Oxidized LDLs alter the activity of the ubiquitin-

proteasome pathway: potential role in oxidized LDL-

induced apoptosis
OTÍLIA VIEIRA,*,† ISABELLE ESCARGUEIL-BLANC,* GÜNTHER JÜRGENS,‡
CHRISTOPH BORNER,§ LEONOR ALMEIDA,† ROBERT SALVAYRE,*,1 AND
ANNE NÈGRE-SALVAYRE*,1
*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

ABSTRACT Oxidized low-density lipoproteins (ox- L., Salvayre, R., Nègre-Salvayre, A. Oxidized LDL
LDL) play a role in the genesis of atherosclerosis. alter the activity of the ubiquitin-proteasome path-
OxLDL are able to induce apoptosis of vascular way: potential role in oxidized LDL-induced apopto-
cells, which is potentially involved in the formation sis. FASEB J. 14, 532–542 (2000)
of the necrotic center of atherosclerotic lesions,
plaque rupture, and subsequent thrombotic events. Key Words: oxidized LDL z 4-hydroxynonenal z proteasome
Because oxLDL may induce structural modifications z ubiquitin z apoptosis
of cell protein and altered proteins may impair cell
viability, the present work aimed to evaluate the Atherosclerosis and subsequent vascular dis-
extent of protein alterations, the degradation of eases are the first cause of morbidity and mortality in
modified proteins through the ubiquitin-proteasome Western countries. Atherosclerotic lesions associate
system (a major degradative pathway for altered and to variable degrees with accumulation of lipid laden
oxidatively modified proteins) and their role during macrophagic cells, proliferating smooth muscle
apoptosis induced by oxLDL. This paper reports the cells, fibrosis, and necrotic areas in the subendothe-
following: 1) oxLDL induce derivatization of cell lial space of the arterial wall (1, 2). Low-density
proteins by 4-hydroxynonenal (4-HNE) and ubiquiti- lipoproteins (LDL) play an important role in athero-
nation. 2) Toxic concentrations of oxLDL elicit a genesis (3) and are thought to become atherogenic
biphasic effect on proteasome activity. An early and after undergoing oxidative modifications (4). LDL
transient activation of endogenous proteolysis is oxidation is mediated by free radicals generated by
followed rapidly by a subsequent decay (resulting vascular cultured cells, transition metals, and heme
probably from the 26S proteasome inhibition) and proteins such as ferrylmyoglobin (5, 8). During the
followed later by the inhibition of the 20S protea- initial steps of the oxidative process, antioxidants are
some (as assessed by inhibition of sLLVY-MCA hy- consumed and lipid peroxidation begins to rise,
drolysis). 3) Specific inhibitors of proteasome (lac- leading to the formation of mildly oxidized LDL
tacystin and proteasome inhibitor I) potentiated (characterized by relatively low levels of lipid peroxi-
considerably the toxicity of oxLDL (nontoxic doses dation products without or only minor changes in
of oxLDL became severely toxic). The defect of the apoB). Later, progression of the oxidative process
ubiquitination pathway (in temperature-sensitive mu- leads to the formation of extensively oxidized LDL
tants) also potentiated the toxicity of oxLDL. This (containing high levels of lipid peroxidation prod-
suggests that the ubiquitin-proteasome pathway plays ucts and severe apoB alterations), which is detected
a role in the cellular defenses against oxLDL-in- in atherosclerotic plaques (4, 9).
duced toxicity. 4) Dinitrophenylhydrazine (DNPH), Oxidized low-density lipoproteins (oxLDL) ex-
an aldehyde reagent, prevented both the oxLDL- hibit a wide spectrum of biological properties and
induced derivatization of cell proteins and subse- are able to induce events potentially involved in
quent cytotoxicity. Altogether, the reported data atherogenesis, such as monocytes chemotaxis,
suggest that both derivatization of cell proteins (by
4-HNE and other oxidized lipids) and inhibition of 1
Correspondence: INSERM U-466 and Biochimie, CHU
the proteasome pathway are involved in the mecha- Rangueil, 1 Avenue Jean Poulhès, 31403 Toulouse Cedex 04,
nism of oxLDL-induced apoptosis.—Vieira, O., Es- France. E-mail: [email protected] or salvayre@
cargueil-Blanc, I., Jürgens, G., Borner, C., Almeida, rangueil.inserm.fr

532 0892-6638/00/0014-0532/$02.25 © FASEB

foam cells formation, endothelial dysfunction, oxLDLs and potentiates their toxic effect to ECV-304
smooth muscle cell proliferation, and cytotoxicity cells, thus suggesting that, in these cells, accumula-
to cultured cells (4 –7, 10). Lipid peroxidation tion of altered proteins is involved in oxidized LDL-
products (e.g., oxysterols and aldehydes) con- induced apoptosis and that proteasome activity may
tained in oxLDL are able to elicit apoptosis or be involved in cellular defenses against oxidized
necrosis of cultured cells (13, 14) through a LDL-induced apoptosis.
calcium-dependent pathway (14, 15). The primary
cellular targets and the precise mechanisms of
toxicity are only poorly understood. MATERIALS AND METHODS
OxLDL contain MDA, 4-HNE, hexanal, and other
aldehydes (6) able to form apoB- and cell proteins- Chemicals and reagents
adducts that are detected in atherosclerotic areas (9,
16 –18). Derivatization may induce dramatic changes [3H] N-succinimidyl propionate (99.0 Ci/mmol), [35S]methi-
onine/cysteine mixture (ProMix [35S] cell labeling kit,
in the functional properties of proteins; for instance, .1000 Ci/mmol), and ECL reagent were obtained from
apoB modifications alter LDL metabolism (4, 8) and Amersham (Les Ulis, France); N-succinyl-L-leucyl-L-leucyl-L-
cell protein derivatization may lead to a loss (19) or valyl-Ltyrosine-7-amido-4-methyl coumarin (sLLVY-MCA)
gain of function (20 –22), which may deregulate cell from Bachem (Voisins-le-Bretonneux, France); Lactacystin
(Lc) and Proteasome Inhibitor I (PSI) from Calbiochem
functions. These events may play a significant role in (Nottingham, UK); anti-ubiquitin (polyclonal) antibody,
atherosclerosis and other pathophysiological pro- horse heart myoglobin, hydrogen peroxide, LLnL (N-acetyl-
cesses (such as inflammatory reactions, aging, and Leucyl-leucyl-norleucinal), and buthionine sulfoximine from
related pathologies) (23, 24). Sigma (St. Louis, Mo.); and RPMI 1640, L-glutamine, penicil-
Cellular defense systems against oxidized proteins lin, and streptomycin from Life Technologies (Cergy-Pon-
toise, France). All other chemicals (grade for biochemical
consist in proteolytic degradation (25, 26) through a analyses) were purchased from Merck (Darmstadt, Germany),
multicatalytic proteinase complex (proteasome), ei- Sigma, or Prolabo (Paris). Before use, metmyoglobin was
ther dependent or independent of the ubiquitin purified by dialysis against phosphate buffered saline (PBS)
system (25–31). The 26S proteasome complex is pH 7.4 containing 50 mM DTPA and Chelex-100. Stock
metmyoglobin and H2O2 solutions were standardized using
constituted by a central 20S proteolytic core (20S
ε632 nm 5 2.1 mM21zcm21 and ε240 nm 5 43.6 mM21zcm21,
proteasome) associated with two regulatory subcom- respectively.
plexes, termed PA700 or 19S and PA28 (or 11S
regulator) (26, 27). Ubiquitination of proteins is LDL isolation and oxidation
performed by the ATP-dependent ubiquitin system,
and polyubiquitinated proteins are targeted to the LDL were isolated from human pooled and heat inactivated
26S proteasome for degradation (28 –31). The ubiq- (1 h at 56°C) sera by ultracentrifugation, under the previously
described conditions (16, 32), sterilized on 0.2 mM Millipore
uitin-dependent proteolytic pathway (26S protea-
membrane, and stored at 4°C under nitrogen (up to 4 wk).
some) is involved in the continuous turnover of The electrophoretic mobility of LDL was evaluated on aga-
regulatory proteins and in the selective degradation rose gel (Hydragel®; Sebia, Paris), and apoB was determined
of misfolded and denatured proteins. It is thought to by immunonephelometry under the previously used condi-
play a major role in regulating numerous cell pro- tions (15, 16).
LDLs were oxidized by incubation with metmyoglobin/
cesses (signal transduction, cell cycle progression, H2O2 (18/27 mM) in PBS, at 37°C for 2 h, under the
transcription, endocytosis, apoptosis) and ‘detoxify- previously used conditions (32). The level of LDL oxidation
ing’ altered proteins (25–31). Misfolded or ubiq- was evaluated by monitoring lipid hydroperoxides (33), thio-
uitin-tagged, or moderately oxidized or otherwise barbituric reactive substances (34), and 4-HNE content (35).
Lipid peroxidation levels of mildly oxLDL used here
structurally altered, proteins are valuable substrates
ranged between 52 and 68 nmol lipid hydroperoxides/mg
for the proteasome, but heavily oxidized proteins are apoB, between 5 and 8 nmol TBARS/mg apoB, and between
no longer degradable, accumulate, and may become 12 and 15 nmol 4-HNE/mg apoB, without major modifica-
toxic (25). tions of apoB (32).
As we have recently shown, 4-HNE contained in
oxLDL was able to derivatize the EGF receptor, and Cell culture
we hypothesized that such a derivatization may mod-
ECV-304 human endothelial cell line (CRL-1998; ATCC,
ify the structure of a number of cell proteins, impair
Rockville, Md.) were grown under the previously described
their functions, and finally alter cell viability. conditions (15, 32). Briefly, all passages were made using a
We report here that incubation of cells with ox- splitting ratio 1:4. Cells were seeded (two 105 cells/ml) in six
LDL induces derivatization of cell proteins by 4-HNE multiwell plates or in falcons (Nunc, Roskilde, Denmark) and
(4-HNE-protein adducts), ubiquitination of cell pro- grown in RPMI 1640 medium containing Glutamax® supple-
mented with 10% heat inactivated fetal calf serum, 100 U/ml
teins, and (de)regulation of proteasome activity (i.e., penicillin, and 100 mg/ml streptomycin (in 5% CO2, at
early activation followed by late inhibition). Inhibi- 37°C). After starved in serum-free medium for 24 h before
tion of proteasome reduces the toxicity threshold of LDL addition, subconfluent cell cultures were incubated with

UBIQUITIN-PROTEASOME PATHWAY IN oxLDL-INDUCED APOPTOSIS 533

oxLDL or native LDL, and inhibitors (PSI dissolved in onto nitrocellulose membranes (Hybond-C, Amersham),
ethanol and DNPH in DMSO— each solvent used at 0.1% probed with an anti-ubiquitin antibody (Sigma) or an anti-4-
final—were added at time 0, just before LDL addition) under HNE-protein antibody (K5– 4412), and revealed by ECL sys-
the conditions indicated below. tem (Amersham) using a peroxidase-coupled secondary anti-
H38 –5 and ts20 fibroblasts were a generous gift of Dr. H. L. body, as previously used (22).
Ozer (36). ts20 is a Balb/C 3T3 clone A31 fibroblast cell line
that is temperature-sensitive for the E1 ubiquitin activating Determination of free amino group content in cell proteins
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 The free amino group content of cell proteins was evaluated
temperature. ts20-pMV12 and ts20Bcl-2#7 cell lines (which on cells homogenates using the amino-reactive probe [3H]N-
exhibit a similar decrease in ubiquitination at 39°C as the succinimidyl propionate ([3H]NSP) (39), under the previ-
parental ts20 cell line) were generated in the laboratory of Dr. ously used conditions (22). Briefly, after incubation, cells
C. Borner (37) by retroviral transduction of the pMV12 hygro were washed three times in PBS and homogenized in 0.5 M
plasmid or the pMV12 hygro plasmid containing the mouse borate buffer pH 8.5 (sonication, two runs of 5 s). An aliquot
Bcl-2 cDNA, respectively. The ts20Bcl-2#7 cell line overex- of the cell suspension was saved for protein determination.
presses Bcl-2 5- to 10-fold over controls. Cells were grown at 34 Cell homogenates were let to react with 10 mCi of [3H]NSP
or 39°C as described previously (36, 37). Subconfluent cells (Amersham, 99.0 Ci/mmol) in 0.5 M borate buffer pH 8.5,
were starved in serum-free RPMI 1640 for 24 h at 34°C before for 15 min, in an ice bath (22). Then, cell proteins were
LDL were added, and the temperature was shifted to 39°C for precipitated by TCA and the radioactivity counted as above
18 h. indicated.

In situ proteolysis measurements and in vitro determination Determination of cytotoxicity and apoptosis
of proteasome activity
The whole cytotoxicity was evaluated by using the MTT test
The degradation of cellular proteins was determined under (40). The number of morphologically apoptotic or/and
the conditions described by Grüne et al. (38). Briefly, ECV- necrotic cells was evaluated concomitantly on intact cultured
304, grown in six multiwell plates, were preincubated with a cells (grown in six multiwell plates) according to the fluores-
[35S]methionine/cysteine mixture (0.5 mCi/ml) in methi- cent double-staining we recently described (41). Briefly, cells
onine-free MEM culture medium for 2 h (short-lived pro- were incubated with two vital fluorescent dyes, 0.6 mM
teins) or 16 h (long-lived proteins). Then, short-lived– and SYTO-13 (a permeant DNA intercalating green-colored
long-lived–labeled cells were chased in standard RPMI 1640 probe) and 15 mM propidium iodide (a nonpermeant inter-
medium containing 10 mmol/l of unlabelled methionine for calating orange probe) and counted by using an inverted
10 min and 2 h, respectively; then, cells were incubated with fluorescence microscope (Fluovert FU; Leitz, Rockleigh,
oxLDL or native LDL, washed twice in PBS, scrapped off and N. J.). Normal nuclei exhibited a loose chromatin colored in
pelleted by centrifugation at 1,500g for 10 min. Cell proteins green by SYTO; apoptotic nuclei exhibited condensed green-
were then precipitated by 10% trichloroacetic acid (TCA) for colored chromatin and/or fragmentation (postapoptotic ne-
30 min at 4°C and after centrifugation (15,000g for 10 min), crosis being characterized by nuclei exhibiting the same
the radioactivity of TCA-soluble and TCA-precipitable frac- apoptotic morphological features but orange-colored); ne-
tions (precipitate dissolved in 50 ml of NaOH 1N) was crotic cells exhibited orange-colored nuclei with loose chro-
counted by liquid scintillation counting (Aquasafe®, Packard matin. It may be noted that necrotic cells (orange-colored by
Tricarb 4530, Downers Grove, Ill.). propidium iodide) were generally stained by trypan blue.
The in vitro activity of the 20S proteasome was determined Alternatively, the morphology was also examined after May-
according to Grüne et al. (38). Cells were harvested, pelleted, Grünwald-Giemsa staining, as previously used (15).
resuspended in PBS containing 0.1% Triton X-100 and 0.5 Biochemical methods were also used in order to evaluate
mM dithiotreitol, homogenized (sonication, two runs of 5 s, the level of apoptosis and necrosis in the whole cell popula-
Bransonic sonicator) and used immediately for determining tion. Chromatin fragmentation, evaluated by the procedure
the enzymatic activity. The assay mixture contained 50 ml of of McConkey et al. (42), and lactate deshydrogenase released
buffer (50 mM Tris-HCl pH 7.8, 20 mM KCl, 5 mM MgCl2, into the culture medium (Roche assay kit, MA kit 10), were
and 0.1 mM DTT), 250 mM of sLLVY-MCA, and 50 ml of cell determined under the previously described conditions (14,
lysates (15 mg of proteins). After 30 min at 37°C, the reaction 15). The results were generally consistent with morphological
was stopped by adding 1 ml of 0.2 M glycine buffer pH 10 and counts (the data presented here were selected in order to
the fluorescence of the liberated 7-amino-4 methylcoumarin avoid redundancy).
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
RESULTS
reagent.

Western-blots experiments oxLDL induce derivatization and ubiquitination of

cellular proteins
ECV-304 cells, treated or not by oxLDL, were washed,
scrapped off in PBS, centrifuged (2,000g for 5 min at 4°C) In ECV-304 cells, oxLDL induced a progressive
and lysed in solubilizing buffer (50 mM Tris pH 7.4, 250 mM time- and dose-dependent decay of the level of
NaCl, 5 mM EDTA, 1 mM Na3VO4, 10 mM Na pyrophos- free [3H]NSP-reactive amino groups, whereas na-
phate, 160 mM NaF, 2.5 mM PMSF, 10 mM leupeptin, 2 mM
pepstatin, 10 mg/ml aprotinin, 1% triton X-100) for 30 min tive LDL did not (Fig. 1A, B). As shown by western
on ice. Fifty micrograms of cell lysates were resolved by blots revealed by anti-4-HNE antibody, the loss of
electrophoresis in a 7.5% SDS-polyacrylamide gel, transferred [3H]NSP-reactive amino groups induced by ox-

534 Vol. 14 March 2000 The FASEB Journal VIEIRA ET AL.

Figure 1. In situ modifications of cell proteins induced by oxLDL in ECV-304 cells. A) Time course of [3H]NSP-reactive amino
groups in cells incubated with 200 mg apoB/ml of native LDL (empty circles) or oxLDL (filled circles) or 1 mM of 4-HNE (filled
squares). B) Effect of increasing concentrations of oxLDL (filled circles) or native LDL (empty circles), incubated for 5 h with
the cells, on the level of [3H]NSP-reactive amino groups. In panels A and B, the results are expressed as percent of the initial
value (control); means 6 se of three separate experiments. C, D) Detection of 4-HNE-adducts (C) or ubiquitinated proteins (D)
in lysates of cells incubated for the indicated time without (Co, control) or with 200 mg/ml of oxLDL (oxL) or native (natL)
or FeMb or 1 mM 4-HNE. Western-blot experiments were done using anti-4-HNE-protein or anti-ubiquitin or anti-b-actin (as
control) antibodies, used under the conditions indicated in Materials and Methods.

LDL was associated with derivatization of cell Biphasic effect of toxic concentrations of oxLDL
proteins by 4-HNE, a lipid peroxidation derivative on the proteasome activity
able to react with free amino groups of proteins
(Fig. 1C). This derivatization was mimicked by OxLDL induced a time- and dose-dependent tran-
oxLDL lipid extracts (data not shown) and by sient activation of in situ (i.e., in intact living cell)
4-HNE (1 mM) (Fig. 1A, C). intracellular proteolysis of both short-lived and long-
In the same time, an increased level of high- lived [35S]-radiolabelled proteins (Figs. 2A, B). In
molecular-mass (HMM) ubiquitin-protein conju- contrast, native (nonoxidized) LDL did not. Toxic
gates (HMM . 200 kDa) was observed in cells concentrations of oxLDL (200 mg apoB/ml) evoked
incubated with oxLDL but not in cells incubated a rapid and transient peak of in situ proteolysis
with native LDL or FeMb (used alone) (Fig. 1D). (maximum between 1 and 3 h) followed by a return
Thus, in cells treated by oxLDL, some proteins are to the ground level at 5–7 h. It may be noted that
probably recognized as structurally abnormal and lower oxLDL concentration (50 mg apoB/ml) also
tagged with ubiquitin probably for subsequent deg- induced a transient peak of intracellular proteolysis
radation through the ubiquitin-dependent proteo- that occurred later (maximum between 5 and 7 h)
lytic pathway. This led us to evaluate the proteasome and then return slowly to the basal level (at 15 h)
activity in cells incubated with oxLDL. (Fig. 2A).

UBIQUITIN-PROTEASOME PATHWAY IN oxLDL-INDUCED APOPTOSIS 535

 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 apo-
ptosis in ECV-304 cells. Because accumulation of
oxidized (or otherwise altered) proteins is poten-
tially 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 re-
lated or parallel unrelated events. This was investi-
gated by using proteasome inhibitors and ubiquiti-
nation-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
Figure 2. Effect of oxLDL on the proteasome activity (i.e., in experiments) but were effective in inhibiting protea-
situ proteolysis and in vitro hydrolysis of sLLVY-MCA) in some activity [i.e., inhibiting both the early peak of
ECV-304 cells. A) Time course of short-lived (squares) and oxLDL-induced autoproteolysis (data not shown)
long-lived (triangles) protein degradation in cells prelabeled and sLLVY-MCA hydrolysis (Fig. 2D)]. This concen-
with [35S]methionine/cysteine for 2 h (short-lived) or 16 h tration of proteasome inhibitors potentiated both
(long-lived) and incubated with 200 or 50 mg/ml of OxLDL
(filled and empty symbols, respectively). B) Effect of increas- the accumulation of ubiquitinated proteins and the
ing concentrations of oxLDL or native LDL (filled and empty oxLDL-induced toxicity. Proteasome inhibitors po-
symbols, respectively) (incubation time 5 h) on short-lived tentiated rapidly the accumulation of ubiquitinated
(squares) and long-lived (triangles) protein degradation. C) proteins (data not shown) probably because of the
Time course of in vitro sLLVY-MCA hydrolysis by lysates from inhibition of oxLDL-induced early peak of protea-
cells incubated for the indicated time with 200 mg/ml of
oxLDL or native LDL (filled circles and empty squares, some activation.
respectively). As a comparison, the in situ proteolysis, trig- The potentiation of the oxLDL-induced toxicity by
gered under the same conditions (i.e., by 200 mg/ml of proteasome inhibitors was obvious at low (non- or
oxLDL), is indicated by the dashed line. D) Effect of protea- slightly toxic) oxLDL concentrations, because, in the
some inhibitors, LLnL (5 mM) and PSI (5 mM) on the in situ presence of inhibitors, the toxic effect of oxLDL to
proteolysis (black bars) and on the in vitro hydrolysis of
LLVY-MCA (hatched bars) (both activities being determined ECV-304 cells occurred faster and at lower concen-
at 3 h under standard conditions). Means 6 se of at least four trations (Figs. 3A, B). For instance, when cells were
separate experiments. incubated with low oxLDL concentration (50 mg
apoB/ml) in the absence of proteasome inhibitor,
Toxic concentrations of oxLDL (200 mg apoB/ml) no significant toxicity was observed (Figs. 3B, C),
induced also a biphasic effect on the in vitro hydro- thus suggesting that the toxic threshold dose of
lysis of sLLVY-MCA (a fluorogenic synthetic peptide oxLDL (43) was not reached. In contrast, when the
substrate for 20S proteasome) (Fig. 2C), but the time same experiment was performed in the presence of
course was different from that of in situ proteolysis. proteasome inhibitors, cells underwent apoptosis
The in vitro hydrolysis of sLLVY-MCA began to rise at (Fig. 3C). This suggests that proteasome inhibition
2 h, then reached a plateau (sustained for 3– 4 h), lowered the threshold of oxLDL concentration in-
and finally decreased toward the baseline (back at ducing the cytotoxicity.
15 h). Proteasome inhibitors did not alter the type of cell
Despite different time courses, both in situ proteoly- death induced by oxLDL (Fig. 3D–G), because ECV-
sis and in vitro hydrolysis of sLLVY-MCA resulted very 304 EC killed by low (not toxic per se) oxLDL
probably from proteasome activity, as assessed by inhi- concentrations in the presence of proteasome inhib-
bition by the cell permeant inhibitors of proteasome, itors exhibited the characteristic features of apopto-
LLnL (5 mM), and PSI (5 mM) (Fig. 2D). sis, quite similarly to cells killed by toxic concentra-
It may be noted that intracellular accumulation of tions of oxLDL (15).
altered or derivatized proteins is obvious as early as These data suggest that proteasome activity may
5 h (Fig. 1) and persisted for the duration of the participate in the cellular defenses against oxLDL-

536 Vol. 14 March 2000 The FASEB Journal VIEIRA ET AL.

Figure 3. Proteasome inhibitors potentiate the effect of oxLDL on ECV-304 cells. A–G) Effect of proteasome inhibitors, LLnL
(5 mM), Lc (5 mM), and PSI (5 mM) on the whole toxicity (A, B) and apoptosis (C–G) induced by oxLDL. A) Time course of
toxicity using 200 mg apoB/ml of LDL or native LDL (empty circles); B) Dose dependence of the toxic effect (determined at
24 h) induced by increasing concentrations of oxLDL (filled symbols) in the absence (filled circles) or presence of proteasome
inhibitors LLnL, Lc, and PSI (filled squares and triangles, respectively). C) Apoptosis evaluated by morphological counting after
18 h incubation with low (nontoxic) concentration (50 mg apoB/ml) of oxLDL in the presence or absence of proteasome
inhibitors LLnL (5 mM), Lc (5 mM), and PSI (5 mM). Means 6 se of three separate experiments. D–G) Double fluorescent
staining of cell nuclei (by SYTO-13 and propidium iodide) discriminating normal cells and cells undergoing apoptosis (A),
primary necrosis and postapoptotic necrosis (AN). E) Untreated cells. E–G) Cells incubated for 18 h with 50 mg apoB/ml of
oxLDL in the absence (E) or presence (F) of 5 mM PSI, or with PSI alone (G). (*P,0.01).

induced toxicity and, conversely, that inhibition of (25), we investigated whether the ubiquitin pathway
endogenous proteolysis lowered the toxic threshold is involved in the cellular defenses against oxLDL
dose of oxLDL. toxicity by using genetically engineered cells (de-
It may be noted that the oxLDL toxicity and its rived from the E1-thermo-sensitive ts20 cell line). As
potentiation by proteasome inhibitors is not restricted previously reported (36, 37), ubiquitination was ef-
to endothelial cells. In rabbit arterial smooth muscle fective at 34°C in the three cell lines, and at 39°C in
cells, after 24 h incubation with 200 mg apoB/ml of the E1-transduced H38 –5, but was defective at 39°C
oxLDL in the presence or absence of 10 mM PSI, the in ts20pMV12 and ts20Bcl-2#7 cell lines (both in the
viability was 33 6 4% and 78 6 8%, respectively. In the presence or absence of oxLDL; Fig. 4A).
U937 monocytic cell line, after 24 h incubation with The relative susceptibility of the three cell lines to
100 mg apoB/ml of oxLDL in the presence or absence the toxic effect of oxLDL was compared at 34 and
of 10 nM PSI (this cell line is very susceptible to PSI), 39°C. The toxicity of oxLDL (200 mg apoB/ml) to
the viability was 30 6 5% and 60 6 7%, respectively (as H38 –5 cells (in which ubiquitination is always work-
assessed by the trypan blue test). ing) was quite similar at 34 and 39°C. In contrast,
both ts20pMV12 and ts20Bcl-2#7 cell lines were
Defective ubiquitination potentiates oxLDL- much more susceptible to the toxic effect of oxLDL
induced apoptosis at 39°C (defective ubiquitination) than at 34°C (ef-
fective ubiquitination) (Fig. 4B). These data suggest
As the degradation of altered proteins by protea- that the ubiquitin pathway plays a role in the cellular
some may be ubiquitin-dependent or -independent defenses against oxLDL-induced toxicity.

UBIQUITIN-PROTEASOME PATHWAY IN oxLDL-INDUCED APOPTOSIS 537

 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 prevent-
ing oxLDL-induced cell death. In contrast, in agree-
ment 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 inhibi-
tion of cell protein derivatization should inhibit the
toxic effect of oxLDL. Dinitrophenylhydrazine, a
well-known reagent of lipid peroxidation-derived al-
dehydes, 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 concentra-
tion) 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.

DISCUSSION

OxLDL toxicity may potentially be involved in the

genesis of the necrotic core and of complicated
atherosclerotic plaques prone to plaque rupture and
thrombosis. The mechanism and type of cell death
occurring in atherosclerotic areas may be of impor-
tance, because (in principle) apoptotic cells are
rapidly engulfed and cleared, whereas necrotic cell
debris may trigger a local inflammatory response. It
Figure 4. Influence of ubiquitination on the cytotoxicity of
oxLDL in ts20 engineered cell lines ts20pMV12. H 38 –5 and is therefore of interest to better understand the
ts20Bcl-2#7 (ubiquitination is effective in the three cell lines cellular mechanisms regulating the susceptibility of
at 34 and 39°C in the H38 –5 cell line, whereas, it is defective cells to oxLDL cytotoxicity.
at 39°C in ts20pMV12 and ts20Bcl-2#7) (37). A) Western blot To our knowledge, this is the first study linking the
showing the ubiquination levels of the three cell lines incu- toxicity induced by oxLDL with cell protein alter-
bated at 39°C for 5 h in the absence or presence of oxLDL
(200 mg apoB/ml). B) Evaluation of the cytotoxicity of oxLDL ations and proteasome inhibition. Conversely, it is
(200 mg apoB/ml for 18 h) at 34 and 39°C. also suggested that the ubiquitin-proteasome path-
way plays a role in the cellular defenses against the
It may be noted that the Bcl-2 overexpressing toxic effect of oxLDL.
ts20Bcl-2#7 was used in order to examine whether, in The reported data show that lipid peroxidation
this model system (ubiquitin-defective), the oxLDL- derivatives (among them 4-HNE) contained in ox-

538 Vol. 14 March 2000 The FASEB Journal VIEIRA ET AL.

Figure 5. DNPH prevents cell protein derivatization and cytotoxicity induced by oxLDL on ECV-304 cells. Cultured cells were
incubated with or without 200 mg apoB/ml oxLDL in the absence (vehicle only) or presence of 100 mM DNPH (dissolved in
DMSO). A, B) 4-HNE-adducts were evaluated at 16 and 24 h on western-blot probed with anti-4-HNE antibody (A), and the
whole derivatization was determined at 24 h by titration of free reactive-amino groups by [3H]NSP (B), under the conditions
of Fig. 1. C) Cytotoxicity was evaluated at 24 h by the MTT test. Means 6 se of three separate experiments.

Figure 6. Role of the ubiquitin-proteasome pathway on cell survival (black). Sites of action and potential interferences of toxic
concentrations of oxLDL (red), proteasome inhibitors, lactacystin and PSI (blue), and ubiquitination defect (temperature
sensitive cells) (green). The colored arrows indicate the observed variations and their relationship with oxLDL or/and
inhibitors.

UBIQUITIN-PROTEASOME PATHWAY IN oxLDL-INDUCED APOPTOSIS 539

LDL are able to derivatize cell proteins, in agree- This transient proteasome activation was followed
ment with the observation of Rosenfeld et al. (18) in by a decay of proteasomal proteolysis toward the
macrophagic cells. Until now, the possible interac- baseline. This decay resulted neither from a sub-
tions between lipid peroxidation end products con- strate (modified proteins) depletion, because high
tained in oxLDL and cell proteins have been only levels of derivatized proteins and ubiquitinated-
poorly investigated in contrast to apoB modifica- HMM still persisted up to 18 h, nor from an irrevers-
tions, which are well documented (see reviews in refs ible inhibition of the 20S proteasome activity, be-
4, 6). Our data suggest that 4-HNE, a major aldehy- cause proteasome was able to hydrolyze in vitro the
dic lipid peroxidation derivative able to form ad- fluorogenic substrate sLLVY-MCA up to 10 –12 h.
ducts with lysine, histidine, or cysteine of proteins Altogether, these data led us to hypothesize a two-
(16, 17), seems to play a major role in oxLDL- step mechanism of inhibition affecting first the 19S
induced derivatization of cell proteins. But, it is not and later the 20S. The rapid decay of endogenous
excluded that other reactive compounds of oxLDL proteolysis may be a result of inhibition of the 19S
[e.g., malondialdehyde, fatty acid peroxides (9)], (thus inhibiting de-ubiquitination and degradation
may also derivatize cell proteins. of ubiquitinated proteins), because 4-HNE cross-
The dramatic accumulation of ubiquitinated pro- linked proteins are resistant to proteolysis and are
teins may result from two additional mechanisms. able to inhibit the 26S proteasome (45) and because
During the early time of incubation (up to 3 h with Reinheckel et al. (46) reported that the 26S protea-
toxic concentrations of oxLDL), the rise of ubiquiti- some is less resistant to H2O2-induced oxidative
nation cannot result from a defect in the de-ubiquiti- stress than the 20S proteolytic core. The second step
nation process (by the 19S complex) because the [i.e., inhibition of the 20S core (involved in sLLVY-
whole activity of the 26S proteasome is high and may MCA hydrolysis)] may be a result of the progressing
result from an activation of the ubiquitination path- intracellular oxidative stress induced by oxLDL (47;
way. This may be because of the oxLDL-induced Fig. 6). At this stage, when the proteasome is com-
protein derivatization, given that 4-HNE is able to pletely inhibited, cells are rapidly dying. This led us
induce both protein derivatization and proteasome to investigate the relationship between proteasome
activation (44). After 5 h, the inhibition of the 26S inhibition and the oxLDL-induced cytotoxicity, be-
proteasome may constitute an additional mechanism cause, according to the model systems, proteasome
of accumulation of ubiquitinated proteins (Fig. 6). may participate in the apoptotic process or prevent it
OxLDLs affect, in a biphasic manner, the protea- (26 –31).
some activity by inducing an early transient activa- The reported data strongly suggest that the active
tion of proteasome followed by a sustained decay. proteasome plays a role in the cellular defenses
The early and transient peak (between 1 and 3 h against the oxLDL-induced cytotoxicity, and, con-
with 200 mg apoB/ml of oxLDL) of proteolysis versely, that proteasome inhibition may be involved
resulted from proteasome activation, as assessed by in the mechanism of cytotoxicity.
the use of proteasome inhibitors (whereas the basal The protective effect of proteasome was (at least in
proteolysis was probably proteasome-independent part) dependent on ubiquitination, because at 39°C
because it was not affected by proteasome inhibi- the ubiquitin-defective ts20pMV12 and ts20Bcl-2#7
tors). This early proteasome activation may be sub- cells were more susceptible to the toxic effect of
sequent to oxLDL-induced structural alterations and oxLDL than the ubiquitin-expressing H38 –5 cells. It
ubiquitination of cellular proteins, which are valu- is noteworthy that the higher susceptibility of the
able substrates for the proteasome (23–25). Activa- ubiquitin-defective cells seems to be independent of
tion of endogenous proteolysis and of sLLVY-MCA heat-shock toxicity because in ts20pMV12, the time
hydrolysis exhibited different time courses (the rise courses of the oxLDL-induced and heat shock-in-
of sLLVY-MCA hydrolysis beginning 2 h after that of duced toxicity are different (beginning at 12–15 and
autoproteolysis). The activation of in vitro degrada- after 24 h, respectively); in ts20Bcl-2#7 cells, the heat
tion of sLLVY-MCA synthetic substrate (cannot be shock-induced apoptosis is blocked by Bcl-2 overex-
attributed to substrate generation) may result from pression (36, 37) in contrast to oxLDL-induced
activation of the 20S proteasomal core by oxLDL. apoptosis. The inefficiency of Bcl-2 in preventing the
OxLDL may act either 1) by generating a cellular oxLDL-induced toxicity to ts20Bcl-2#7 cells is quite
oxidative stress (47), which is known to activate the consistent with our recently reported data, which
20S proteasome (and subsequently, NFkB) (48, 49), show that Bcl-2 overexpression alters only the bal-
or 2) by activating protein kinases (22, 51, 52), some ance between apoptosis and necrosis but does not
of them being potentially able to phosphorylate and prevent cell death triggered by oxLDL (41). Alto-
activate (50) the PA28 proteasome activator (which gether, these data strongly suggest that ubiquitina-
can in turn stimulate synthetic substrate hydrolysis by tion plays a role in the cellular resistance against the
the 20S proteasome). toxicity of oxLDL.

540 Vol. 14 March 2000 The FASEB Journal VIEIRA ET AL.

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542 Vol. 14 March 2000 The FASEB Journal VIEIRA ET AL.