Original Paper
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Acta Biochim Biophys
Sin 2008, 40: 339-348
doi:10.1111/j.1745-7270.2008.00409.x
Glucose-regulated protein 75
suppresses apoptosis induced by glucose deprivation in PC12 cells through
inhibition of Bax conformational change
Ling Yang, Xiaoyu Liu, Jinyu Hao,
Yunlong Yang, Mingxia Zhao, Ji Zuo, and Wen Liu*
Department of
Cellular and Genetic Medicine, Shanghai Medical College, Fudan University,
Shanghai 200032, China
Received: December
28, 2007
Accepted: February
20, 2008
This work was supported
by a grant from the Specialized Research Fund for the Doctoral Program of
Higher Education of China (No. 20050246079)
*Corresponding
author: Tel, 86-21-54237311; Fax, 86-21-54237091; E-mail, [email protected]
Glucose-regulated
protein 75 (Grp75) is an important molecular chaperone that belongs to the heat
shock protein 70 family and resides predominantly in mitochondria. Grp75 can
protect cells from glucose deprivation (GD) injury. However, the molecular
mechanisms by which it carries out this function are unknown. Here we report
that Grp75 could delay the release of cytochrome c and reduce apoptosis induced
by GD, and we also found that Grp75 could decrease Bax/Bcl-2 gene
expression ratio and inhibit the conformational change of Bax during this
process. In conclusion, these findings suggested that Grp75 overexpression was
able to inhibit apoptosis induced by GD. Grp75 inhibited Bax conformational
change to delay the release of cytochrome c, thus providing protection to PC12
cell which
was used primarily as a neuron model against GD toxicity.
Keywords glucose-regulated protein 75; glucose deprivation; apoptosis;
cytochrome c release; Bax conformational change
Apoptosis is essential for normal development and tissue homeostasis
of all multicellular organisms. Two distinct pathways of apoptosis have been
described, the death receptor pathway (the extrinsic apoptotic pathway) and the
mitochondrial pathway (the intrinsic apoptotic pathway) [1]. Through various
stresses, cells have the option of actively engaging the intrinsic apoptotic
pathway, which subsequently leads to their death [2]. Previous studies have
shown that release of cytochrome c and other pro-apoptogenic factors from
mitochondria to cytosol is an important step in the intrinsic pathway of
apoptosis [3]. The release of cytochrome c is regulated by a complex balance of
pro-apoptotic and anti-apoptotic proteins of the Bcl-2 family [4]. Studies suggested that the relative ratios of Bcl-2 to Bax,
rather than the single expression of either protein, can influence survival or
death to diverse apoptotic stimuli [5]. The conformational change of Bax is
also important to regulate cytochrome c release and induce apoptosis in
stressed cells. Bax is a monomeric and predominant cytosolic protein in
unstressed
cells. Apoptotic signals induce conformational change
of Bax, inducing its translocation to outer mitochondrial membrane, where its
oligomerization leads to membrane permeabilization and cytochrome c release
[6].Members of the heat shock protein family of molecular chaperones can
suppress the apoptotic program and attempt to repair the damage. These proteins
play essential roles in regulating protein conformation by preventing protein
misfolding and aggregation as well as assisting the productive folding of
proteins to their native state [7]. Glucose-regulated protein 75
(Grp75/mortalin/PBP74/mthsp7) is an important molecular chaperone belonging to
the heat shock protein 70 (HSP70) family [8]. It is located in multiple
subcellular organs, dominantly in mitochondria [9]. Grp75 achieves multiple
functions including mitochondrial import, intracellular trafficking, receptor
internalization, and inactivation of tumor suppressor protein p53 [10]. It can
also respond to many forms of stress including glucose deprivation (GD),
oxidative injury, and ultraviolet (UV) irradiation [11]. Our previous
observation showed that Grp75 overexpression provides protection against injuries
induced by GD [12]. However, the mechanisms underpinning this protection are
not fully understood. In this study we examined the mechanism through which Grp75 protect
cells from GD injury. The results indicated that Grp75 could protect PC12 cell
which was used primarily as a neuron model from apoptosis through the
inhibition of Bax conformational change and cytochrome c release. And Grp75
decreased the Bax/Bcl-2 gene expression ratio during the apoptotic
process.
Materials and Methods
Cell culture and antibodies
Rat adrenal pheochromocytoma (PC12) cell line was purchased from
American Type Culture Collection (Manassas, USA). Cells were grown in
Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine
serum, 100 mg/ml streptomycin, and 100 u/ml
penicillin in a 5% CO2 humidified atmosphere at 37 ?C.The following primary antibodies were used for Western blot and
immunofluorescence experiments: anti-actin antibody (Sigma-Aldrich, St. Louis,
USA), anti-Grp75 antibody (Abcam, Cambridge, UK), anti-Bcl-2 antibody (Cell
Signaling Technology, Beverly, USA), anti-Bax antibody (Cell Signaling
Technology), anti-Bax 6A7 antibody (Trevigen, Gaithersburg, usa), and anti-cytochrome c antibody
(BD Pharmingen, San Diego, USA).
Transfection and Grp75
overexpression in PC12 cells
The pcDNA3/Grp75 containing Grp75 full-length cDNA or pcDNA3 (empty
vector) was transfected into PC12 cells using Lipofectamine 2000 (Invitrogen,
Carlsbad, USA). Neomycin-resistant colonies were isolated in the medium
supplemented with neomycin analog G418-sulfate (1 mg/ml; Amresco, Solon, USA).
Single cell cloned was cultured further in the presence of G418-sulfate (600 mg/ml). The
expression of Grp75 in PC12 cells, pcDNA3-transfected cells and pcDNA3/Grp75-transfected
cells was confirmed by Western blot analysis.
GD
Exponentially growing cells plated on dishes or wells were gently
washed twice with glucose-free DMEM medium then incubated in glucose-free DMEM
medium for 6, 12, 24, and 48 h.
3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium
bromide (MTT) reduction assay for estimating cell viability
The determination of cell viability was carried out by MTT reduction
assay, based on the reduction of tetrazolium salt. In brief, MTT (Pufei,
Shanghai, China) was added to the culture medium at the end of incubation.
After further incubation at 37 ?C for 4 h, media were removed. Cells were lysed
and formazan was dissolved with 150 ml dimethyl sulfoxide. Absorbance at 492 nm was
measured with a microplate reader (Thermo Scientific, Anaheim, USA) and results
were expressed as the percentage of MTT reduction, assuming the absorbance of
control cells as 100%.
Determination of apoptosis
Giemsa and Hoechst 33258 staining Cell morphology was studied by Giemsa and
Hoechst 33258 staining. In brief, cells were seeded on poly-L-lysine-coated
coverslips for 1 d. Slides were treated with GD medium for the indicated time,
then fixed with 4% paraformaldehyde. Cells were stained with Giemsa or Hoechst
33258. Five hundred cells from each slide were examined and counted under a
fluorescence microscope (Leica, Wetzlar, Germany). Annexin V/propidium iodide (PI) staining An early indicator of apoptosis is the
rapid translocation and accumulation of the membrane phospholipid phosphatidylserine
from the cytoplasmic interface of the membrane to the extracellular surface.
This disruption of membrane asymmetry can be detected using the binding
properties of annexin V. To confirm apoptosis, an annexin
V-fluorescein-isothiocyanate (FITC) apoptosis detection kit (R&D Systems,
Minneapolis, USA) was used according to the manufacturer’s instructions, and
analyzed by flow cytometry (Beckman Coulter, Miami, USA).
Whole cell protein extraction
and Western blot analysis
Cells were lysed in RIPA buffer [150 mM NaCl, 1% NP-40, 0.5% Doc,
0.1% sodium dodecyl sulfate, and 50 mM Tris/HCl (pH 8.0)] supplemented with 1 mg/ml aprotinin
and 100 mg/ml phenylmethylsulfonyl fluoride. The cell suspension was
incubated on ice for 30 min then centrifuged at 20,000 g for 15 min at 4
?C. The supernatants were collected for further analysis. The protein concentration of the samples was determined by Bradford
assay. A total of 20 mg proteins was separated by 10% or 15% sodium dodecyl sulfate-polyacrylamide
gels and transferred onto nitrocellulose membranes (Amersham Pharmacia Biotech,
Little Chalfont, UK). Membranes were blocked with 5% (w/v)
not-fat dry milk in TBS-T buffer (20 mM Tris-HCl, pH 7.6, 137 mM NaCl, and
0.05% Tween-20) and incubated overnight at 4 ?C with relevant primary
antibodies, followed by washing and incubation with appropriate horseradish
peroxidase-conjugated secondary antibodies. Immunocomplexes were visualized
using the enhanced chemiluminescence Western blotting detection system (Cell
Signaling Technology) with exposure of the membranes to X-ray film (Eastman
Kodak, Rochester, USA). The signal intensity of the respective bands was
quantified by a scanning densitometer using an image analysis system with Scion
Image version 4.03 software (http://www.bbioo.com/Soft/2006/305.htm).
Reverse
transcription-polymerase chain reaction (RT-PCR) and real-time quantitative
RT-PCR
Total RNA was isolated from cells after GD for the indicated times
using TRIzol reagent (Invitrogen) according to the manufacturer’s instructions.
The first-strand cDNA was synthesized with a RevertAid first strand cDNA
synthesis kit (MBI Fermentas, Vilnius, Lithuania). cDNA amplification was
carried out according to the following temperature profile: 95 ?C for 30 s, 58
?C for 1 min, and 72 ?C for 1 min. At the end of 28 cycles, the reaction was
prolonged for 8 min at 72 ?C, then 5 ml mixture was analyzed on a 1% agarose gel.
The primers used in this study were: b-actin, 5‘-AACCGTGAAAAGATGACCCAG-3‘ and
5‘-CTCCTGCTTGCTGATCCACAT-3‘; Bcl-2, 5‘-ACTTTGCAGAGATGTCCAGTCAG-3‘
and 5‘-GTTCAGGTACTCAGTCATCCACAG-3‘; and bax, 5‘-GGAGGAAGTCCAGTGTCCAG-3‘
and 5‘-TGCAGAGGATGATTGCTGAC-3‘.Real-time quantitative RT-PCR was carried out on cDNA generated as
described above. cDNA, primers, and iQ SYBR Green SuperMix (Bio-Rad, Hercules,
USA) in a final volume of 25 ml were used for PCR. Amplification and detection of specific
products were carried out in a Mini opticon real-time PCR system (Bio-Rad)
under the following conditions: at 94 ?C for 30 s, 58 ?C for 30 s, and 72 ?C
for 40 s, 40 cycles. As an internal control, 18s rRNA (5‘-CGGCTACCACATCCAAGGAA-3‘ and 5‘-GCTCGAATTACCGCGGCT-3‘)
was used for template normalization. Following amplification, a melting curve
analysis was carried out to verify the correct product by its specific melting
temperature (Tm). The 2-DDCt method was used to analyze the relative changes in expression of the
target gene [13]. The relative expression level between treatments can be
calculated using the following equation:
Eq.
Immunofluorescence
Slides were air-dried and cells were fixed in 4% paraformaldehyde
(Sigma-Aldrich) for 10 min. After washing with phosphate-buffered saline, the
slides were treated by 0.1% Triton X-100 or 0.01% saponin, blocked with 10%
normal goat serum, and incubated overnight at 4 ?C with anti-cytochrome c or
anti-Bax 6A7 antibody. After washing with phosphate-buffered saline, slides
were then incubated with FITC-conjugated goat anti-mouse immunoglobulin G
(Sigma-Aldrich) for 1 h. The slides were then incubated with Hoechst 33258 for
5 min. Slides were examined under a fluorescence microscope (Leica) and all
pictures were taken by a Cool Snap CCD camera (Leica) attached to the
microscope.
Statistical analysis
Data were expressed as the mean±EM from at least three independent
experiments. Statistical significance analysis was carried out using Student’s t-test
or ANOVA. P<0.05 was considered significant.
Results
Inhibition of GD injury by
Grp75
PC12 cells were transfected with pcDNA3/Grp75 or control pcDNA3
vector, and selected for stable clones. Western blot analysis revealed a higher
expression level of Grp75 in clone 1 and clone 2 (transfected with pcDNA3/Grp75)
compared with vector-transfected cells [Fig. 1(A)]. We selected the
cells from clone 1 (Grp75 overexpressing) and vector-transfected cells
(control) for further experiments. To investigate the influence of Grp75 on PC12 cells under GD, the
MTT method was used to monitor the cell viability. Corresponding to the reduced
cell viability, cell growth was inhibited in a time-dependent manner in control
cells after glucose withdrawal [Fig. 1(B)]. After exposed to
glucose-free medium for 12 h, the cell viability of the control group decreased
by 55.1%. However, no obvious decrease in cell viability was found in Grp75
overexpressing cells after 12 h. The cell viability decreased to 73.8% in the
Grp75 group after GD treatment for 24 h. The cell viability in control cells
was only 41.6%. Even though the cell viability in the Grp75 overexpressing
group clearly decreased after 48 h, it was significantly higher than in the
control group (p<0.05). All the data indicated that Grp75 could protect PC12 cells from GD injury.
Grp75 suppresses apoptosis
induced by GD
To elucidate whether the decreased rate of cell viability was caused
by apoptosis and whether the apoptosis could be inhibited by Grp75, cell
morphology and annexin V-FITC/PI double staining were analyzed. Morphological
changes were examined with Giemsa and Hoechst 33258 staining. The results of
Giemsa and Hoechst 33258 staining were consistent. After treatment with
glucose-free medium for 6 h, approximately 10% of control cells showed
characteristic morphological changes of apoptosis, such as shrunken cytoplasm
and nuclear fragmentation with an intact cell membrane [Fig. 2(A)] or
contracted nucleus and condensed chromatin fragments [Fig. 2(C)].
Apoptotic cells were progressively increased in the control group induced by
GD. After 48 h, almost all the control cells showed typical apoptotic
morphological changes, however, only 45% of Grp75 overexpressing cells
underwent apoptosis. Compared with the control group at the indicated times,
the numbers of apoptotic cells in the Grp75 overexpressing group were
significantly decreased (p<0.05) [Fig. 2(B,D)]. We quantified the apoptotic cells after GD treatment by flow
cytometry analysis using annexin V-PI staining, a hallmark for apoptosis [14].
The results (Fig. 3) showed that early apoptotic cells increased after
GD treatment for 6 h in the control group and for 12 h in the Grp75
overexpressing group. After 24 h, the number of early apoptotic cells decreased
and the number of late apoptotic and necrotic cells increased in both groups.
Although the apoptotic course of cells was similar, the apoptotic cell numbers
in the Grp75 overexpressing group showed a significant decrease compared with
that in the control group. All of these results suggested that GD treatment suppresses
the growth of PC12 cells by inducing apoptosis, which can be inhibited by
Grp75.
Grp75 delays release of
cytochrome c from mitochondria following GD
Cytochrome c release from mitochondria to cytosol is a key event in
the intrinsic apoptotic pathway, induced by various cell stresses. We therefore
examined whether the apoptosis of PC12 cells induced by GD uses the
mitochondrial-mediated apoptotic pathway, and whether Grp75 plays a role in
regulating the release of cytochrome c. Immunofluorescence staining of normal
cells with an anti-cytochrome c antibody gave a punctate staining pattern of
mitochondrial localization (Fig. 4). After GD for 6 h, individual cells
of the control group showed diffuse cytoplasmic staining, indicating that
cytochrome c was released from mitochondria (Fig. 4). Most cells lost
mitochondrial cytochrome c staining after 12 h in the control group. Almost all
the control cells lost mitochondrial cytochrome c staining after 24 h and, at
the same time point, most cells showed cytoplastic shrinkage, nuclear
fragmentation, and chromation condensation. However, Grp75 overexpressing cells
showed loss of mitochondrial cytochrome c staining at 12 h, and most cells
showed diffuse cytoplasmic staining after 24 h. Although some cells failed to
show dominant apoptotic nuclei, almost all cells in the Grp75 overexpressing
group showed loss of mitochondrial cytochrome c staining after 48 h. So we
concluded that Grp75 overexpression delayed cytochrome c release induced by GD.
Grp75 inhibits increase of Bax/Bcl-2
ratio
The Bcl-2 family is an important regulator in cytochrome c release,
so changes in the expression of either the pro-apoptotic or anti-apoptotic
Bcl-2 family members can affect the execution of apoptosis. Because the
relative ratios of Bcl-2 to Bax, rather than the single expression of either
protein, can prescribe survival or death to diverse apoptotic stimuli [5], we
analyzed the expression of both Bcl-2 and Bax. RT-PCR results
revealed that the Bcl-2 mRNA expression was decreased under GD, but
there was no significant difference in Bax mRNA expression between the
two groups [Fig. 5(A,B)]. Compared with control cells, the
down-regulation of Bcl-2 mRNA levels in Grp75 overexpressing cells was
slightly inhibited. These RT-PCR results were confirmed by real-time
quantitative RT-PCR [Fig. 5(C)]. The level of Bcl-2 mRNA in the
control group began to decrease after 12 h; in the Grp75 overexpressing group,
the decrease began after 24 h [Fig. 5(D)]. There was no change in Bax
mRNA expression (data not shown). An increase in the Bax/Bcl-2 ratio was
observed in the control group after 12 h and in the Grp75 overexpressing group
after 24 h [Fig. 5(E)]. At the same time point, the increased Bax/Bcl-2
ratio was slightly inhibited by Grp75 overexpression.To further analyze the changes of Bax and Bcl-2 protein levels,
Western blot analysis was carried out [Fig. 6(A)]. Normalized by b-actin, the
Bcl-2 protein levels were slightly decreased under GD in control cells, whereas
Bax protein levels remained unchanged [Fig. 6(A,B)]. Consistent with the
PCR results, the Bax/Bcl-2 ratio of control cells was increased after glucose
withdrawal [Fig. 6(C)]. In the Grp75 overexpressing cells, the
down-regulation of Bcl-2 and increased Bax/Bcl-2 ratio were inhibited. All the
results indicated that Grp75 overexpression slightly inhibited the increased
Bax/Bcl-2 ratio both at the mRNA and protein levels.
Grp75 inhibits Bax
conformational change induced by GD
Apoptotic signals induce conformational change in Bax, leading to
Bax translocation to the outer mitochondrial membrane, where it serves as a
critical gateway to cytochrome c release [15]. As no change was detected in the
cellular content of Bax in GD-treated cells, we suggested that there might be
some change in Bax conformation, which could be examined by immunofluorescence.
The analysis was carried out using a Bax conformation-specific antibody (6A7)
that recognizes only the form that is competent for membrane insertion [16].
Normal cells in both groups were negative for Bax staining with Bax (6A7)
antibody [Fig. 7(A)]. After 6 h of glucose withdrawal, nearly 35% of
control cells were strongly stained by the anti-Bax antibody, indicating that
Bax underwent a conformational change [Fig. 7(B)]. The numbers of
Bax-positive cells increased in a time-dependent manner in the control group.
In the Grp75 overexpressing group, Bax-positive cells increased to
approximately 23% after 12 h, compared to approximately 50% in the control
group. In addition, some Bax-positive cells also showed typical apoptotic
nuclear morphology. Compared with the control group under the same conditions,
the numbers of apoptotic cells in the Grp75 overexpressing group were
significantly decreased (p<0.05). Therefore, Grp75 appeared to prevent GD-induced apoptosis by inhibiting the conformational change of Bax.
Discussion
Grp75 can be found in multiple subcellular sites [17] and is a
highly conserved member of the HSP70 family. Grp75 is involved in multiple
physiological functions, such as mitochondrial import, regulation of glucose
responses, antigen processing, control of cellular proliferation, and
differentiation [18–20]. Previous studies showed that Grp75 could
rescue some cells from apoptosis induced by various stresses, such as serum
starvation, UV irradiation, and g-irradiation [21]. Grp75 can also protect the
brain against focal cerebral ischemia in vivo [22], and our previous
studies indicated that overexpression of Grp75 could protect Chinese hamster
fibroblast cells and PC12 cells from damage induced by GD in vitro [11,12].
However, the critical mechanisms by which Grp75 exerts its cytoprotective
activity have not yet been clearly elucidated. In this study, our results showed
that the injury of PC12 cells induced by GD treatment was caused by apoptosis,
which was significantly reduced by Grp75 overexpression. Although the increased
expression of Grp75 failed to fully protect the cells from GD-induced damage,
Grp75 overexpression could suppress apoptosis at least for 48 h after GD. There are two overlapping signaling pathways that lead to apoptosis,
termed the intrinsic and extrinsic pathways. Mitochondria play a critical role
during apoptosis [23] and the key element in the intrinsic pathway is the
release of cytochrome c from mitochondria into the cytosol. Cell damage induced
by many stresses involves the intrinsic pathway of apoptosis, such as UV
irradiation, serum deprivation, and ischemia/reperfusion injury [24,25]. Our
data showed that the release of cytochrome c began at 6 h after GD in the
control group and the overproduction of Grp75 delayed the release of cytochrome
c release into the cytosol. Compared with the time-course of the appearance of
apoptosis, we concluded that the mitochondrial pathway was involved in
apoptosis induced by GD, and Grp75 decreased apoptosis through the regulation
of cytochrome c release. To assess whether Grp75 affected events occurring upstream of
cytochrome c release, we focused on the Bcl-2 protein family, known to
constitute a central checkpoint during apoptosis [26]. Bcl-2, one of the most
important anti-apoptotic proteins of the Bcl-2 family, can prevent cytochrome c
release [27], whereas pro-apoptotic family members such as Bax can promote
cytochrome c release. Released cytochrome c contributes to apoptosis through
binding Apaf-1 and caspase-9, whereas anti-apoptotic Bcl-2 binds to Bax and can
form heterodimers that block cellular apoptosis [28]. The relative ratio of
Bcl-2 to Bax has been reported to be associated with apoptosis [5]. Our data
clearly showed that PC12 cells treated with GD did not result in Bax
gene expression change at either the mRNA or protein level. So the
time-dependent decrease of Bcl-2 gene expression level resulted in the
increase of the Bax/Bcl-2 ratio. But the increased Bax/Bcl-2
ratio failed to precede the morphological appearance of apoptosis already
apparent at 6 and 12 h, so it is probably not the major cause of apoptosis by GD,
or it is just a consequence of apoptosis. Although Grp75 overexpression could
slightly inhibit the increase of the Bax/Bcl-2 gene expression ratio, it
was not the major reason for Grp75 protecting PC12 cells from GD injury.However, the conformational change of Bax and its translocation to
mitochondria are responsible for cytochrome c release from mitochondria during
apoptosis [29]. We found that Grp75 could also inhibit Bax conformational
change induced by GD. At the same time, the time-course of Bax conformational
change inhibition was consistent with cytochrome c release in both groups. So
we concluded that Grp75 blocks GD-induced apoptosis, at least partially, due to
the inhibition of Bax conformational change, thereby preventing the release of
cytochrome c from the mitochondrial intermembrane space. HSPs are reported to promote cell survival by inhibiting apoptosis
at several levels [30]. How can Grp75, a molecular chaperone, affect the
process of apoptosis? One possibility is the chaperone function of Grp75 for
Bax. Grp75, as a molecular chaperone, plays an essential role in the
restoration of structure and function of denatured proteins [31,32]. Bax
activation is associated with exposure of the C-terminal transmembrane region
by an unknown mechanism, and this conformational change might be expected to
provide an ideal target to interact with Grp75. Another aspect of Grp75
protection is its interaction with other components of the tightly regulated
apoptosis machinery upstream of mitochondrial events. Our research also
indicated that Grp75 could influence changes in the mitogen-activated protein
kinase/extracellular regulated kinase and AKT pathways induced by GD (data not
shown). Members of the Bcl-2 family have been shown to be targets of the kinases
that are activated in stressed cells [4]. Wadhwa et al [33]
showed that Grp75 could inactivate p53 through binding to its cytoplasmic
sequestration domain. And p53 can function analogously to the BH3-only subset
of pro-apoptotic Bcl-2 proteins to activate Bax and trigger apoptosis [34].
Based on these facts, Grp75 might also affect the Bcl-2 family members through
its interaction with the members of mitogen-activated protein
kinase/extracellular regulated kinase and AKT pathways or p53. All of these
hypotheses require further study. In summary, our results showed that overexpression of Grp75 could
inhibit Bax conformational change, delay the release of cytochrome c, and
suppress apoptosis, thus providing protection for PC12 cells against GD
toxicity.
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