteasome by accumulating
oxidized/cross-linked proteins as discussed by us and others (12
, 27
, 29)
. Therefore, it seems possible that proteasome inhibition
by accumulating protein oxidation products further inhibits the
degradation of cellular proteins in a gradually worsening ‘vicious
aging spiral.’
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ACKNOWLEDGMENTS |
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Received for publication March 28, 2000. Accepted for publication June 6, 2000.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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This article has been cited by other articles:
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  S. MARTIEN and C. ABBADIE Acquisition of Oxidative DNA Damage during Senescence: The First Step toward Carcinogenesis? Ann. N.Y. Acad. Sci., November 1, 2007; 1119(1): 51 - 63. [Abstract] [Full Text] [PDF]
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 O. Zolk, C. Schenke, and A. Sarikas The ubiquitin-proteasome system: Focus on the heart Cardiovasc Res, June 1, 2006; 70(3): 410 - 421. [Abstract] [Full Text] [PDF]
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 K. J.A. Davies and R. Shringarpure Preferential degradation of oxidized proteins by the 20S proteasome may be inhibited in aging and in inflammatory neuromuscular diseases Neurology, January 24, 2006; 66(1_suppl_1): S93 - S96. [Abstract] [Full Text] [PDF]
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 N. Chondrogianni, C. Tzavelas, A. J. Pemberton, I. P. Nezis, A. J. Rivett, and E. S. Gonos Overexpression of Proteasome {beta}5 Assembled Subunit Increases the Amount of Proteasome and Confers Ameliorated Response to Oxidative Stress and Higher Survival Rates J. Biol. Chem., March 25, 2005; 280(12): 11840 - 11850. [Abstract] [Full Text] [PDF]
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 D. A. Gray and J. Woulfe Lipofuscin and Aging: A Matter of Toxic Waste Sci. Aging Knowl. Environ., February 2, 2005; 2005(5): re1 - re1. [Abstract] [Full Text] [PDF]
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 R. Kiffin, C. Christian, E. Knecht, and A. M. Cuervo Activation of Chaperone-mediated Autophagy during Oxidative Stress Mol. Biol. Cell, November 1, 2004; 15(11): 4829 - 4840. [Abstract] [Full Text] [PDF]
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 S. Judge, A. Judge, T. Grune, and C. Leeuwenburgh Short-term CR decreases cardiac mitochondrial oxidant production but increases carbonyl content Am J Physiol Regulatory Integrative Comp Physiol, February 1, 2004; 286(2): R254 - R259. [Abstract] [Full Text] [PDF]
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N. Chondrogianni, F. L. L. Stratford, I. P. Trougakos, B. Friguet, A. J. Rivett, and E. S. Gonos Central Role of the Proteasome in Senescence and Survival of Human Fibroblasts: INDUCTION OF A SENESCENCE-LIKE PHENOTYPE UPON ITS INHIBITION AND RESISTANCE TO STRESS UPON ITS ACTIVATION J. Biol. Chem., July 18, 2003; 278(30): 28026 - 28037. [Abstract] [Full Text] [PDF]
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 T. GRUNE, T. REINHECKEL, J. A. NORTH, R. LI, P. B. BESCOS, R. SHRINGARPURE, and K. J. A. DAVIES Ezrin turnover and cell shape changes catalyzed by proteasome in oxidatively stressed cells FASEB J, October 1, 2002; 16(12): 1602 - 1610. [Abstract] [Full Text] [PDF]
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H. Atamna, J. Liu, and B. N. Ames Heme Deficiency Selectively Interrupts Assembly of Mitochondrial Complex IV in Human Fibroblasts. RELEVANCE TO AGING J. Biol. Chem., December 14, 2001; 276(51): 48410 - 48416. [Abstract] [Full Text] [PDF]
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 T. Grune, R. Shringarpure, N. Sitte, and K. Davies Age-Related Changes in Protein Oxidation and Proteolysis in Mammalian Cells J. Gerontol. A Biol. Sci. Med. Sci., November 1, 2001; 56(11): B459 - 467. [Abstract] [Full Text] [PDF]
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N. SITTE, K. MERKER, T. VON ZGLINICKI, T. GRUNE, and K. J. A. DAVIES Protein oxidation and degradation during cellular senescence of human BJ fibroblasts: part I--effects of proliferative senescence FASEB J, December 1, 2000; 14(15): 2495 - 2502. [Abstract] [Full Text]
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