Enhancement of oxidative stress-induced apoptosis by Hsp105ain mouse embryonal F9 cells Nobuyuki Yamagishi, Youhei Saito, Keiichi Ishihara and Takumi Hatayama Department of Biochemistry,
Trang 1Enhancement of oxidative stress-induced apoptosis by Hsp105a
in mouse embryonal F9 cells
Nobuyuki Yamagishi, Youhei Saito, Keiichi Ishihara and Takumi Hatayama
Department of Biochemistry, Kyoto Pharmaceutical University, Japan
Hsp105a is one of the major mammalian heat shock proteins
that belongs to the HSP105/110 family, and is expressed at
especially high levels in the brain as compared with other
tissues in mammals Previously, we showed that Hsp105a
prevents stress-induced apoptosis in neuronal PC12 cells,
and is a novel anti-apoptotic neuroprotective factor in
the mammalian brain On the other hand, we have also
demonstrated that Hsp105a is expressed transiently at
high levels during mouse embryogenesis and is found not
only in various tissues but also in apoptotic cells In the
present study, to elucidate the role of Hsp105a during
mouse embryogenesis, we established mouse embryonal F9
cell lines that constitutively over-express Hsp105a
Over-expression of Hsp105a enhanced hydrogen
peroxide-induced apoptosis by enhancing the activation of caspase-3, poly(ADP-ribose)polymerasecleavage,cytochrome crelease and activation of p38 mitogen-activated protein kinase (p38) Furthermore, oxidative stress-induced apoptosis was suppressed by SB202190, a potent inhibitor of p38, in F9 cells These findings indicated that the activation of p38 is an essential step for apoptosis in F9 cells and that Hsp105a enhances activation of p38, release of cytochrome c and caspase activation Hsp105a may play important roles in organogenesis, during which marked apoptosis occurs, by enhancing apoptosis during mouse embryogenesis Keywords: apoptosis; F9 cells; Hsp105; p38 MAPK; oxida-tive stress
Cell death is classified into two major morphologically and
biochemically distinct modes, necrosis and apoptosis [1]
Necrosis is characterized by swelling of organelles and cells,
followed by lysis of the plasma membrane and random
DNA degradation In contrast, apoptosis is a process that is
characterized by cell shrinkage, plasma membrane blebbing,
nuclear condensation and endonucleolytic cleavage of DNA
into fragments of oligonucleosomal length, and is a
funda-mental and indispensable process during normal embryonic
development, tissue homeostasis and regulation of the
immune system [2–4] In addition, environmental stresses
such as heat shock, radiation, chemical agents and oxidative
stress can also induce apoptosis
Heat shock proteins (Hsps) are a set of highly conserved
proteins that are induced in response to physiological and
environmental stress, and are classified into several families
on the basis of their apparent molecular weights, such as
HSP105/110, HSP90, HSP70, HSP60, HSP40 and HSP27
[5,6] Several studies have shown that Hsp70, Hsp90 and
Hsp27 protect against cell death through apoptosis by a
variety of stressors, such as heat shock, oxidative stress and
chemotherapeutic agents [7–9] In addition, recent studies
have demonstrated that Hsp70, Hsp90, Hsp60 and Hsp27
can modulate the functions of several major components of apoptotic processes, including the caspase cascade and the c-Jun N-terminal kinase (JNK) signalling pathway [10–19]
We have previously characterized two heat shock pro-teins, Hsp105a and Hsp105b, which belong to the HSP105/
110 family and are expressed in various mammals including human, mouse and rat [20–22] Hsp105a is a constitutively expressed 105-kDa stress protein and is induced by a variety
of stressors, whereas Hsp105b is an alternatively spliced form of Hsp105a that is specifically induced by heat shock at
42C These proteins exist as complexes associated with Hsp70/Hsc70 [23,24], and negatively regulate Hsp70/Hsc70 chaperone activity [25] In addition, our recent study demon-strated that Hsp105a protects neuronal cells against the apoptosis induced by various stresses [26] On the other hand, we have shown previously that the level of Hsp105a increases transiently in most tissues of mouse embryos from gestational day 9–11, and that Hsp105a is localized not only
in various tissues, but also in apoptotic cells and apoptotic bodies at the interdigital regions of limbs, suggesting that Hsp105a may play important roles in apoptosis dur-ing mouse embryogenesis [27]
In the present study, to examine the role of Hsp105a during mouse embryogenesis, we established mouse embry-onal F9 cells that constitutively express Hsp105a, and showed that Hsp105a enhanced the oxidative stress-induced apoptosis at or upstream of p38 mitogen-activated protein kinase (p38) activation
E X P E R I M E N T A L P R O C E D U R E S
Cell culture Mouse teratocarcinoma F9 cells were obtained from the Japanese Cancer Research Resources Bank and maintained
Correspondence to T Hatayama, Department of Biochemistry,
Kyoto Pharmaceutical University, 5 Nakauchi-cho, Misasagi,
Yamashina-ku, Kyoto 607-8414, Japan.
Fax: + 81 75 595 4758, Tel.: + 81 75 595 4653,
E-mail: hatayama@mb.kyoto-phu.ac.jp
Abbreviations: HSP, heat shock protein; JNK, c-Jun N-terminal
kinase; PARP, poly (ADP-ribose) polymerase; Ac-DEVD-pNA,
N-acetyl-Asp-Glu-Val-Asp-p-nitroanilide.
(Received 19 March 2002, revised 19 June 2002,
accepted 11 July 2002)
Trang 2in Dulbecco’s modified Eagle’s minimal essential medium
(Nissui Pharmaceutical) supplemented with 10% foetal
bovine serum (Life Technologies) in a humidified
atmo-sphere of 5% CO2in air at 37C To induce cell
differen-tiation, cells grown on collagen-coated culture dishes were
incubated in the presence of 100 nMretinoic acid or 1 mM
dibutyryl-cAMP/100 nMretinoic acid at 37C for 6 days
Cell morphology was examined using a difference
interfer-ence contrast microscope
Construction of mouse Hsp105a expression plasmid
and isolation of Hsp105a-over-expressing cells
Plasmid pcDNA105a was u sed to express mou se Hsp105a
in F9 cells To construct this plasmid, the mouse Hsp105a
cDNA derived from pB105-1 plasmid [21] was subcloned
into EcoRV–XbaI sites of the mammalian expression vector
pcDNA3 (Invitrogen)
F9 cells were transfected with pcDNA105a or pcDNA3
empty vector by lipofection using Superfect reagent (Qiagen)
according to the manufacturer’s instructions Forty-eight
hours after transfection, the cells were maintained in
complete medium containing 400 lgÆmL)1 geneticin (Life
Technologies) for 3 weeks to select geneticin-resistant cells
The surviving cell clones were isolated, grown in complete
medium containing 200 lgÆmL)1geneticin, and the
expres-sion levels of Hsp105a were analysed by Western blotting
using anti-mouse Hsp105 Ig [28]
Oxidative stress treatment
Cells were grown exponentially on culture dishes at 37C
for 24 h and then treated with 0.25–2 mM hydrogen
peroxide in NaCl/Picontaining 0.9 mMCaCl2and 0.5 mM
MgCl2at 37C for 30–60 min, washed with NaCl/Piand
further incubated in fresh medium at 37C for 24 h
Cell viability assay
After stress treatment, cells were incubated in medium
containing 50 lgÆmL)1neutral red at 37C for 3 h, then
fixed with 1% formaldehyde containing 1% CaCl2 for
1 min The dye incorporated into viable cells was extracted
with 50% ethanol containing 1% acetic acid, and
absorb-ance at 540 nm was measured
DNA fragmentation analysis
DNA fragmentation was analysed essentially as described
by Ishizawa et al [29] Cells were lysed at 37C for 30 min
in 200 lL lysis bu ffer (10 mM Tris/HCl pH 8.0, 150 mM
NaCl, 10 mM EDTA, 0.1% SDS, 0.5 mgÆmL)1
ribonuc-lease A, 0.5 mgÆmL)1 proteinase K), and the cell lysates
were mixed with 300 lL NaI solu tion (6M NaI, 10 mM
Tris/HCl pH 8.0, 13 mMEDTA, 0.5% sodium
N-lauroyl-sarcosine, 30 lgÆmL)1glycogen) and incubated at 60C for
15 min An equal volume of isopropanol was added to the
mixtures, which were shaken vigorously and kept for
15 min at room temperature After centrifugation at
15 000 g for 15 min, the precipitate was washed successively
with 50% and 100% isopropanol, dried in air, and resolved
in Tris/HCl/EDTA buffer (10 mMTris/HCl pH 8.0, 1 mM
EDTA) Aliquots of 5 lg of DNA were electrophoresed on
2% agarose gels and stained with 1 lgÆmL)1 ethidium bromide
Morphological examination of apoptotic cells Cells were plated onto coverslips at a density of 1· 105 cellsÆcm)2and grown at 37C for 24 h After treatment, cells were washed with NaCl/Pi, fixed with 3.7% formalde-hyde for 30 min at room temperature, and stained with
10 lMHoechst 33342 for 10 min in the dark After washing with NaCl/Pi, the stained cells were observed using a fluorescence microscope (Zeiss) Cells were scored as apoptotic if they displayed nuclear fragmentation and/or chromatin condensation
Assay for poly (ADP-ribose) polymerase (PARP) cleavage Cells (1· 106 cells) were lysed with 200 lL lysis bu ffer (50 mMTris/HCl pH 8.0, 150 mMNaCl, 1% NP-40, 0.1% SDS, 5 lgÆmL)1aprotinin, 5 lgÆmL)1leupeptin, 2 lgÆmL)1 pepstatin A, 1 mMphenylmethanesulfonyl fluoride) on ice for 1 h The lysate was then sonicated for 10 s and centrifuged at 20 000 g for 15 min at 4C The supernatant was recoverd as the cell extract Aliquots (20 lg protein) of cell extracts in SDS sample buffer containing urea (62.5 mM Tris/HCl pH 6.8, 6Murea, 10% glycerol, 2% SDS, 5% 2-mercaptoethanol, 0.00125% Bromophenol blue) were sub-jected to 7.5% SDS/PAGE, then transferred onto nitrocel-lulose membranes by electrotransfer The membranes were blocked with 10% skim milk in NaCl/Tris (20 mMTris/HCl
pH 7.6, 137 mMNaCl) containing 0.1% Tween 20 (NaCl/ Tris/Tween), and incubated with anti-PARP Ig (Santa Cruz) Then, the membranes were incubated with horse-radish peroxidase-conjugated anti-rabbit IgG, and the antibody–antigen complexes were detected using the ECL-Western blot detection system (Amersham Pharmacia Biotech)
Measurement of caspase-3 activity Caspase-3 activity was measured using the colorimetric CaspACE assay system according to the manufacturer’s instructions (Promega) Briefly, cells (1· 106 cells) were suspended in 50 lL cell lysis buffer on ice for 10 min, and lysed by freezing and thawing After centrifugation at
20 000 g for 20 min at 4C, the supernatants were recov-ered as cell extracts Cell extracts (50 lg protein) were incubated in caspase assay buffer (100 mMHepes pH 7.5,
10 mM sucrose, 0.1% Chaps, 10 mM dithiothreitol) con-taining 200 lM N-acetyl-Asp-Glu-Val-Asp-p-nitroanilide (Ac-DEVD-pNA) at 25C for 2 h, and then absorbance
at 405 nm was measured using a microplate reader Release of cytochromec from mitochondria Release of cytochrome c was analysed essentially as des-cribed by Yano et al [30] Briefly, cell suspensions were mixed with an equal volume of 100 lgÆmL)1digitonin in NaCl/Pi, and incubated at 25C for 5 min After centrif-ugation at 15 000 g for 2 min, supernatants were recovered
as cytosolic fractions Pellets were dissolved in NaCl/Pi containing 0.5% Triton X-100, and centrifuged, then supernatants were recovered as mitochondrial fractions
Trang 3Both fractions were subjected to 15% SDS/PAGE, and
analysed by Western blotting using anti-cytochrome c Ig
(Santa Cruz)
Activation of JNK and p38
Phosphoryled JNK and p38 were detected by Western
blotting using PhosphoPlus JNK (Thr183/Tyr185) and
PhosphoPlus p38 (Thr180/Tyr182) antibody kits (Cell
Signaling Technology, Inc.), respectively Cell extracts
(20 lg protein) were separated by SDS/PAGE (10%
polyacrylamide), and analysed by Western blotting using
phosphorylation state-specific anti-JNK Ig or anti-p38 Ig
Then, the membranes were incubated at 50C for 30 min in
stripping buffer (62.5 mM Tris/HCl pH 6.7, 2% SDS,
100 mM2-mercaptoethanol), and total JNK or p38 on the
same membranes were detected using JNK Ig or
anti-p38 Ig, respectively
Kinase activity of p38 was assayed by its ability to
phosphorylate MAPKAPK-2 Cell extracts (20 lg protein)
were separated by SDS/PAGE (10% polyacrylamide), and
analysed by Western blotting using phosphorylation
state-specific anti-(MAPKAPK-2) Ig (Cell Signaling Technology,
Inc.)
R E S U L T S
Characterization of stable transfectants over-expressing
Hsp105a
To determine the role of Hsp105a in embryonal cells, we
established mouse embryonal F9 cell clones that express
Hsp105a at high levels In this study, we used two Hsp105a over-expressing F9 cell lines, S3 and S23, in which the expression levels of Hsp105a were about two-and threefold higher than that in parental F9 cells or controls transfected with empty pcDNA3 vector (V1), respectively (Fig 1A) The growth rates of S3 and S23 cells were not significantly different from those of F9 and V1 cells (Fig 1B) F9 cells can be differentiated toward primitive like and parietal endoderm-like cells by exposure to retinoic acid and dibutyryl-cAMP/retinoic acid, respectively [31,32] Upon exposure
to retinoic acid, S3 and S23 cells showed an enlarged and flattened morphology as seen in F9 or V1 cells, which is characteristic of primitive endoderm-like cells (Fig 1C, e–h) In addition, the Hsp105a over-expressing cells were also induced to differentiate toward parietal endo-derm-like cells by treatment with retinoic acid and dibutyryl-cAMP; they also showed the typical changes
in morphology (Fig 1C, i–l) Thus, over-expression of Hsp105a did not affect cell growth and differentiation toward primitive like and parietal endoderm-like phenotypes in F9 cells
Enhancement of oxidative stress-induced apoptosis
by over-expression of Hsp105a in F9 cells
As Hsp105a is expressed transiently at high levels during mouse embryogenesis and is localized in apop-totic cells [27], we next examined the effects of over-expression of Hsp105a on stress-induced cell death in embryonal F9 cells When F9, V1, S3 and S23 cells were treated with 0.25–1 m hydrogen peroxide for 1 h,
Fig 1 Over-expression of Hsp105a in F9 cells and its effects on growth and differentiation (A) Cell extracts (20 lg protein) from parental F9 cells (F9) and cell clones stably transfected with either pcDNA3 vector (V1) or pcDNA105a (S3 and S23) were separated by SDS/PAGE, and the levels
of Hsp105a were determined by Western blotting using anti-mouse Hsp105 Ig (B) F9 V1, S3 and S23 cells were plated into 35 mm culture dishes at
a density of 1 · 10 4
cells per dish, and cultured for 6 days at 37 C At the indicated times, cell numbers were counted (C) Cells (1 · 10 5
cells per dish) were cultured in the presence of 100 n M retinoic acid (+ RA) or 1 m M dibutyryl-cAMP/100 n M retinoic acid (+ RA/cAMP) at 37 C for
6 days Cell morphology was observed using a difference interference contrast microscope Scale bars ¼ 10 lm.
Trang 4S3 and S23 cells were more sensitive to the oxidative
stress than F9 or V1 cells (Fig 2A) Therefore, we
further analysed the hydrogen peroxide-induced cell
death of F9 cells
Cell death is classified into two morphologically and
biochemical distinct modes, apoptosis and necrosis [1]
To characterize the hydrogen peroxide-induced cell
death in F9 cells, we examined whether DNA
fragmen-tation, characteristic of apoptosis, occurred in these cells
As shown in Fig 2B, nucleosomal-length DNA
frag-mentation was observed in the hydrogen
peroxide-treated F9 cells, and the amounts of fragmented DNA
caused by low doses of hydrogen peroxide were
increased in the Hsp105a over-expressing cells as
compared with those in S3 and S23 cells In addition,
apoptotic morphology such as nuclear condensation and
chromatin fragmentation was also prominently observed
by Hoechst 33342 staining in these cells (Fig 3A), and
the rate of apoptotic cells was approximately threefold
higher in the Hsp105a over-expressing cells than in F9
or V1 cells (Fig 3B) However, these morphological
changes were suppressed by treatment with a
cell-permeable caspase inhibitor zVAD-fmk Thus, Hsp105a
was demonstrated to enhance oxidative stress-induced
apoptosis in F9 cells
Over-expression of Hsp105a enhances caspase-3 activity and PARP cleavage after treatment with hydrogen peroxide
A common event in the apoptotic pathway is the activation of caspases These enzymes participate in a cascade that is triggered in response to pro-apoptotic signals and results in cleavage of a set of proteins, resulting in disassembly of the cells Caspase-3 is a major effector caspase, and induces cleavage of several substrate proteins and is responsible for several apoptotic processes
We next assayed caspase-3 activity in extracts from cells treated with oxidative stress As shown in Fig 4A, although caspase-3 activity increased slightly in F9 and V1 cells treated with hydrogen peroxide, its activity was increased markedly in S3 and S23 cells by this treatment
In addition, the activation of caspase-3 was suppressed by the treatment with a cell-permeable caspase inhibitor zVAD-fmk
PARP, a DNA repair-related enzyme, is an important substrate of caspase-3, and is cleaved from a 116-kDa protein to an 85-kDa fragment and inactivated during apoptosis [33,34] Western blotting analysis clearly revealed cleavage of PARP in F9 cells treated with hydrogen peroxide (Fig 4B) Furthermore, in accordance with the
Fig 2 Effects of Hsp105a over-expression on the sensitivityof F9 cells to hydrogen peroxide F9, V1, S3 and S23 cells were exposed to 0.1–1 m M hydrogen peroxide for 1 h, and further incubated for 24 h at 37 C (A) Cell viability was then determined by neutral red assay Experiments were repeated at least three times and essentially the same results were obtained each time (B) DNA was extracted from the cells treated with hydrogen peroxide, and aliquots (5 lg each) of DNA were electrophoresed on a 2% agarose gel and visualized by staining with ethidium bromide.
Trang 5enhancement of caspase-3 activity, the cleavage of PARP
by hydrogen peroxide was more intense in S3 and S23 cells than in F9 or V1 cells Thus, over-expression of Hsp105a was shown to markedly enhance the activation
of procaspase-3 during apoptosis induced by hydrogen peroxide
Over-expression of Hsp105a enhances release
of cytochromec from mitochondria after treatment with hydrogen peroxide
In mammalian cells, one of the main pathways that activates caspase-3 is via mitochondria When mitochondria receive appropriate signals from a variety of stresses or are damaged irreversibly, pro-apoptotic molecules such as cytochrome c are released from mitochondria into the cytosol [35–37] In the cytosol, cytochrome c forms a complex with Apaf-1 and procaspase-9, and activates caspase-9, which in turn converts procaspase-3 into its active form, resulting in apoptosis [38–40] We next analysed whether cytochrome c is released from mitochondria in F9 cells by oxidative stress When F9 cells were fractionated into mitochondrial and cytosolic fractions and cytochrome c was examined by Western blotting, a large amount of cytochrome c was found in mitochondria with only a small amount in the cytosolic fraction of control cells under these experimental conditions (Fig 5A) However, although the amounts of cytochrome c released by hydrogen peroxide increased slightly in F9 or V1 cells, the release was increased
to a greater extent in S3 and S23 cells than in control and V1 cells (Fig 5B) Thus, over-expression of Hsp105a seemed to enhance the release of cytochrome c from mitochondria in F9 cells by hydrogen peroxide
Fig 3 Enhancement of oxidative stress-induced apoptosis byover-expression of Hsp105a (A) F9, V1, S3 and S23 cells were grown on coverslips, exposed to 1 m M hydrogen peroxide for 1 h, and further incubated at 37 C for 24 h Ten l M z-VAD-fmk was added to the medium 1 h before treatment Cells were washed with NaCl/P i , fixed with 3.7% formaldehyde, and stained with 10 l M Hoechst 33342 Nuclear morphology of cells was observed using a fluorescence microscope (B) Rates of apoptotic cells were obtained from at least 200 cells in each experiment Data are shown
as the means ± SD of three independent experiments The significance of differences was assessed by unpaired Student’t t-test.
Fig 4 Effects of over-expression of Hsp105a on caspase-3 activity F9,
V1, S3 and S23 cells were exposed to 1 m M hydrogen peroxide for 1 h,
and further incubated for 24 h at 37 C (A) Caspase-3 activity in cell
extracts was measured with caspase-3 substrate, Ac-DEVD-pNA.
Data represent the means ± SD of three independent experiments (B)
Aliquots (20 lg protein) of cell extracts were separated by SDS/
PAGE, and PARP (116 kDa) and the cleaved fragment (85 kDa) were
detected by Western blotting using anti-PARP Ig.
Trang 6Over-expression of Hsp105a enhances activation
of p38 after treatment with hydrogen peroxide JNK and p38 pathways are activated by cellular stresses and inflammatory cytokines, resulting in growth arrest and apoptosis, and have been implicated as key regulators of stress-induced apoptosis in many cell types [41,42] Fur-thermore, mitochondria are influenced by proapoptotic signals through the JNK pathway [43] To determine the effects of Hsp105a on JNK and p38 signalling pathways, we examined the activation of JNK and p38 in F9 cells by oxidative stress As shown in Fig 6A, JNK was not activated in control F9 or V1 cells by treatment with hydrogen peroxide In contrast, hydrogen peroxide-treat-ment induced marked activation of JNK within 30 min in S23 cells, but not in S3 cells Therefore, the enhancement of the oxidative stress-induced JNK-activation seemed not to
be solely due to the over-expression of Hsp105a in F9 cells
On the other hand, p38 was activated at low levels within 1–2 h in F9 and V1 cells after treatment with hydrogen peroxide, and the activation of p38 by hydrogen peroxide was enhanced in both S3 and S23 cells compared with F9 and V1 cells (Fig 6B) Thus, over-expression of Hsp105a seemed to enhance the oxidative stress-induced activation of p38 but not JNK in F9 cells
As Hsp105a enhances the activation of p38 induced by oxidative stress, we further examined whether its activation
is responsible for the induction of apoptosis in F9 cells using SB202190, a potent inhibitor of p38 As shown in Fig 7A, although hydrogen peroxide-treatment induced apoptosis in F9 cells as described above, the apoptosis was significantly suppressed by SB202190 Under these conditions, although phosporylation of MAPKAPK-2, a substrate of p38, was enhanced by the hydrogen peroxide treatment, it was suppressed to basal level by treatment with 10 lMSB202190 (Fig 7B) These findings indicate that the activation of p38
is an essential step for induction of apoptosis by hydrogen peroxide, and Hsp105a is suggested to enhance the
oxida-Fig 6 Effects of Hsp105a on activation of JNK and p38 byoxidative stress F9, V1, S3 and S23 cells were exposed to 1 m M hydrogen peroxide for 30 min and further incubated for 0.5, 1 or 2 h at 37 C Aliquots (20 lg protein)
of cell extracts were separated by SDS/PAGE Activated and total JNK or p38 were detected
by Western blotting, as described in Experi-mental procedures (A) JNK, (B) p38 Upper and lower panels represent activated and total JNK or p38, respectively.
Fig 5 Effects of Hsp105a over-expression on release of cytochrome c
from mitochondria F9, V1, S3 and S23 cells were exposed to 1 m M
hydrogen peroxide for 45 min, and further incubated for 24 h at
37 C Cells were then fractionated into cytosolic and mitochondrial
fractions, as described in Experimental procedures (A) Both fractions
were subjected to 15% SDS/PAGE, and analysed by Western blotting
using anti-cytochrome c Ig C, cytosolic fraction; M, mitochondrial
fraction (B) The densities the cytochrome c bands were quantified by
densitometry, and the rates release into the cytosol are shown Data
represent the means ± SD of three independent experiments Filled
bars, untreated cells; open bars, hydrogen peroxide-treated cells.
Trang 7tive stress-induced apoptosis at or upstream of p38
activa-tion in F9 cells
D I S C U S S I O N
Hsp105a is expressed in most tissues, but its levels are
especially high in the brain of adult mammals such as rats,
mice and humans [21–23] We have shown that Hsp105a
plays an important role in protection of neuronal cells
against stress-induced apoptosis [26] In accordance with
these findings, ischemia/reperfusion in the rat forebrain
induces the expression of HSP105/110 family proteins,
Hsp105a, APG-1 (testis-specific homologue of Hsp105) and
APG-2 [44,45], and Hsp110 (hamster homologue of
Hsp105a) confers heat resistance on rat fibroblasts and
human epithelial carcinoma cells [46] On the other hand, we
have also shown that the levels of Hsp105a increase
transiently in embryonic tissues during mouse
embryogen-esis, and this protein is localized not only in various tissues,
but also in apoptotic cells and apoptotic bodies at the
interdigital regions of limbs [27] In the present study, to
explore the function of Hsp105a in embryogenesis, we
established mouse embryonal F9 cell lines that constitutively
express Hsp105a Although growth rate and differentiation
of F9 cells were not affected by the over-expression of
Hsp105a, the sensitivity of cells to oxidative stress was
enhanced by the over-expression of Hsp105a, and these
findings were in clear contrast with those in neuronal PC12
cells However, as sensitivity of F9 cells to stresses such as heat shock, etoposide, actinomycin D and serum depriva-tion was also enhanced by over-expression of Hsp105a (unpublished data), the enhancement of cell death by Hsp105a seemed to be a general phenomenon in embryonal F9 cells The present findings together with previous observations in neuronal PC12 cells suggested that Hsp105a has a pro-apoptotic effect in embryonal cells and an anti-apoptotic effect in neuronal cells Thus, these observations provide the first evidence that Hsp105a can function as an enhancer or suppressor of apoptosis depending on the cell type in mammals
Apoptosis is an active process resulting in characteristic morphological changes such as cell shrinkage, condensation
of chromatin and membrane blebbing [2–4] The common pathway of apoptosis involves a family of proteases known
as the caspases, which are activated in a proteolytic cascade
to cleave specific substrates The release of cytochrome c from mitochondria triggers the formation of apoptosome complex with Apaf-1 and pro-caspase-9, and activates caspase-9, which then in turn activates downstream effector caspases such as caspase-3 [38–40] Active caspase-3 cleaves several substrates such as PARP [33,34], and activates death effector molecules or triggers the structural changes char-acteristic of apoptotic cells Here, we showed that over-expression of Hsp105a enhances PARP cleavage, caspase-3 activation and release of cytochrome c from mitochondria
in F9 cells exposed to hydrogen peroxide, and our results
Fig 7 Suppression of oxidative stress-induced
apoptosis in F9 cells bya potent p38 inhibitor.
F9, V1, S3 and S23 cells were grown on
coverslips, treated with or without 10 l M
SB202190 for 1 h before and during the
1 h-treatment with 1 m M hydrogen peroxide,
and further incubated at 37 C for 24 h (A) or
2 h (B) (A) Cells were then washed with
NaCl/P i , fixed with 3.7% formaldehyde, and
stained with 10 l M Hoechst 33342 Nuclear
morphology of cells was observed using a
fluorescence microscope Rates of apoptotic
cells were obtained from at least 300 cells in
each experiment Data are shown as the means
± SD of at least three independent
experi-ments The significance of differences was
assessed by unpaired Student’t t-test (B) Cell
extracts (20 lg proteins) of S23 cells were
separated by SDS/PAGE, and
phosphory-lated MAPKAPK-2 was detected by Western
blotting using anti-phospho-MAPKAPK-2
Ig.
Trang 8suggested that Hsp105a enhances the apoptosis at or
upstream of cytochrome c release from mitochondria
Furthermore, the transmission of signals from external
stresses is accompanied by the activation of a family of
stress-activated protein kinases, JNK and p38 Activation of
these signalling pathways leads to apoptosis [41,42], and
mitochondria is influenced by proapoptotic signal
trans-duction through the JNK pathway [43] As the activation of
p38 is an essential step for apoptosis induced by hydrogen
peroxide in F9 cells as shown in Fig 7, Hsp105a was
suggested to enhance the oxidative stress-induced apoptosis
directly or indirectly at or upstream of activation of p38 In
contrast, although Hsp105a prevents the apoptosis induced
by several stresses including hydrogen peroxide in neuronal
PC12 cells, p38 is not activated by these stresses in neuronal
cells [26] The p38 signalling pathway may be a possible
target at which Hsp105a enhances the apoptosis induced by
hydrogen peroxide in embryonal cells
Several Hsps have been shown to modulate the pathway
of apoptosis positively or negatively Hsp60 with or without
Hsp10 directly stimulates apoptosis by promoting the
proteolytic maturation of caspase-3 [17,18] In contrast,
Hsp70, Hsp90 and Hsp27 exert negative influences on
apoptotic signalling In particular, Hsp70 has been shown to
protect against apoptosis by a variety of stressors through
suppression of JNK activation [10–13] and apoptosome
formation [14,15] Interestingly, a recent study
demonstra-ted that the chaperone activity of Hsp70 is required for
protection against heat-induced apoptosis [47] In contrast,
we cannot detect the chaperone activity of Hsp105a, but the
protein exists as complexes associated with Hsp70/Hsc70
[23,24] and suppresses the Hsc70 chaperone activity [25]
Therefore, it is possible that Hsp105a may stimulate
stress-induced apoptosis by negative regulation of Hsp70/Hsc70
chaperone that is required for suppression of apoptosis
In conclusion, we showed here that Hsp105a enhances
apoptosis at or upstream of p38 activation The
apoptosis-enhancing activity of Hsp105a may play important roles in
organogenesis, in which marked apoptosis occurs during
mouse embryogenesis, although further studies are
neces-sary to understand the precise mechanism by which Hsp105a
enhances stress-induced apoptosis
A C K N O W L E D G M E N T S
This study was supported in part by grant from the Ministry of
Education, Science, Sports and Culture of Japan (T H.).
R E F E R E N C E S
1 Kerr, J.F., Wyllie, A.H & Currie, A.R (1972) Apoptosis: a basic
biological phenomenon with wide-ranging implications in tissue
kinetics Br.J.Cancer 26, 239–257.
2 Wyllie, A.H., Kerr, J.F & Currie, A.R (1980) Cell death: the
significance of apoptosis Int.Dev.Cytol.68, 251–306.
3 Steller, H (1995) Mechanisms and genes of cellular suicide
Sci-ence 267, 1445–1449.
4 White, E (1996) Life, death, and the pursuit of apoptosis Genes
Dev 10, 1–15.
5 Hendrick, J.P & Hartl, F.-U (1993) Molecular chaperone
func-tions of heat-shock proteins Annu.Rev.Biochem.62, 349–384.
6 Craig, E.A., Weissman, J.S & Horwich, A.L (1994) Heat shock
proteins and molecular chaperones: mediators of protein
con-formation and turnover in the cell Cell 78, 365–372.
7 Buzzard, K.A., Giaccia, A.J., Killender, M & Anderson, R.L (1998) Heat shock protein 72 modulates pathways of stress-induced apoptosis J.Biol.Chem.273, 17147–17153.
8 Samali, A & Cotter, T.G (1996) Heat shock proteins increase resistance to apoptosis Exp.Cell Res.223, 163–170.
9 Mosser, D.D., Caron, A.W., Bourget, L., Denis-Larose, C & Massie, B (1997) Role of the human heat shock protein hsp70 in protection against stress-induced apoptosis Mol.Cell Biol.17, 5317–5327.
10 Mehlen, P., Schulze-Osthoff, K & Arrigo, A.-P (1996) Small stress proteins as novel regulators of apoptosis Heat shock protein
27 blocks Fas/APO-1- and staurosporine-induced cell death J.Biol.Chem.271, 16510–16514.
11 Gabai, V.L., Meriin, A.B., Mosser, D.D., Caron, A.W., Rits, S., Shifrin, V.I & Sherman, M.Y (1997) Hsp70 prevents activation of stress kinases A novel pathway of cellular thermotolerance J.Biol.Chem.272, 18033–18037.
12 Meriin, A.B., Yaglom, J., Gabai, V.L., Zon, L., Ganiatsas, S., Mosser, D.D., Zon, L & Sherman, M.Y (1999) Protein-dama-ging stresses activate c-Jun N-terminal kinase via inhibition of its dephosphorylation: a novel pathway controlled by HSP72 Mol Cell Biol 19, 2547–2555.
13 Park, H.-S., Lee, J.-S., Hu h, S.-H., Seo, J.-S & Choi, E.-J (2001) Hsp72 functions as a natural inhibitory protein of c-Jun N-terminal kinase EMBO J 20, 446–456.
14 Beere, H.M., Wolf, B.B., Cain, K., Mosser, D.D., Mahboubi, A., Kuwana, T., Tailor, P., Morimoto, R.I., Cohen, G.M & Green, D.R (2000) Heat-shock protein 70 inhibits apoptosis by pre-venting recruitment of procaspase-9 to the Apaf-1 apoptosome Nat.Cell Biol.2, 469–475.
15 Saleh, A., Srinivasula, S.M., Balkir, L., Robbins, P.D & Alnemri, E.S (2000) Negative regulation of the Apaf-1 apoptosome by Hsp70 Nat.Cell Biol.2, 476–483.
16 Pandey, P., Saleh, A., Makazawa, A., Kumar, S., Srinivasula, S.M., Kumar, V., Weichselbaum, R., Nalin, C., Alnemri, E.S., Kufe, D & Kharbanda, S (2000) Negative regulation of cyto-chrome c-mediated oligomerization of Apaf-1 and activation of procaspase-9 by heat shock protein 90 EMBO J 19, 4310–4322.
17 Samali, A., Cai, J., Zhivotovsky, B., Jones, D.P & Orrenius, S (1999) Presence of a pre-apoptotic complex of pro-caspase-3, Hsp60 and Hsp10 in the mitochondrial fraction of jurkat cells EMBO J 18, 2040–2048.
18 Xanthoudakis, S., Roy, S., Rasper, D., Hennessey, T., Aubin, Y., Cassady, R., Tawa, P., Ruel, R., Rosen, A & Nicholson, D.W (1999) Hsp60 accelerates the maturation of pro-caspase-3 by upstream activator proteases during apoptosis EMBO J 18, 2049–2056.
19 Bruey, J.-M., Ducasse, C., Bonniaud, P., Ravagnan, L., Susin, S.A., Diaz-Latoud, C., Gurbuxani, S., Arrigo, A.-P., Kroemer, G., Solary, E & Garrido, C (2000) Hsp27 negatively regulates cell death by interacting with cytochrome c Nat.Cell Biol.2, 645–652.
20 Hatayama, T., Yasuda, K & Nishiyama, E (1994) Character-ization of high-molecular-mass heat shock proteins and 42 degrees C-specific heat shock proteins of murine cells Biochem.Biophys Res.Commun.204, 357–365.
21 Yasuda, K., Nakai, A., Hatayama, T & Nagata, K (1995) Cloning and expression of murine high molecular mass heat shock proteins, HSP105 J.Biol.Chem.270, 29718–29723.
22 Ishihara, K., Yasu da, K & Hatayama, T (1999) Molecu lar cloning, expression and localization of human 105 kDa heat shock protein, hsp105 Biochim.Biophys.Acta 1444, 138–142.
23 Wakatsuki, T & Hatayama, T (1998) Characteristic expression of 105-kDa heat shock protein (HSP105) in various tissues of non-stressed and heat-non-stressed rats Biol.Pharm.Bull.21, 905–910.
24 Hatayama, T., Yasuda, K & Yasuda, K (1998) Association of HSP105 with HSC70 in high molecular mass complexes in mouse FM3A cells Biochem.Biophys.Res.Commun.248, 395–401.
Trang 925 Yamagishi, N., Nishihori, H., Ishihara, K., Ohtsu ka, K &
Hatayama, T (2000) Modulation of the chaperone activities of
Hsc70/Hsp40 by Hsp105alpha and Hsp105beta
Biochem.Bio-phys.Res.Commun.272, 850–855.
26 Hatayama, T., Yamagishi, N., Minobe, E & Sakai, K (2001)
Role of hsp105 in protection against stress-induced apoptosis in
neuronal PC12 cells Biochem.Biophys.Res.Commun.288, 528–
534.
27 Hatayama, T., Takigawa, T., Takeuchi, S & Shiota, K (1997)
Characteristic expression of high molecular mass heat shock
protein HSP105 during mouse embryo development Cell Struct.
Funct 22, 517–525.
28 Honda, K., Hatayama, T & Yukioka, M (1989) Common
anti-genicity of mouse 42 degrees C-specific heat-shock protein with
mouse HSP 105 Biochem.Biophys.Res.Commun.160, 60–66.
29 Ishizawa, M., Kobayashi, Y., Miyamu ra, T & Matsu u ra, S.
(1991) Simple procedure of DNA isolation from human serum.
Nucleic Acids Res 19, 5792.
30 Yano, M., Kanazawa, M., Terada, K., Namchai, C.,
Yamaiz-umi, M., Hanson, B., Hoogenraad, N & Mori, M (1997)
Visualization of mitochondrial protein import in cultured
mam-malian cells with green fluorescent protein and effects of
over-expression of the human import receptor Tom20 J.Biol.Chem.
272, 8459–8465.
31 Strickland, S & Mahdavi, V (1978) The induction of
differ-entiation in teratocarcinoma stem cells by retinoic acid Cell 15,
393–403.
32 Strickland, S., Smith, K.K & Marotti, K.R (1980) Hormonal
induction of differentiation in teratocarcinoma stem cells:
gen-eration of parietal endoderm by retinoic acid and dibutyryl cAMP.
Cell 21, 347–355.
33 Nicholson, D.W., Ali, A., Thornberry, N.A., Valliancourt, J.P.,
Ding, C.K., Gallant, M., Gareau, Y., Griffin, P.R., Labelle, M.,
Lazebnik, Y.A et al (1995) Identification and inhibition of the
ICE/CED-3 protease necessary for mammalian apoptosis Nature
376, 37–43.
34 Tewari, M., Quan, L.T., O’Rourke, K., Desnoyers, S., Zeng, Z.,
Beidler, D.R., Poirier, G.G., Salvesen, G.S & Dixit, V.M (1995)
Yama/CPP32 beta, a mammalian homolog of CED-3, is a
CrmA-inhibitable protease that cleaves the death substrate poly
(ADP-ribose) polymerase Cell 81, 801–809.
35 Yang, J., Liu, X., Bhalla, K., Kim, C.N., Ibrado, A.M., Cai, J.,
Peng, T.I., Jones, D.P & Wang, X (1997) Prevention of apoptosis
by Bcl-2: release of cytochrome c from mitochondria blocked.
Science 275, 1129–1132.
36 Kluck, R.M., Bossy-Wetzel, E., Green, D.R & Newmeyer, D.D (1997) The release of cytochrome c from mitochondria: a primary site for Bcl-2 regulation of apoptosis Science 275, 1132–1136.
37 Zou, H., Henzel, W.J., Liu, X., Lutschg, A & Wang, X (1997) Apaf-1, a human protein homologous to C.elegans CED-4, par-ticipates in cytochrome c-dependent activation of caspase-3 Cell
90, 405–413.
38 Li, P., Nijhawan, D., Budihardjo, I., Srinivasula, S.M., Ahmad, M., Alnemri, E.S & Wang, X (1997) Cytochrome c and dATP-dependent formation of Apaf-1/caspase-9 complex initiates an apoptotic protease cascade Cell 91, 479–489.
39 Zou, H., Li, Y., Liu, X & Wang, X (1999) An APAF-1 cyto-chrome c multimeric complex is a functional apoptosome that activates procaspase-9 J.Biol.Chem.274, 11549–11556.
40 Xia, Z., Dickens, M., Raingeau d, J., Davis, R.J & Greenberg, M.E (1995) Opposing effects of ERK and JNK-p38 MAP kinases
on apoptosis Science 270, 1326–1331.
41 Kyriakis, J.M & Avruch, J (1996) Sounding the alarm: protein kinase cascades activated by stress and inflammation J.Biol Chem 271, 24313–24316.
42 Chen, Y., Wang, X., Templeton, D., Davis, R.J & Tan, T.H (1996) The role of c-Jun N-terminal kinase (JNK) in apoptosis induced by ultraviolet C and gamma radiation Duration of JNK activation may determine cell death and proliferation J.Biol Chem 271, 31929–31936.
43 Tournier, C., Hess, P., Yang, D.D., Xu, J., Turner, T.K., Nimnual, A., Bar-Sagi, D., Jones, S.N., Flavell, R.A & Davis, R.J (2000) Requirement of JNK for stress-induced activation of the cytochrome c-mediated death pathway Science 288, 870–874.
44 Xue, J.H., Fukuyama, H., Nonoguchi, K., Kaneko, Y., Kido, T., Fukumoto, M., Fujibayashi, Y., Itoh, K & Fujita, J (1998) Induction of Apg-1, a member of the heat shock protein 110 family, following transient forebrain ischemia in the rat brain Biochem.Biophys.Res.Commun.247, 796–801.
45 Yagita, Y., Kitagawa, K., Ohtsuki, T., Tanaka, S., Hori, M & Matsumoto, M (2001) Induction of the HSP110/105 family in the rat hippocampus in cerebral ischemia and ischemic tolerance J.Cereb.Blood Flow Metab.21, 811–819.
46 Oh, H.J., Chen, X & Subjeck, J.R (1997) Hsp110 protects heat-denatured proteins and confers cellular thermoresistance J.Biol Chem 272, 31636–31640.
47 Mosser, D.D., Caron, A.W., Bourget, L., Meriin, A.B., Sherman, M.Y., Morimoto, R.I & Massie, B (2000) The chaperone func-tion of hsp70 is required for protecfunc-tion against stress-induced apoptosis Mol.Cell.Biol.20, 7146–7159.