© 2001 American Heart Association, Inc.
Vascular Biology |
Overexpression of Inducible Heat Shock Protein 70 in COS-1 Cells Fails to Protect From Cytotoxicity of Oxidized LDLs
From the Institute of Pharmacological Sciences (A.P., G.D.N., T.Z., A.L.C.), University of Milano, and Centro per lo Studio (A.L.C.), la Prevenzione e la Terapia delle Vasculopatie Arteriosclerotiche, Ospedale Bassini, Milano, Italy.
Correspondence to Prof Alberico L. Catapano, Institute of Pharmacological Sciences, Via Balzaretti, 9, 20133 Milano, Italy. E-mail alberico.catapano{at}unimi.it
 |
Abstract |
|---|
 Top Abstract IntroductionMethodsResultsDiscussionReferences |
|---|
Abstract—Oxidized low density lipoproteins (OxLDLs) are believed to play a central role in atherogenesis and to possess a wide variety of biological properties; among them, OxLDLs are cytotoxic to cultured vascular cells in that they induce necrosis and apoptosis. Moreover, OxLDLs are known to induce the expression of heat shock protein 70 (Hsp70), a protein that protects cells from several cytotoxic stimuli. To determine whether Hsp70 can protect cells against OxLDL-induced cytotoxicity, COS-1 cells were transfected with a construct containing human Hsp70. A number of cell lines permanently expressing Hsp70 were obtained, 1 of which (cos-Hsp70/10, with high Hsp70 expression) was selected for further studies. Hsp70 overexpression protected cells from toxic stimuli, such as H2O2, UV irradiation, and heat shock, suggesting that the overexpressed protein was functional. When incubated with OxLDLs, however, the clone overexpressing Hsp70 showed a significant decrease in viability, as determined by the [3H]adenine release assay (319.8±3.16% of control for transfected cells versus 217.6±6.08% for control cells exposed to 100 µg protein/mL of OxLDL), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay (12.5±0.9% versus 28.9±1.99% of control, respectively), and LDH release (48.4±0.04% versus 15.2±0.06% of control cells). The increased expression of Bax and the decreased expression of Bcl-2 (a proapoptotic and an antiapoptotic protein, respectively) in cos-Hsp70/10 cells and in control cells on incubation with OxLDLs suggested that overexpression of Hsp70 did not confer protection against apoptosis induced by OxLDLs. The analysis of nucleosome content and the nuclear staining with Hoechst 33258 confirmed this finding. These data suggest that overexpression of Hsp70 not only fails to protect COS-1 cells against OxLDL-induced apoptosis but rather confers a higher sensitivity to the cytotoxic action of these lipoproteins. Thus, the Hsp70 response, although induced by OxLDLs, cannot protect cells from lipoprotein toxicity.
Key Words: low density lipoproteins • apoptosis • necrosis • Bcl-2 • Bax
|
Introduction |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
When exposed to stresses, such as high temperature or other toxic stimuli, cells react by synthesizing stress proteins, also named heat shock proteins (Hsps).1 2 3 4 5 6 Hsps include constitutive and inducible forms. Constitutive Hsps play important biological roles, including facilitation of the correct folding of newly synthesized polypeptides7 and their transport across cellular membranes8 9 10 ; inducible forms, expressed by cells under stress conditions, are believed to protect cellular proteins from denaturation.11 Hsps show a very high degree of homology among different organisms12 ; Hsp70, the major Hsp, is the most conserved throughout evolution13 and plays a key role in protecting cells from environmental stresses.14 In fact, overexpression of Hsp70 confers thermotolerance and increases cell survival in the presence of noxious stimuli.15 16 17 18 19 Interestingly, the cellular content of Hsp70 is directly related to the degree of myocardial protection from ischemia in rat hearts20 and in hearts from transgenic mice overexpressing Hsp70.21
Oxidized LDL (OxLDL), a modified lipoprotein that is toxic to the cells of the arterial wall,22 23 probably because it induces apoptosis,24 25 increases the expression of Hsp70 in human endothelial cells26 and in smooth muscle cells,27 suggesting the possibility that Hsp70 may represent a cellular defense against OxLDL toxicity.28 These observations raise the question of whether high levels of Hsp70 expression could protect cells from OxLDL cytotoxicity. To address this issue, we have produced stable transfectants overexpressing the inducible form of the human Hsp70 and investigated whether high intracellular levels of Hsp70 may protect cells from the apoptosis induced by OxLDL.
|
Methods |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Cells
The COS-1 cell line29 was grown at 37°C in a humidified atmosphere of 5% CO2 in MEM (Sigma Chemical Co) supplemented with 10% FCS (Sigma), 100 U/mL penicillin, 0.1 mg/mL streptomycin, 20 mmol/L tricine buffer, and 1% (vol/vol) nonessential amino acid solution.
Cell Transfection
Hsp70 (plasmid pH 2.3, kindly provided by Dr R.
Morimoto)13 was
subcloned into the pBK-CMV vector (Stratagene), which allows
eukaryotic and prokaryotic expression. In the former case,
expression is driven by the cytomegalovirus (CMV) immediate-early
promoter, and transfected cells are selected in the presence of G418
(neomycin, 800 µg/mL, Life Technologies Italia). COS-1 cells were
transfected by the calcium coprecipitation
method.30 Cells were seeded
at 5x105 cells per 10-cm Petri dishes in 10
mL of complete medium the day before transfection. Plasmid DNA (20
µg) was diluted to 1 µg/µL with TE buffer (10 mmol/L Tris
and 1 mmol/L EDTA, pH 8.0). The DNA solution was mixed with 500
µL of 0.25 mol/L CaCl2, and then 500 µL of
2x
N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic
acid (pH 6.95) was added, followed by a 20-minute incubation at room
temperature. The calcium-phosphate-DNA solution was added dropwise to
the medium in the dishes. Cells were incubated for 24 hours at 35°C
in 3% CO2. At the end of the incubation, cells
were washed twice in PBS (pH 7.4) and incubated overnight in complete
medium at 37°C in 5% CO2. Cells were then
divided 1:10, and permanently transfected cells were selected by growth
in 800 mg/mL of the antibiotic G418, resistance to which is dependent
on the neomycin resistance gene on the plasmid vector. Neomycin (400
µg/mL) was used to maintain the clones in culture. Cells transfected
by use of a plasmid DNA in which the Hsp70 gene was lacking were used
as controls.
[3H]Thymidine
Incorporation
The growth of transfected cells was evaluated by the
[3H]thymidine incorporation
assay.31 Cells were plated in
35-mm wells at different densities (3125 and 6250
cells/cm2) in MEM+10% FCS. After 20 hours,
1 µCi/mL of [3H]thymidine in fresh
medium was added, and the incubation was carried out for 3 hours. At
the end of the incubation, the media were removed, and the cells were
washed 3 times with PBS and incubated with 10% trichloroacetic acid
(TCA) for 5 minutes. Free [3H]thymidine
(non–TCA precipitable) was removed by washing the cell layer once with
10% TCA and once with distilled H2O. After the
washing, 0.75 mL of 0.1N NaOH was added to each well. Aliquots (200
µL) were used to measure the cell-associated radioactivity, and the
remaining part of the sample was used to measure the protein
content.32 The
cell-associated radioactivity was normalized for cellular protein
content (counts per minute per microgram
protein).
Lipoproteins
Human LDL (1.019 to 1.063 g/mL) was isolated by
sequential
ultracentrifugation33
from freshly isolated human plasma from normolipidemic volunteers by
use of a Beckman 50.2 Ti rotor at 4°C, dialyzed against 0.15 mol/L
NaCl and 0.01% EDTA, and sterilely filtered by passage through a
0.45-µm filter (Corning). Before oxidation, EDTA was removed from LDL
by a passage on a Sephadex G25 column (PD-10, Pharmacia) in PBS. LDLs
were oxidized under sterile conditions at a concentration of 0.2 mg
protein/mL with 20 µmol/L CuSO4 at 37°C for
24 hours.26 The oxidation was
blocked by the addition of 40 µmol/L butylated hydroxytoluene. Under
these conditions, we have previously shown that LDL oxidation does not
proceed further at 4°C. LDLs were used within 2 days from preparation
because the experiments were strongly oxidized, as shown by the level
of thiobarbituric acid–reactive substances (0.99 versus 0.043 µg
MDA/µg protein of native LDL), and had an electrophoretic mobility
relative to native LDL of 2.4.
Immunoblotting
To verify the overexpression of Hsp70, cells were
plated in 6-well plates for 24 hours and then lysed with the use of a
Tris-glycine buffer (0.25 mol/L Tris and 0.173 mol/L glycine)
containing 3% SDS and 1 mmol/L phenylmethylsulfonyl
fluoride, and the protein content was evaluated by the method
of Lowry et al,32 with BSA
used as a standard. Aliquots of the samples (10 µg) were diluted in a
2% ß-mercaptoethanol buffer containing glycerol and bromophenol blue
and electrophoresed on 8%
SDS-PAGE34 and then
transferred onto a nitrocellulose
membrane35 with use of a
Trans Blot Cell (Hoefer Scientific Instrument). The membrane was
saturated at 25°C in PBS containing 3% BSA for 1 hour, washed with
PBS containing 0.1% Tween 20, and then incubated for 1 hour at 25°C
with a mixture of mouse monoclonal antibody specific for the inducible
form of Hsp70 (Hsp72, 1:1500, C92F3A-5, StressGen) and anti–ß-actin
antibody (1:5000, Sigma). An anti-mouse IgG peroxidase conjugate
(1:5000, Sigma) was used as a second antibody; enhanced
chemiluminescence (Amersham) and autoradiography
(X-OMAT, Kodak) followed. The bands were quantified by a
computer-assisted system for image analysis (ISF Image 1.47),
and the expression of Hsp70 was evaluated as the ratio of Hsp70 to
ß-actin to correct for cell number.
To verify the expression of Bax and Bcl-2, cellular proteins were subjected to Western blotting as described above. The membrane was saturated with 3% BSA/PBS and then incubated with a polyclonal antibody specific for Bax (1:200, Amersham) or a polyclonal antibody specific for Bcl-2 (1:200, Amersham) and with an anti–ß-actin antibody (1:5000, Sigma) for 1 hour. As a second antibody, an anti-rabbit or anti-mouse IgG peroxidase conjugate (Bio-Rad) was used; enhanced chemiluminescence and autoradiography followed.
Cell Viability
Cell viability was evaluated after exposure to toxic
agents by a [3H]adenine release
assay,36
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
test,37 and LDH release
assay.38 For
[3H]adenine release, cells were seeded in
12-well plates (6x104 cells per well) in
MEM containing 10% FCS; after 24 hours, the cells were incubated with
[3H]adenine (1 µCi/mL) in adenine-free
medium for 3 hours, washed 3 times with PBS, and incubated overnight in
1 mL of serum-free medium containing OxLDL (up to 100 µg of LDL
protein/mL). The incubation medium was collected and
centrifuged at 800 rpm, and 200-µL aliquots were counted in a
ß-counter (Packard) to quantify the radioactivity released from the
cells into the medium. One milliliter of 0.25N NaOH was added to the
cell monolayers for 3 hours at room temperature, and 200-µL aliquots
were used to determine the cell-associated radioactivity. The
[3H]adenine release was calculated as the
ratio of radioactivity released in the medium to the medium plus cell
(total)
radioactivity.39
For the MTT assay, cells in 12-well plates (6x104 cells per well) were exposed to different stresses. At the end of the experiment, the media were removed, and the cells were incubated for a further 3 hours in RPMI 1640 medium (red phenol free, Sigma) containing MTT (1 mg/mL, Sigma). Isopropanol was used to dissolve the formazan salt crystals, and the optical density at 550 nm was read by an automatic plate reader (Titertek Multiscan 2, Bio-Rad). Cell viability was determined as the ratio of the stress-exposed cells versus control cells. OxLDLs were added in serum-free medium at increasing concentrations (100 to 400 µg protein/mL) for 18 hours. To evaluate the effect of different types of stress on cell viability, MTT was added immediately after heat shock (45°C, 40 to 80 minutes), and the incubation continued for 3 hours. MTT was added again at the end of the recovery time (16 hours), after UV irradiation (10 to 30 minutes), and at the end of the incubation with H2O2 (18 hours).
For LDH release assay, at the end of the experiment, the culture supernatants were collected and centrifuged at 800 rpm for 10 minutes. Aliquots (100 µL) of the supernatants were transferred into wells of a 96-well flat-bottomed microtiter plate, and 100 µL reaction mixture (Cytotoxicity Detection Kit [LDH], Boehringer-Mannheim) was added to each well. After a 30-minute incubation at room temperature in the dark, the absorbance was read at 490 nm.
Immunocytochemistry
Cells were cultured on coverslips in 24-well plates
(1.5x104 cells per well). After incubation
with OxLDL, the media were removed, the cells were fixed with 3%
paraformaldehyde for 20 minutes and washed twice with
PBS, and a solution of 0.2% Triton X-100 was added for 2 minutes at
4°C. After they were washed twice with PBS, the fixed cells were
incubated with a polyclonal antibody for
Bax or
Bcl-2 (1:20) for 1 hour at room
temperature. As a second antibody, an anti-rabbit IgG biotin conjugate
(1:200, Sigma) was used for 1 hour; incubation with streptavidin-FITC
(1:200, Sigma) followed for 30 minutes. After they were washed, the
coverslips were mounted with Vectashield (Vector Laboratories Inc) and
analyzed with a Zeiss Axioscop microscope at x400
magnification and photographed with 400 ASA Kodak Elite
film.
Nuclear morphology was analyzed with the fluorescent dye bis-benzimide (Hoechst 33258). Cells cultured on coverslips were incubated with medium control and OxLDL, the media were then removed, and the cells were fixed with 3% paraformaldehyde for 20 minutes. The fixed cells were incubated with a 1:10.000 solution of Hoechst 33258 for 30 minutes and washed twice with PBS. The coverslips were mounted with a solution of water and glycerol (1:1), analyzed under UV light with a Zeiss Axioscop microscope at x400 and x1000 magnifications, and photographed with 400 ASA Kodak Elite film.
Nucleosome ELISA
The nucleosome ELISA (Oncogene) allows the
quantification of apoptotic cells in vitro by DNA
affinity-mediated capture of free nucleosomes, followed by their
antihistone-facilitated detection. At the end of the incubation with
OxLDL, cells were lysed and frozen for 18 hours at -20°C. After
appropriate dilutions, aliquots of the samples were added to each well
and incubated at room temperature for 3 hours. The wells were washed 3
times with wash buffer and incubated for 1 hour at room temperature
with a biotinylated polyclonal anti–histone 3 antibody, followed by a
30-minute incubation with peroxidase streptavidin conjugate and a
30-minute incubation with a chromogenic substrate. After
addition of the stop solution, the absorbance was measured by using a
spectrophotometric plate reader at dual wavelengths of 450/595
nm.
Statistical Analysis
Data presented in the text and figures are
mean±SD and are representative of 4 different
experiments. Statistical analysis was performed by ANOVA with
use of the Statsoft Statistica
Package.
|
Results |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Expression of Hsp70 in transfected cells was determined by immunoblotting, and clones were chosen on the basis of their relative content in Hsp70 normalized for ß-actin content. Ten clones permanently transfected with Hsp70 were obtained; among them, 1 was chosen as a clone with the highest expression (cos-Hsp70/10, 0.639±0.19 Hsp70/ß-actin) as determined by immunocytochemistry and immunoblotting (Figure 1
). The expression of Hsp70 remained constant with
culture time and passages (up to 15). Control cells
(pBk2, 0.345±0.11
Hsp70/ß-actin) were obtained by transfection of COS-1 cells with the
same plasmid devoid of the Hsp70 gene; the transfection with the pBk2
plasmid did not result in a higher expression of Hsp70 compared with
that in nontransfected cells (not shown).
|
To verify that the overexpression of Hsp70 could
protect cells from the classic stimuli known to induce the expression
of this stress protein, transfected cells were exposed to toxic agents
such as H2O2, heat, and
UV irradiation. Compared with
pBk2 cells,
cos-Hsp70/10 cells were
protected against the toxicity of
H2O2 as determined by the
MTT test
(Figure 2a).
Cos-Hsp70/10 cells were also
protected from UV irradiation
(Figure 2b) and from heat shock
(Figure 2c).
|
Because OxLDLs are toxic to cells in
culture22 40 and
because Hsp70 plays a protective role against a number of toxic
stimuli,1 2 3 4 5 6 14
we addressed the question of whether Hsp70 overexpression could protect
cells from OxLDL-mediated toxicity. In a preliminary set of
experiments, transfected COS-1 cells
(pBk2 and
cos-Hsp70/10 clones) were
incubated in the presence of increasing concentrations of native LDL,
CuSO4, and butylated hydroxytoluene in the same
conditions used for the experiments with OxLDLs. As shown in
Figure 3, we demonstrated that under these experimental
conditions, there was no LDH release, and only a minor decrease of the
cell number (MTT test), suggesting a very limited, if any, toxicity.
Moreover, under these experimental conditions, the expression of
Bax and
Bcl-2 was not affected
(Figure 3). The same results were obtained with control cells
(pBk2, data not
shown).
|
In cells overexpressing Hsp70, the incubation with
OxLDLs induced a significant increase of
[3H]adenine release
(Figure 4a). The MTT test and LDH release assay confirmed
this finding: after incubation with OxLDLs,
cos-Hsp70/10 cells were less
viable than were the control cells
(Figure 4b and 4c). These data suggest not only that the
overexpression of Hsp70 fails to protect cells against OxLDL toxicity
but that it may confer a higher sensitivity to the cytotoxic action of
OxLDLs.
|
We have previously reported that OxLDLs are cytotoxic to proliferating cells but not to quiescent cells41 ; therefore, we sought to determine whether Hsp70 overexpression affected cell proliferation, as determined by the [3H]thymidine incorporation assay. Growth rates for all clones were similar to the control condition (data not shown). Therefore, different proliferation rates may not explain differences of sensitivity of the different clones to the OxLDL cytotoxicity.
It is known that OxLDLs induce apoptosis in cells of
the vascular
wall24 42 ; for this
reason, we sought to determine whether the
cos-Hsp70/10 cell line
undergoes apoptosis while incubated in the presence of OxLDLs.
Several lines of evidence suggest that this is the case: (1) For cell
morphology, cells incubated with OxLDL (80 µg/mL) showed shrinkage
and blebs, characteristic alterations of apoptotic cells
(Figure 5A and 5B). (2) For nuclei morphology, the staining
with Hoechst 33258 showed, in cells incubated with OxLDLs (80 µg/mL),
condensed nuclei and the appearance of 
1 apoptotic body
containing DNA fragments
(Figure 5C through 5F), with no differences between
pBk2 and
cos-Hsp70/10 cells. (3) To
confirm these findings, we studied the expression of
Bax and
Bcl-2, 2 proteins involved in
the apoptotic
pathway.43 44 In
cos-Hsp70/10 cells incubated
with OxLDLs (80 µg/mL), an increased expression of
Bax, a proapoptotic
protein,44 was detected
(Figure 6A and 6B), whereas
Bcl-2 expression, an
antiapoptotic
protein,44 decreased
(Figure 6D and 6E) compared with that in control cells. This
finding was confirmed by immunoblotting
analysis of cell protein extracts
(Figure 6C and 6F), with a clear increase of
Bax in both clones but a more
evident decrease of Bcl-2 in
the transfected cells. In pBk2
cells incubated with 80 µg OxLDL/mL, the ratio of
Bax to
Bcl-2 in cells increased from
0.22 (basal value) to 0.99 and from 0.32 to 1.91 in
cos-Hsp70/10 cells, further
stressing the proapoptotic state in these conditions. (4) The
analysis of the nucleosome content, an index of
apoptotic death, showed a larger and significant increase in
nucleosomes in cos-Hsp70/10
cells incubated with OxLDL (60 µg/mL) compared with
pBk2 cells under the same
experimental conditions
(Table,
P<0.005).
|
|
|
|
Discussion |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
The inducible form of Hsp70 is produced by cells in the presence of toxic stimuli, such as oxidative injury,1 2 3 4 5 6 and Hsp70 overexpression elicits a cytoprotective function in response to several stresses.1 2 3 4 5 6 14 OxLDLs represent an oxidative stress to cells, and this effect could account for their ability to induce Hsp70 in cultured cells.26 27 Because OxLDLs are cytotoxic to several cell types, we speculated that the Hsp response to OxLDLs could represent a protective response. To address this issue, we permanently transfected COS-1 cells with Hsp70. COS-1 cells are widely used in transfection experiments, including transfection with Hsp.45 46 From our transfectants, 1 clone with a high expression of Hsp70 was selected. This clone, as expected, was protected against toxic stimuli such as H2O2, heat shock, and UV irradiation, in agreement with in vitro and in vivo data on the role of this protein in protecting from cellular damage.15 16 17 18 19 20 21 However, when transfected cells were incubated with OxLDL, overexpression of Hsp70 not only failed to protect against OxLDL cytotoxicity but rather conferred a higher sensitivity. This may be due to either necrotic or apoptotic cell death. The latter is more likely, inasmuch as several in vitro observations suggest that OxLDLs induce apoptotic death in vascular wall cells.24 25 42
Why Hsp70 overexpression confers hypersensitivity to OxLDL cytotoxicity is unclear. In vivo, the Hsp70 inducible form has been detected in areas of the arterial wall exposed to stresses, such as high blood pressure or oxidative burst.47 48 This evidence, although circumstantial, suggests a protective role for Hsp70. Paradoxically the stress response, although cytoprotective against these stimuli, appears to "sensitize" cells to the cytotoxic action of OxLDL. We have recently shown that the Hsp response to OxLDL follows a specific pathway that can be differentiated from that of other stimuli (including heat shock and H2O2) and is mevalonate dependent.49 Hsp70 may interact with specific cytoplasmic proteins essential to protect cells from OxLDL, thus favoring their cytotoxicity. Alternatively, the interaction of Hsp70 with cellular proteins may affect cell membrane properties, which, in turn, could account for an increased uptake of OxLDLs or of some components derived from these lipoproteins. For instance, an increased uptake of oxysterols, the major toxic components of OxLDLs,50 could justify the rise in OxLDL cytotoxicity in this clone. Interestingly, oxysterols, although extremely toxic, fail to induce the Hsp response in human endothelial cells51 and induce apoptotic cell death in bovine endothelial cells.52 Furthermore, we have shown that the uptake and degradation of 125I-OxLDL does not differ between control and transfected cells (the uptake and degradation were 97±11% and 93±17% of control cells, respectively).
Alternatively, permanently overexpressed Hsp70 in transfected cells may be interacting with some cytoplasm proteins that are essential to counteract the cytotoxicity of OxLDL and, therefore, may not be available for "protecting" newly synthesized proteins. However, this possibility is unlikely because we have shown that Hsp70 overexpression protects from other toxic stimuli, such as heat shock or UV irradiation.
The role of Hsp70 in preventing apoptosis is still under debate: it has been reported that Hsp70 inhibits caspase-dependent and -independent apoptosis in Jurkat T cells53 and prevents the recruitment and association of procaspase-9 to the Apaf-1 apoptosome in cell-free systems.54 55 Yet a mild heat shock can protect vascular smooth muscle cells against necrosis but not against apoptosis.56 Moreover, it has been reported that overexpression of Hsp70 is unable to protect neuronal cells from apoptosis57 and that the overexpression of Hsp70 enhances the Fas/apo-1/CD95–mediated apoptotic cell death in Jurkat T cells.58 Therefore, it appears that stable overexpression of Hsp70 may protect cells from cytotoxic stimuli leading to necrosis but not to apoptosis driven by Fas. Sata and Walsh59 have recently suggested that OxLDLs activate the Fas-mediated apoptotic pathway by sensitizing endothelial cells to the death signal from the Fas receptor; perhaps in cells overexpressing Hsp 70 this pathway is further upregulated. It is tempting to speculate that during LDL oxidation substances that interact with the Fas receptors are formed. Whatever the mechanism involved, several hallmarks of apoptosis are present in COS-1 cells exposed to OxLDLs, including nuclear condensation and a prevalence of Bax over Bcl-2. Increased apoptosis is also suggested by the increase of nucleosome cellular content. Whether these findings bear in vivo relevance remains to be addressed in further studies.
In summary, we have shown that the overexpression of Hsp70 fails to protect transfected COS-1 cells from OxLDL cytotoxicity but that it confers a higher sensitivity. This effect is specific for OxLDLs (inasmuch as transfected cells are protected from other toxic stimuli) and appears to be related to the ability of OxLDLs to induce apoptosis.
|
Acknowledgments |
|---|
This work was supported in part by a research grant from the European Community (BMH4-CT98-3191) and Ministero Pubblica Istruzione to A.L.C.
Received July 27, 2000; accepted November 24, 2000.
|
References |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
1. Hightower LE. Heat shock, stress proteins, chaperones and proteotoxicity. Cell. 1991;66:191–197.[Medline] [Order article via Infotrieve]
2.
Wu BJ,
Morimoto RI. Transcription of the human Hsp70 gene is induced by serum
stimulation. Proc Natl Acad Sci
U S A. 1985;82:6070–6074.
3. Suzuki K, Watanabe M. Augmented expression of HSP72 protein in normal human fibroblasts irradiated with ultraviolet light. Biochem Biophys Res Commun. 1992;186:1257–1264.[Medline] [Order article via Infotrieve]
4.
Ananthan J,
Goldberg AL, Voellmy R. Abnormal proteins serve as
eukaryotic stress signal and trigger activation of heat
shock genes. Science. 1986;232:522–524.
5. Hightower LE. Cultured animal cells exposed to amino acid analogues or puromycin rapidly synthesize several polypeptides. J Cell Physiol. 1980;102:407–427.[Medline] [Order article via Infotrieve]
6.
Benjamin IJ,
Kroger B, Williams RS. Activation of the heat shock transcription
factor by hypoxia in mammalian cells.
Proc Natl Acad Sci
U S A. 1990;87:6263–6267.
7. Hendrick JP, Hartl FU. Molecular chaperon functions of heat-shock proteins. Annu Rev Biochem. 1993;62:349–384.[Medline] [Order article via Infotrieve]
8. Chirico WJ, Waters MG, Blobel G. 70K heat shock related proteins stimulate protein translocation into microsomes. Nature. 1988;332:805–810.[Medline] [Order article via Infotrieve]
9.
Beckmann RP,
Mizzen LA, Welch WJ. Interaction of Hsp70 with newly synthesized
proteins: implications for protein folding and assembly.
Science. 1990;248:850–854.
10. Kang PJ, Ostermann J, Shilling J, Neupert W, Craig EA, Pfanner N. Requirement for Hsp70 in the mitochondrial matrix for translocation and folding of precursor protein. Nature. 1990;348:137–143.[Medline] [Order article via Infotrieve]
11. Ang D, Liberek K, Skowyra D, Zylicz M, Georgopoulos C. Biological role and regulation of the universally conserved heat shock proteins. J Biol Chem. 1991;266:4233–4236.
12. Lindquist S. The heat shock response. Annu Rev Biochem. 1986;55:1151–1191.[Medline] [Order article via Infotrieve]
13.
Hunt C,
Morimoto RI. Conserved features of eukaryotic Hsp70 genes
revealed by comparison with the nucleotide sequence of
human Hsp70. Proc Natl Acad Sci
U S A. 1985;82:6455–6459.
14. Morimoto RI, Tiesseres A, Georgopoulos C. The stress response, function of the proteins, and perspectives. In: Morimoto RI, Tiesseres A, Georgopoulos C, eds. Stress Proteins in Biology and Medicine. Cold Spring Harbor, NY: Cold Spring Harbor Press; 1990:1–37.
15. Angelidis CE, Lazaridis I, Pagoulatos GN. Constitutive expression of heat-shock protein 70 in mammalian cells confers thermotolerance. Eur J Biochem. 1991;199:35–39.[Medline] [Order article via Infotrieve]
16.
Li GC, Li
LG, Liu YK, Mak JY, Chen LL, Lee WM. Thermal response of rat
fibroblasts stably transfected with the human 70KDa heat shock
protein-encoding gene. Proc Natl Acad Sci
U S A. 1991;88:1681–1685.
17. Lowenstein DH, Chan PH, Miles MF. The stress protein response in cultured neurons: characterization and evidence for a protective role in excitotoxicity. Neuron. 1991;17:1053–1060.
18. Jäättelä M, Wissing D, Bauer PA, Li GC. Major heat shock protein 70 protects tumor cells from tumor necrosis factor cytotoxicity. EMBO J. 1992;11:3507–3512.[Medline] [Order article via Infotrieve]
19. Williams RS, Thomas JA, Fina M, German Z, Benjamin IJ. Human heat shock protein 70 (Hsp70) protects murine cells from injury during metabolic stress. J Clin Invest. 1993;92:503–508.
20.
Hutter MM,
Sievers RE, Barbosa V, Wolfe CL. Heat shock protein induction in rat
hearts: a direct correlation between the amount of heat-shock protein
induced and the degree of myocardial protection.
Circulation. 1994;89:355–360.
21. Marber MS, Mestril R, Chi SH, Sayen MR, Yellon DM, Dillmann WH. Overexpression of the rat inducible 70-kD heat stress protein in a transgenic mouse increases the resistance of the heart to ischemic injury. J Clin Invest. 1995;95:1854–1860.
22.
Hessler JR,
Morel DW, Lewis LJ, Chisolm GM. Lipoprotein oxidation and
lipoprotein-induced cytotoxicity.
Arteriosclerosis. 1993;3:215–222.
23. Steinberg D, Parthasarathy S, Carew TE, Khoo JC, Witztum JL. Beyond cholesterol: modifications of low-density lipoprotein that increase its atherogenicity. N Engl J Med. 1989;320:915–924.[Medline] [Order article via Infotrieve]
24.
Dimmeler S,
Haendeler J, Galle J, Zeiher AM. Oxidized low-density lipoprotein
induces apoptosis of human endothelial cells by
activation of CPP32-like proteases: a mechanistic clue to the
"response to injury" hypothesis.
Circulation. 1997;95:1760–1763.
25. Bennett MR, Evan GI, Schwartz SM. Apoptosis of human vascular smooth cells derived from normal vessel and coronary atherosclerotic plaques. J Clin Invest. 1995;95:2266–2274.
26. Zhu W, Roma P, Pellegatta F, Catapano AL. Oxidized low density lipoprotein induce the expression of heat shock protein 70 in human endothelial cells. Biochem Biophys Res Commun. 1994;200:389–394.[Medline] [Order article via Infotrieve]
27. Zhu WM, Roma P, Pirillo A, Pellegatta F, Catapano AL. Oxidized LDL induce Hsp70 expression in human smooth muscle cells. FEBS Lett. 1995;372:1–5.[Medline] [Order article via Infotrieve]
28. Roma P, Catapano AL. Stress proteins and atherosclerosis. Atherosclerosis. 1996;127:147–154.[Medline] [Order article via Infotrieve]
29. Gluzman Y. SV40-transformed simian cells support the replication of early SV40 mutants. Cell. 1981;23:175–182.[Medline] [Order article via Infotrieve]
30. Chen CA, Okayama H. Calcium phosphate-mediated gene transfer: a highly efficient system for stably transforming cells with plasmid DNA. Biotechniques. 1988;6:632–638.[Medline] [Order article via Infotrieve]
31. Piotrowicz RS, Weber LE, Hichey E, Levin EG. Accelerated growth and senescence of arterial endothelial cells expressing the small molecular weight heat-shock protein HSP27. FASEB J. 1995;9:1079–1084.[Abstract]
32.
Lowry OH,
Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin
reagent. J Biol Chem. 1951;193:265–275.
33. Havel RJ, Eder HA, Bragdon JH. The distribution and chemical composition of ultracentrifugally separated lipoproteins in human serum. J Clin Invest. 1955;34:1345–1353.
34. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970;227:680–685.[Medline] [Order article via Infotrieve]
35.
Towbin H,
Staehelin T, Gordon J. Electrophoretic transfer of proteins from
polyacrylamide gels to nitrocellulose sheets: procedure and
some applications. Proc Natl Acad Sci
U S A. 1979;76:4350–4354.
36. Kishi Y, Numano F. In vitro study of vascular endothelial injury by activated platelets and its prevention. Atherosclerosis. 1989;76:95–101.[Medline] [Order article via Infotrieve]
37. Denizot F, Lang R. Rapid colorimetric assay for cell growth and survival: modifications to tetrazolium dye procedure giving improved sensitivity and reliability. J Immunol Methods. 1986;89:271–277.[Medline] [Order article via Infotrieve]
38. Lactate dehydrogenase. In: Windholz M, eds. The Merck Index. Rahway, NJ: Merck; 1983:767.
39. Andreoli SP, Baehmer RL, Bergstein JM. In vitro detection of cell damage using 2-deoxy-D-3H-glucose: comparison with chromium 51, 3H-leucine, 3H-adenine and lactate dehydrogenase. J Lab Clin Med. 1985;106:253–261.[Medline] [Order article via Infotrieve]
40. Coffey MD, Cole RA, Colles SM, Chisolm GM. In vitro cell injury by oxidized low density lipoprotein involves lipid hydroperoxide-induced formation of alkoxyl, lipid and peroxyl radicals. J Clin Invest. 1995;96:1866–1873.
41.
Zhu W, Roma
P, Pirillo A, Pellegatta F, Catapano AL. Human
endothelial cells exposed to oxidized LDL express Hsp70
only when proliferating. Arterioscler
Thromb Vasc Biol. 1996;16:1104–1111.
42.
Björkerud
B, Björkerud S. Contrary effects of lightly and strongly oxidized LDL
with potent promotion of growth versus apoptosis on
arterial smooth muscle cells, macrophages and
fibroblasts. Arterioscler Thromb Vasc
Biol. 1996;16:416–424.
43.
Vaux DL,
Weissman IL, Kim SK. Prevention of programmed cell death in
Caenorhabditis elegans by human
Bcl-2. Science. 1992;258:1955–1957.
44. Oltvai ZN, Milliman CL, Korsmeyer SJ. Bcl-2 heterodimerizes in vivo with a conserved homolog, Bax, that accelerates programmed cell death. Cell. 1993;74:609–619.[Medline] [Order article via Infotrieve]
45. Chong KY, Lai CC, Lille S, Chang C, Su CY. Stable overexpression of the constitutive form of heat shock protein 70 confers oxidative protection. J Mol Cell Cardiol. 1998;30:599–608.[Medline] [Order article via Infotrieve]
46. Mehlen P, Briolay J, Smith L, Diaz-Latoud C, Fabre N, Pauli D, Arrigo AP. Analysis of the resistance to heat and hydrogen peroxide stresses in Cos cells transiently expressing wild type or deletion mutants of the Drosophila 27-kDa heat shock protein. Eur J Biochem. 1993;215:277–284.[Medline] [Order article via Infotrieve]
47.
Koane DS,
Sarzani R, Schwartz JH, Chobanian AV, Brecher P. Stress-induced
proteins in aortic smooth muscle cells and aorta of hypertensive rats.
Am J Physiol. 1990;258:H1699–H1705.
48.
Kukreja RC,
Kontos MC, Loesser KE, Batra SK, Qian YZ, Gbur CJ, Naseem SA, Jesse RL,
Hess ML. Oxidant stress increases heat shock protein 70 mRNA in
isolated perfused rat heart. Am J
Physiol. 1994;267:H2213–H2219.
49. Pirillo A, Jacoviello C, Longoni C, Radaelli A, Catapano AL. Simvastatin modulates the heat shock response and cytotoxicity mediated by oxidized LDL in cultured human endothelial and smooth muscle cells. Biochem Biophys Res Commun. 1997;231:437–441.[Medline] [Order article via Infotrieve]
50.
Hughes H,
Mathews B, Lenz ML, Guyton JR. Cytotoxicity of oxidized LDL to porcine
aortic smooth muscle cells is associated with the oxysterols
7-ketocholesterol and 7-hydroxycholesterol.
Arterioscler Thromb Vasc Biol. 1994;14:1177–1185.
51. Pirillo A, Zhu W, Roma P, Galli G, Caruso D, Pellegatta F, Catapano AL. Oxysterols from oxidized LDL are cytotoxic but fail to induce Hsp70 expression in endothelial cells. FEBS Lett.. 1999;462:113–116.[Medline] [Order article via Infotrieve]
52. Lizard G, Deckert V, Dubrez L, Moisant M, Gambert P, Lagrost L. Induction of apoptosis in endothelial cells treated with cholesterol oxides. Am J Pathol. 1996;148:1625–1638.[Abstract]
53. Creagh EM, Carmody RJ, Cotter TG. Heat shock protein 70 inhibits caspase-dependent and -independent apoptosis in Jurkat T cells. Exp Cell Res. 2000;257:58–66.[Medline] [Order article via Infotrieve]
54. Beere HM, Wolf BB, Cain K, Mosser DD, Mahboubi A, Kuwana T, Tailor P, Morimoto RI, Cohen GM, Green DR. Heat-shock protein 70 inhibits apoptosis by preventing recruitment of pro-caspase-9 to the Apaf-1 apoptosome. Nat Cell Biol. 2000;2:469–475.[Medline] [Order article via Infotrieve]
55. Saleh A, Srinivasula SM, Balkir L, Robbins PD, Alnemri ES. Negative regulation of the Apaf-1 apoptosome by Hsp 70. Nat Cell Biol. 2000;2:476–483.[Medline] [Order article via Infotrieve]
56.
Champagne
MJ, Dumas P, Orlov SN, Bennet MR, Hamet P, Tremblay J. Protection
against necrosis but not apoptosis by heat-stress proteins in
vascular smooth muscle cells.
Hypertension. 1999;33:906–913.
57. Mailhos C, Howard MK, Latchman DS. Heat shock proteins Hsp90 and Hsp70 protect neuronal cells from thermal stress but not from programmed cell death. J Neurochem. 1994;63:1787–1795.[Medline] [Order article via Infotrieve]
58. Liossis SNC, Ding XZ, Kiang JG, Tsokos GC. Overexpression of the heat shock protein 70 enhances the TCR/CD3- and Fas/Apo-1/CD95-mediated apoptotic cell death in Jurkat T cells. J Immunol. 1997;158:5668–5675.[Abstract]
59. Sata M, Walsh K. Oxidized LDL activates Fas-mediated endothelial cell apoptosis. J Clin Invest. 1998;102:1682–1689. [Medline] [Order article via Infotrieve]
This article has been cited by other articles:
 |
|
 |
|
  G. D. Norata, G. Tibolla, P. M. Seccomandi, A. Poletti, and A. L. Catapano Dihydrotestosterone Decreases Tumor Necrosis Factor-{alpha} and Lipopolysaccharide-Induced Inflammatory Response in Human Endothelial Cells J. Clin. Endocrinol. Metab., February 1, 2006; 91(2): 546 - 554. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G.D. Norata, A. Pirillo, E. Callegari, A. Hamsten, A.L. Catapano, and P. Eriksson Gene expression and intracellular pathways involved in endothelial dysfunction induced by VLDL and oxidised VLDL Cardiovasc Res, July 1, 2003; 59(1): 169 - 180. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||