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* INSERM U.466, Biochemistry Department, University Paul Sabatier, Toulouse, France;
Laboratorio de Bioquimica, Faculdade de Farmacia and Centro de Neurociências, Universidade de Coimbra, 3000 Coimbra, Portugal;
Institute of Medical Biochemistry, Karl-Franzen Universität Graz, Austria; and
§ Institute of Biochemistry, University of Fribourg, Switzerland
1Correspondence: INSERM U-466 and Biochimie, CHU Rangueil, 1 Avenue Jean Poulhès, 31403 Toulouse Cedex 04, France. E-mail: anesalv{at}rangueil.inserm.fr or salvayre{at}rangueil.inserm.fr
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: oxidized LDL • 4-hydroxynonenal • proteasome • ubiquitin • apoptosis
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 2)
. Low-density lipoproteins (LDL) play
an important role in atherogenesis (3)
and are thought to
become atherogenic after undergoing oxidative modifications
(4)
. LDL oxidation is mediated by free radicals generated
by vascular cultured cells, transition metals, and heme proteins such
as ferrylmyoglobin (5
, 8)
. During the initial steps of the
oxidative process, antioxidants are consumed and lipid peroxidation
begins to rise, leading to the formation of mildly oxidized LDL
(characterized by relatively low levels of lipid peroxidation products
without or only minor changes in apoB). Later, progression of the
oxidative process leads to the formation of extensively oxidized LDL
(containing high levels of lipid peroxidation products and severe apoB
alterations), which is detected in atherosclerotic plaques (4
, 9)
.
Oxidized low-density lipoproteins (oxLDL) exhibit a wide spectrum
of biological properties and are able to induce events potentially
involved in atherogenesis, such as monocytes chemotaxis, foam cells
formation, endothelial dysfunction, smooth muscle cell proliferation,
and cytotoxicity to cultured cells (4
5
6
7
, 10)
. Lipid
peroxidation products (e.g., oxysterols and aldehydes) contained in
oxLDL are able to elicit apoptosis or necrosis of cultured cells
(13
, 14)
through a calcium-dependent pathway (14
, 15)
. The primary cellular targets and the precise mechanisms of
toxicity are only poorly understood.
OxLDL contain MDA, 4-HNE, hexanal, and other aldehydes (6)
able to form apoB- and cell proteins-adducts that are detected in
atherosclerotic areas (9
, 16
17
18)
. Derivatization may
induce dramatic changes in the functional properties of proteins; for
instance, apoB modifications alter LDL metabolism (4
, 8)
and cell protein derivatization may lead to a loss (19)
or
gain of function (20
21
22)
, which may deregulate cell
functions. These events may play a significant role in atherosclerosis
and other pathophysiological processes (such as inflammatory reactions,
aging, and related pathologies) (23
, 24)
.
Cellular defense systems against oxidized proteins consist in
proteolytic degradation (25
, 26)
through a multicatalytic
proteinase complex (proteasome), either dependent or independent of the
ubiquitin system (25
26
27
28
29
30
31)
. The 26S proteasome complex is
constituted by a central 20S proteolytic core (20S proteasome)
associated with two regulatory subcomplexes, termed PA700 or 19S and
PA28 (or 11S regulator) (26
, 27)
. Ubiquitination of
proteins is performed by the ATP-dependent ubiquitin system, and
polyubiquitinated proteins are targeted to the 26S proteasome for
degradation (28
29
30
31)
. The ubiquitin-dependent proteolytic
pathway (26S proteasome) is involved in the continuous turnover of
regulatory proteins and in the selective degradation of misfolded and
denatured proteins. It is thought to play a major role in regulating
numerous cell processes (signal transduction, cell cycle progression,
transcription, endocytosis, apoptosis) and ‘detoxifying’ altered
proteins (25
26
27
28
29
30
31)
. Misfolded or ubiquitin-tagged, or
moderately oxidized or otherwise structurally altered, proteins are
valuable substrates for the proteasome, but heavily oxidized proteins
are no longer degradable, accumulate, and may become toxic
(25)
.
As we have recently shown, 4-HNE contained in oxLDL was able to derivatize the EGF receptor, and we hypothesized that such a derivatization may modify the structure of a number of cell proteins, impair their functions, and finally alter cell viability.
We report here that incubation of cells with oxLDL induces derivatization of cell proteins by 4-HNE (4-HNE-protein adducts), ubiquitination of cell proteins, and (de)regulation of proteasome activity (i.e., early activation followed by late inhibition). Inhibition of proteasome reduces the toxicity threshold of oxLDLs and potentiates their toxic effect to ECV-304 cells, thus suggesting that, in these cells, accumulation of altered proteins is involved in oxidized LDL-induced apoptosis and that proteasome activity may be involved in cellular defenses against oxidized LDL-induced apoptosis.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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632 nm =
2.1 mM-1·cm-1 and 240 nm =
43.6 mM-1·cm-1, respectively.
LDL isolation and oxidation
LDL were isolated from human pooled and heat inactivated (1 h at
56°C) sera by ultracentrifugation, under the previously described
conditions (16
, 32)
, sterilized on 0.2 µM Millipore
membrane, and stored at 4°C under nitrogen (up to 4 wk). The
electrophoretic mobility of LDL was evaluated on agarose gel
(Hydragel®; Sebia, Paris), and apoB was
determined by immunonephelometry under the previously used conditions
(15
, 16)
.
LDLs were oxidized by incubation with
metmyoglobin/H2O2 (18/27
µM) in PBS, at 37°C for 2 h, under the previously used
conditions (32)
. The level of LDL oxidation was evaluated
by monitoring lipid hydroperoxides (33)
, thiobarbituric
reactive substances (34)
, and 4-HNE content
(35)
.
Lipid peroxidation levels of mildly oxLDL used here ranged between 52
and 68 nmol lipid hydroperoxides/mg apoB, between 5 and 8 nmol TBARS/mg
apoB, and between 12 and 15 nmol 4-HNE/mg apoB, without major
modifications of apoB (32)
.
Cell culture
ECV-304 human endothelial cell line (CRL-1998; ATCC, Rockville,
Md.) were grown under the previously described conditions (15
, 32)
. Briefly, all passages were made using a splitting ratio
1:4. Cells were seeded (two 105 cells/ml)
in six multiwell plates or in falcons (Nunc, Roskilde, Denmark) and
grown in RPMI 1640 medium containing Glutamax®
supplemented with 10% heat inactivated fetal calf serum, 100 U/ml
penicillin, and 100 µg/ml streptomycin (in 5%
CO2, at 37°C). After starved in serum-free
medium for 24 h before LDL addition, subconfluent cell cultures
were incubated with oxLDL or native LDL, and inhibitors (PSI dissolved
in ethanol and DNPH in DMSO—each solvent used at 0.1% final—were
added at time 0, just before LDL addition) under the conditions
indicated below.
H38–5 and ts20 fibroblasts were a generous gift of Dr. H. L. Ozer
(36)
. ts20 is a Balb/C 3T3 clone A31 fibroblast cell line
that is temperature-sensitive for the E1 ubiquitin activating enzyme
(i.e., E1 activity and ubiquitination are drastically decreased at
39°C). H38–5 is an E1-transfected (corrected) ts20 derivative that
expresses E1 and ubiquitinates at all temperature. ts20-pMV12 and
ts20Bcl-2#7 cell lines (which exhibit a similar decrease in
ubiquitination at 39°C as the parental ts20 cell line) were generated
in the laboratory of Dr. C. Borner (37)
by retroviral
transduction of the pMV12 hygro plasmid or the pMV12 hygro plasmid
containing the mouse Bcl-2 cDNA, respectively. The ts20Bcl-2#7 cell
line overexpresses Bcl-2 5- to 10-fold over controls. Cells were grown
at 34 or 39°C as described previously (36
, 37)
.
Subconfluent cells were starved in serum-free RPMI 1640 for 24 h
at 34°C before LDL were added, and the temperature was shifted to
39°C for 18 h.
In situ proteolysis measurements and in
vitro determination of proteasome activity
The degradation of cellular proteins was determined under the
conditions described by Grüne et al. (38)
. Briefly,
ECV-304, grown in six multiwell plates, were preincubated with a
[35S]methionine/cysteine mixture (0.5 µCi/ml)
in methionine-free MEM culture medium for 2 h (short-lived
proteins) or 16 h (long-lived proteins). Then, short-lived– and
long-lived–labeled cells were chased in standard RPMI 1640 medium
containing 10 mmol/l of unlabelled methionine for 10 min and 2 h,
respectively; then, cells were incubated with oxLDL or native LDL,
washed twice in PBS, scrapped off and pelleted by centrifugation at
1,500g for 10 min. Cell proteins were then precipitated by
10% trichloroacetic acid (TCA) for 30 min at 4°C and after
centrifugation (15,000g for 10 min), the radioactivity of
TCA-soluble and TCA-precipitable fractions (precipitate dissolved in 50
µl of NaOH 1N) was counted by liquid scintillation counting
(Aquasafe®, Packard Tricarb 4530, Downers Grove,
Ill.).
The in vitro activity of the 20S proteasome was determined
according to Grüne et al. (38)
. Cells were
harvested, pelleted, resuspended in PBS containing 0.1% Triton X-100
and 0.5 mM dithiotreitol, homogenized (sonication, two runs of 5 s, Bransonic sonicator) and used immediately for determining the
enzymatic activity. The assay mixture contained 50 µl of buffer (50
mM Tris-HCl pH 7.8, 20 mM KCl, 5 mM MgCl2, and
0.1 mM DTT), 250 µM of sLLVY-MCA, and 50 µl of cell lysates (15
µg of proteins). After 30 min at 37°C, the reaction was stopped by
adding 1 ml of 0.2 M glycine buffer pH 10 and the fluorescence of the
liberated 7-amino-4 methylcoumarin was measured (spectrofluorometer
Jobin-Yvon, excitation 365 nm, emission 460 nm). An aliquot of the cell
homogenate was used for protein determination using the biscinchoninic
reagent.
Western-blots experiments
ECV-304 cells, treated or not by oxLDL, were washed, scrapped
off in PBS, centrifuged (2,000g for 5 min at 4°C) and
lysed in solubilizing buffer (50 mM Tris pH 7.4, 250 mM NaCl, 5 mM
EDTA, 1 mM Na3VO4, 10 mM Na
pyrophosphate, 160 mM NaF, 2.5 mM PMSF, 10 µM leupeptin, 2 µM
pepstatin, 10 µg/ml aprotinin, 1% triton X-100) for 30 min on ice.
Fifty micrograms of cell lysates were resolved by electrophoresis in a
7.5% SDS-polyacrylamide gel, transferred onto nitrocellulose membranes
(Hybond-C, Amersham), probed with an anti-ubiquitin antibody (Sigma) or
an anti-4-HNE-protein antibody (K5–4412), and revealed by ECL system
(Amersham) using a peroxidase-coupled secondary antibody, as previously
used (22)
.
Determination of free amino group content in cell proteins
The free amino group content of cell proteins was evaluated on
cells homogenates using the amino-reactive probe
[3H]N-succinimidyl propionate
([3H]NSP) (39)
, under the
previously used conditions (22)
. Briefly, after
incubation, cells were washed three times in PBS and homogenized in 0.5
M borate buffer pH 8.5 (sonication, two runs of 5 s). An aliquot
of the cell suspension was saved for protein determination. Cell
homogenates were let to react with 10 µCi of [3H]NSP (Amersham,
99.0 Ci/mmol) in 0.5 M borate buffer pH 8.5, for 15 min, in an ice bath
(22)
. Then, cell proteins were precipitated by TCA and the
radioactivity counted as above indicated.
Determination of cytotoxicity and apoptosis
The whole cytotoxicity was evaluated by using the MTT test
(40)
. The number of morphologically apoptotic or/and
necrotic cells was evaluated concomitantly on intact cultured cells
(grown in six multiwell plates) according to the fluorescent
double-staining we recently described (41)
. Briefly, cells
were incubated with two vital fluorescent dyes, 0.6 µM SYTO-13 (a
permeant DNA intercalating green-colored probe) and 15 µM propidium
iodide (a nonpermeant intercalating orange probe) and counted by using
an inverted fluorescence microscope (Fluovert FU; Leitz, Rockleigh,
N. J.). Normal nuclei exhibited a loose chromatin colored in green
by SYTO; apoptotic nuclei exhibited condensed green-colored chromatin
and/or fragmentation (postapoptotic necrosis being
characterized by nuclei exhibiting the same apoptotic morphological
features but orange-colored); necrotic cells exhibited orange-colored
nuclei with loose chromatin. It may be noted that necrotic cells
(orange-colored by propidium iodide) were generally stained by trypan
blue. Alternatively, the morphology was also examined after
May-Grünwald-Giemsa staining, as previously used
(15)
.
Biochemical methods were also used in order to evaluate the level of
apoptosis and necrosis in the whole cell population. Chromatin
fragmentation, evaluated by the procedure of McConkey et al.
(42)
, and lactate deshydrogenase released into the culture
medium (Roche assay kit, MA kit 10), were determined under the
previously described conditions (14
, 15)
. The results were
generally consistent with morphological counts (the data presented here
were selected in order to avoid redundancy).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, B
). As shown by western blots revealed by anti-4-HNE
antibody, the loss of [3H]NSP-reactive amino
groups induced by oxLDL was associated with derivatization of cell
proteins by 4-HNE, a lipid peroxidation derivative able to react with
free amino groups of proteins (Fig. 1C
). This derivatization
was mimicked by oxLDL lipid extracts (data not shown) and by 4-HNE (1
µM) (Fig. 1A
, C
).
|
In the same time, an increased level of high-molecular-mass (HMM)
ubiquitin-protein conjugates (HMM > 200 kDa) was observed in
cells incubated with oxLDL but not in cells incubated with native LDL
or FeMb (used alone) (Fig. 1D
). Thus, in cells treated by
oxLDL, some proteins are probably recognized as structurally abnormal
and tagged with ubiquitin probably for subsequent degradation through
the ubiquitin-dependent proteolytic pathway. This led us to evaluate
the proteasome activity in cells incubated with oxLDL.
Biphasic effect of toxic concentrations of oxLDL on the proteasome
activity
OxLDL induced a time- and dose-dependent transient activation of
in situ (i.e., in intact living cell) intracellular
proteolysis of both short-lived and long-lived
[35S]-radiolabelled proteins (Figs. 2A
, B
). In contrast, native (nonoxidized) LDL did not. Toxic
concentrations of oxLDL (200 µg apoB/ml) evoked a rapid and transient
peak of in situ proteolysis (maximum between 1 and 3 h)
followed by a return to the ground level at 5–7 h. It may be noted
that lower oxLDL concentration (50 µg apoB/ml) also induced a
transient peak of intracellular proteolysis that occurred later
(maximum between 5 and 7 h) and then return slowly to the basal
level (at 15 h) (Fig. 2A
).
|
Toxic concentrations of oxLDL (200 µg apoB/ml) induced also a
biphasic effect on the in vitro hydrolysis of sLLVY-MCA (a
fluorogenic synthetic peptide substrate for 20S proteasome) (Fig. 2C
), but the time course was different from that of in
situ proteolysis. The in vitro hydrolysis of sLLVY-MCA
began to rise at 2 h, then reached a plateau (sustained for 3–4
h), and finally decreased toward the baseline (back at 15 h).
Despite different time courses, both in situ
proteolysis and in vitro hydrolysis of sLLVY-MCA resulted
very probably from proteasome activity, as assessed by inhibition by
the cell permeant inhibitors of proteasome, LLnL (5 µM), and PSI (5
µM) (Fig. 2D
).
It may be noted that intracellular accumulation of altered or
derivatized proteins is obvious as early as 5 h (Fig. 1)
and
persisted for the duration of the experiment (18 h) (i.e., after the
decay of in situ proteolysis) (Fig. 2)
. This suggests that
accumulation of derivatized and ubiquitinated proteins may result from
alteration of the proteasome activity.
Toxic concentrations of oxLDL induced both the accumulation of altered
cellular proteins and apoptosis in ECV-304 cells. Because accumulation
of oxidized (or otherwise altered) proteins is potentially toxic to the
cell (24
, 25)
and proteasome is involved in the
degradation of altered proteins, this led us to investigate whether
proteasome inhibition and apoptosis induced by oxLDL were causally
related or parallel unrelated events. This was investigated by using
proteasome inhibitors and ubiquitination-deficient cells.
Proteasome inhibition potentiates the oxLDL-induced toxicity
The concentrations of proteasome inhibitors used here were not
toxic per se (over the 24 h of the experiments) but
were effective in inhibiting proteasome activity [i.e., inhibiting
both the early peak of oxLDL-induced autoproteolysis (data not shown)
and sLLVY-MCA hydrolysis (Fig. 2D
)]. This concentration of
proteasome inhibitors potentiated both the accumulation of
ubiquitinated proteins and the oxLDL-induced toxicity. Proteasome
inhibitors potentiated rapidly the accumulation of ubiquitinated
proteins (data not shown) probably because of the inhibition of
oxLDL-induced early peak of proteasome activation.
The potentiation of the oxLDL-induced toxicity by proteasome inhibitors
was obvious at low (non- or slightly toxic) oxLDL concentrations,
because, in the presence of inhibitors, the toxic effect of oxLDL to
ECV-304 cells occurred faster and at lower concentrations (Figs. 3A
, B
). For instance, when cells were incubated with low oxLDL
concentration (50 µg apoB/ml) in the absence of proteasome inhibitor,
no significant toxicity was observed (Figs. 3B
, C
), thus
suggesting that the toxic threshold dose of oxLDL (43)
was
not reached. In contrast, when the same experiment was performed in the
presence of proteasome inhibitors, cells underwent apoptosis (Fig. 3C
). This suggests that proteasome inhibition lowered the
threshold of oxLDL concentration inducing the cytotoxicity.
|
Proteasome inhibitors did not alter the type of cell death induced by
oxLDL (Fig. 3D-G
), because ECV-304 EC killed by low (not
toxic per se) oxLDL concentrations in the presence of
proteasome inhibitors exhibited the characteristic features of
apoptosis, quite similarly to cells killed by toxic concentrations of
oxLDL (15)
.
These data suggest that proteasome activity may participate in the cellular defenses against oxLDL-induced toxicity and, conversely, that inhibition of endogenous proteolysis lowered the toxic threshold dose of oxLDL.
It may be noted that the oxLDL toxicity and its potentiation by proteasome inhibitors is not restricted to endothelial cells. In rabbit arterial smooth muscle cells, after 24 h incubation with 200 µg apoB/ml of oxLDL in the presence or absence of 10 µM PSI, the viability was 33 ± 4% and 78 ± 8%, respectively. In the U937 monocytic cell line, after 24 h incubation with 100 µg apoB/ml of oxLDL in the presence or absence of 10 nM PSI (this cell line is very susceptible to PSI), the viability was 30 ± 5% and 60 ± 7%, respectively (as assessed by the trypan blue test).
Defective ubiquitination potentiates oxLDL-induced apoptosis
As the degradation of altered proteins by proteasome may be
ubiquitin-dependent or -independent (25)
, we investigated
whether the ubiquitin pathway is involved in the cellular defenses
against oxLDL toxicity by using genetically engineered cells (derived
from the E1-thermo-sensitive ts20 cell line). As previously reported
(36
, 37)
, ubiquitination was effective at 34°C in the
three cell lines, and at 39°C in the E1-transduced H38–5, but was
defective at 39°C in ts20pMV12 and ts20Bcl-2#7 cell lines (both in
the presence or absence of oxLDL; Fig. 4A
).
|
The relative susceptibility of the three cell lines to the toxic effect
of oxLDL was compared at 34 and 39°C. The toxicity of oxLDL (200 µg
apoB/ml) to H38–5 cells (in which ubiquitination is always working)
was quite similar at 34 and 39°C. In contrast, both ts20pMV12 and
ts20Bcl-2#7 cell lines were much more susceptible to the toxic effect
of oxLDL at 39°C (defective ubiquitination) than at 34°C (effective
ubiquitination) (Fig. 4B
). These data suggest that the
ubiquitin pathway plays a role in the cellular defenses against
oxLDL-induced toxicity.
It may be noted that the Bcl-2 overexpressing ts20Bcl-2#7 was used in
order to examine whether, in this model system (ubiquitin-defective),
the oxLDL-induced apoptosis was counterbalanced by Bcl-2 overexpression
or not. At 39°C, the ubiquitination-defective two cell lines
ts20pMV12 (not expressing Bcl-2) and ts20Bcl-2#7 (overexpressing Bcl-2)
were similarly susceptible to the toxic effect of oxLDL [over the time
of the experiment (i.e., 18–24 h)], thus suggesting that Bcl-2 is not
effective in preventing oxLDL-induced cell death. In contrast, in
agreement with Monney et al. (37)
, overexpression of Bcl-2
in ts20Bcl-2#7 increased the resistance of these cells against heat
shock-induced cell death (data not shown).
DNPH prevents cell protein derivatization and oxLDL-induced
cytotoxicity
As it resulted implicitly from the above reported data, that lipid
peroxidation of oxLDL (e.g., 4-HNE) generates cell protein adducts that
are potentially involved in the toxicity, we hypothesized that
inhibition of cell protein derivatization should inhibit the toxic
effect of oxLDL. Dinitrophenylhydrazine, a well-known reagent of lipid
peroxidation-derived aldehydes, was used in order to scavenge
oxLDL-generated aldehydic compounds. As expected, DNPH reduced both the
level of 4-HNE-derivatized proteins (Figs. 5A
) and, at a lesser extent, the level of the whole derivatization of cell
proteins (evaluated by [3H]NSP-reactive free
amino groups) (Fig. 5B
). Concomitantly, DNPH (used at
nontoxic concentration) was also able to inhibit the toxic effect
induced by oxLDL (Fig. 5C
), thus supporting the hypothesis
that cell protein derivatization induced by oxLDL is involved in their
toxicity.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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To our knowledge, this is the first study linking the toxicity induced by oxLDL with cell protein alterations and proteasome inhibition. Conversely, it is also suggested that the ubiquitin-proteasome pathway plays a role in the cellular defenses against the toxic effect of oxLDL.
The reported data show that lipid peroxidation derivatives (among them
4-HNE) contained in oxLDL are able to derivatize cell proteins, in
agreement with the observation of Rosenfeld et al. (18)
in
macrophagic cells. Until now, the possible interactions between lipid
peroxidation end products contained in oxLDL and cell proteins have
been only poorly investigated in contrast to apoB modifications, which
are well documented (see reviews in refs 4
, 6
). Our data
suggest that 4-HNE, a major aldehydic lipid peroxidation derivative
able to form adducts with lysine, histidine, or cysteine of proteins
(16
, 17)
, seems to play a major role in oxLDL-induced
derivatization of cell proteins. But, it is not excluded that other
reactive compounds of oxLDL [e.g., malondialdehyde, fatty acid
peroxides (9)
], may also derivatize cell proteins.
The dramatic accumulation of ubiquitinated proteins may result from two
additional mechanisms. During the early time of incubation (up to
3 h with toxic concentrations of oxLDL), the rise of
ubiquitination cannot result from a defect in the de-ubiquitination
process (by the 19S complex) because the whole activity of the 26S
proteasome is high and may result from an activation of the
ubiquitination pathway. This may be because of the oxLDL-induced
protein derivatization, given that 4-HNE is able to induce both protein
derivatization and proteasome activation (44)
. After
5 h, the inhibition of the 26S proteasome may constitute an
additional mechanism of accumulation of ubiquitinated proteins
(Fig. 6)
.
|
OxLDLs affect, in a biphasic manner, the proteasome activity by inducing an early transient activation of proteasome followed by a sustained decay.
The early and transient peak (between 1 and 3 h with 200 µg
apoB/ml of oxLDL) of proteolysis resulted from proteasome activation,
as assessed by the use of proteasome inhibitors (whereas the basal
proteolysis was probably proteasome-independent because it was not
affected by proteasome inhibitors). This early proteasome activation
may be subsequent to oxLDL-induced structural alterations and
ubiquitination of cellular proteins, which are valuable substrates for
the proteasome (23
24
25)
. Activation of endogenous
proteolysis and of sLLVY-MCA hydrolysis exhibited different time
courses (the rise of sLLVY-MCA hydrolysis beginning 2 h after that
of autoproteolysis). The activation of in vitro degradation
of sLLVY-MCA synthetic substrate (cannot be attributed to substrate
generation) may result from activation of the 20S proteasomal core by
oxLDL. OxLDL may act either 1) by generating a cellular
oxidative stress (47)
, which is known to activate the 20S
proteasome (and subsequently, NF
B) (48
, 49)
, or
2) by activating protein kinases (22
, 51
, 52)
,
some of them being potentially able to phosphorylate and activate
(50)
the PA28 proteasome activator (which can in turn
stimulate synthetic substrate hydrolysis by the 20S proteasome).
This transient proteasome activation was followed by a decay of
proteasomal proteolysis toward the baseline. This decay resulted
neither from a substrate (modified proteins) depletion, because high
levels of derivatized proteins and ubiquitinated-HMM still persisted up
to 18 h, nor from an irreversible inhibition of the 20S proteasome
activity, because proteasome was able to hydrolyze in vitro
the fluorogenic substrate sLLVY-MCA up to 10–12 h. Altogether, these
data led us to hypothesize a two-step mechanism of inhibition affecting
first the 19S and later the 20S. The rapid decay of endogenous
proteolysis may be a result of inhibition of the 19S (thus inhibiting
de-ubiquitination and degradation of ubiquitinated proteins), because
4-HNE cross-linked proteins are resistant to proteolysis and are able
to inhibit the 26S proteasome (45)
and because Reinheckel
et al. (46)
reported that the 26S proteasome is less
resistant to H2O2-induced
oxidative stress than the 20S proteolytic core. The second step [i.e.,
inhibition of the 20S core (involved in sLLVY-MCA hydrolysis)] may be
a result of the progressing intracellular oxidative stress induced by
oxLDL (47
; Fig. 6
). At this stage, when the proteasome is
completely inhibited, cells are rapidly dying. This led us to
investigate the relationship between proteasome inhibition and the
oxLDL-induced cytotoxicity, because, according to the model systems,
proteasome may participate in the apoptotic process or prevent it
(26
27
28
29
30
31)
.
The reported data strongly suggest that the active proteasome plays a role in the cellular defenses against the oxLDL-induced cytotoxicity, and, conversely, that proteasome inhibition may be involved in the mechanism of cytotoxicity.
The protective effect of proteasome was (at least in part) dependent on
ubiquitination, because at 39°C the ubiquitin-defective ts20pMV12 and
ts20Bcl-2#7 cells were more susceptible to the toxic effect of oxLDL
than the ubiquitin-expressing H38–5 cells. It is noteworthy that the
higher susceptibility of the ubiquitin-defective cells seems to be
independent of heat-shock toxicity because in ts20pMV12, the time
courses of the oxLDL-induced and heat shock-induced toxicity are
different (beginning at 12–15 and after 24 h, respectively); in
ts20Bcl-2#7 cells, the heat shock-induced apoptosis is blocked by Bcl-2
overexpression (36
, 37)
in contrast to oxLDL-induced
apoptosis. The inefficiency of Bcl-2 in preventing the oxLDL-induced
toxicity to ts20Bcl-2#7 cells is quite consistent with our recently
reported data, which show that Bcl-2 overexpression alters only the
balance between apoptosis and necrosis but does not prevent cell death
triggered by oxLDL (41)
. Altogether, these data strongly
suggest that ubiquitination plays a role in the cellular resistance
against the toxicity of oxLDL.
Potentiation of the oxLDL-induced toxicity by proteasome
inhibitors may involve various mechanisms. The ubiquitin-proteasome
pathway is probably involved in cellular defenses against oxLDL by
degrading altered proteins generated by oxLDL [e.g., proteins
derivatized by 4-HNE (this paper and ref 22
) or other
lipid peroxidation products]. Therefore, the inhibition of the
ubiquitin-proteasome pathway (by specific proteasome inhibitors, by
genetic alterations or by oxLDL) results in the accumulation of
structurally and functionally altered cell proteins that impair cell
functions and viability (31
, 53)
. Moreover, proteasome
inhibition may also impair cell cycle regulation and alter cell
viability through the defect of its other housekeeping functions
involved in the regulation of proteins that play a role in the balance
between cell death and survival (29
30
31)
. For instance,
because proteasome is involved in the degradation of p53 and of IB
(and NFB activation), proteasome inhibition may lead to accumulate
the proapoptotic p53 protein and block the activation of NFB [a
survival factor (54)
] (29
30
31
; Fig. 6
).
In conclusion, the reported data may be summarized by the following
hypothetical scenario: Aldehydes (and possibly other compounds
transported by oxLDL) are able to derivatize cell proteins (this paper
and ref 22
) and alter cell signaling (22
, 51
, 52)
. At low (nontoxic) concentrations of oxLDL, the proteasome
acts as a cellular defense system by degrading these structurally
(e.g., derivatized) and functionally altered proteins. At high (toxic)
concentrations of oxLDL, after a transient activation, the proteasome
activity is inhibited (the 26S complex at 5 h and the 20S
proteolytic core at 14 h) by oxLDL, thus leading to the
accumulation of altered cell proteins. The subsequent cell injury
becomes irreversible and leads to cell death. This role of proteasome
in the oxLDL-induced toxicity seems to be a relatively general
mechanism, because proteasome inhibition potentiates the effect of
oxLDL in various cell types present in atherosclerotic areas (e.g.,
endothelial, monocytic, smooth muscle cells, and fibroblasts).
|
ACKNOWLEDGMENTS |
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FOOTNOTES |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-tocopherol and rutin prevent the glutathione and ATP depletion induced by oxidized LDL in cultured endothelial cells. Brit. J. Pharmacol. 16,1985-1990
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