Article

Inhibition of the JNK signalling pathway enhances proteasome inhibitor-induced apoptosis of kidney cancer cells by suppression of BAG3 expression

Department of Biochemistry & Molecular Biology, China Medical University, Shenyang, China.
British Journal of Pharmacology (Impact Factor: 4.84). 09/2009; 158(5):1405-12. DOI: 10.1111/j.1476-5381.2009.00455.x
Source: PubMed
ABSTRACT
Proteasome inhibitors represent a novel class of anti-tumour agents that have clinical efficacy against haematological and solid cancers. The anti-apoptotic protein BAG3 is a member of the Bcl-2-associated athanogene family. We have previously shown that BAG3 is up-regulated after exposure to proteasome inhibitors and that inhibition of BAG3 sensitized cells to apoptosis induced by proteasome inhibition. However, the mechanisms by which proteasome inhibition induced BAG3 expression remained unclear and the present experiments were designed to elucidate these mechanisms.
Effects of the proteasome inhibitor MG132 on activation of mitogenic signalling pathways were evaluated in kidney cancer cells (A498, Caki1, Caki2), with Western blotting. Specific inhibitors against individual mitogenic signalling pathways, real-time reverse transcription-polymerase chain reaction and luciferase reporter assays were used to investigate the roles of mitogenic signalling pathways in BAG3 induction after proteasome inhibition. Cell death was evaluated using Annexin V/propidium iodide staining and subsequent FACS.
MG132 activated several key mitogenic signalling pathways including extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK) activities. Induction of BAG3 by MG132 was inhibited by blocking JNK, but not ERK1/2 and p38 MAPK signalling pathways. In addition, SP600125 and dominant-negative JNK1 suppressed BAG3 promoter-driven reporter gene expression. Furthermore, activation of the JNK pathway induced BAG in kidney cancer cells after treatment with MG132.
Our results suggested that the JNK pathway was associated with the protective response against proteasome inhibition, by mediating induction of BAG3.

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Available from: Xin Meng, Feb 07, 2015
RESEARCH PAPER
Inhibition of the JNK signalling pathway enhances
proteasome inhibitor-induced apoptosis of kidney
cancer cells by suppression of BAG3 expression
bph_455 1405..1412
Hua-Qin Wang
1
, Bao-Qin Liu
1
, Yan-Yan Gao
1
, Xin Meng
1
, Yifu Guan
1
, Hai-Yan Zhang
2
and
Zhen-Xian Du
3
1
Department of Biochemistry & Molecular Biology, China Medical University, Shenyang, China,
2
Department of Geriatrics, the
1
st
Affiliated Hospital, China Medical University, Shenyang, China, and
3
Department of Endocrinology and Metabolism, the 1
st
Affiliated Hospital, China Medical University, Shenyang, China
Background and purpose: Proteasome inhibitors represent a novel class of anti-tumour agents that have clinical efficacy
against haematological and solid cancers. The anti-apoptotic protein BAG3 is a member of the Bcl-2-associated athanogene
family. We have previously shown that BAG3 is up-regulated after exposure to proteasome inhibitors and that inhibition of
BAG3 sensitized cells to apoptosis induced by proteasome inhibition. However, the mechanisms by which proteasome
inhibition induced BAG3 expression remained unclear and the present experiments were designed to elucidate these
mechanisms.
Experimental approach: Effects of the proteasome inhibitor MG132 on activation of mitogenic signalling pathways were
evaluated in kidney cancer cells (A498, Caki1, Caki2), with Western blotting. Specific inhibitors against individual mitogenic
signalling pathways, real-time reverse transcription-polymerase chain reaction and luciferase reporter assays were used to
investigate the roles of mitogenic signalling pathways in BAG3 induction after proteasome inhibition. Cell death was evaluated
using Annexin V/propidium iodide staining and subsequent FACS.
Key results: MG132 activated several key mitogenic signalling pathways including extracellular signal-regulated kinase (ERK),
c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK) activities. Induction of BAG3 by MG132 was
inhibited by blocking JNK, but not ERK1/2 and p38 MAPK signalling pathways. In addition, SP600125 and dominant-negative
JNK1 suppressed BAG3 promoter-driven reporter gene expression. Furthermore, activation of the JNK pathway induced BAG
in kidney cancer cells after treatment with MG132.
Conclusions and implications: Our results suggested that the JNK pathway was associated with the protective response
against proteasome inhibition, by mediating induction of BAG3.
British Journal of Pharmacology (2009) 158, 1405–1412; doi:10.1111/j.1476-5381.2009.00455.x; published online 13
August 2009
Keywords: BAG3; MAPK; MG132; JNK; proteasome; cancer; apoptosis
Abbreviations: AMC, 7-amino-4-methylcoumarin; bp, base pair; DN, dominant-negative; ERK, extracellular signal-regulated
kinase; IP, immunoprecipitation; JNK, c-Jun N-terminal kinase; MAPK, mitogen-activated protein kinase; PCR,
polymerase chain reaction; RNAi, RNA interference; RT-PCR, reverse transcription-polymerase chain reaction;
WT, wild type
Introduction
Proteasome inhibitors represent a new class of drugs that have
anti-tumour activity through a number of mechanisms,
including interfering with cell cycle progression, inducing
apoptosis and inhibiting angiogenesis (Voorhees et al., 2003;
Rajkumar et al., 2005). However, it is increasingly recognized
that, as is the case for other drugs, they may also activate
some anti-apoptotic survival pathways that limit their own
anti-tumour efficacy. The anti-apoptotic protein BAG3 is such
an example and this protein was induced by proteasome
inhibition and impaired tumour response to proteasome
inhibitor treatment (Wang et al., 2008). BAG3, also known as
Bis or CAIR-1, binds to the ATPase domain of heat shock
protein (Hsp)70 with high affinity and inhibits its chaperone
Correspondence: Hua-Qin Wang, Department of Biochemistry & Molecular
Biology, China Medical University, Shenyang 110001, China. E-mail:
wanghq_doctor@hotmail.com
Received 9 April 2009; revised 30 May 2009; accepted 1 July 2009
British Journal of Pharmacology (2009), 158, 1405–1412
© 2009 The Authors
Journal compilation © 2009 The British Pharmacological Society All rights reserved 0007-1188/09
www.brjpharmacol.org
Page 1
activity (Takayama et al., 1999; Rosati et al., 2007a). BAG3 has
also been reported to bind with Bcl-2 and have a synergistic
effect on the anti-apoptotic activity of Bcl-2 (Lee et al., 1999).
With regard to cancer, expression of BAG3 is elevated in
leukaemia and some solid tumours (Liao et al., 2001; Romano
et al., 2003; Chiappetta et al., 2007). Notably, BAG3 expres-
sion can be induced by some stressful agents such as high
temperature and heavy metal exposure (Liao et al., 2001; Pag-
liuca et al., 2003), HIV-1 infection (Rosati et al., 2007b), and
proteasome inhibition (Wang et al., 2008), suggesting that it
is a protein inducible by stress stimuli.
The mitogen-activated protein kinase (MAPK) family of
proteins includes p38 MAPK, c-Jun N-terminal kinase (JNK)
and extracellular signal-regulated kinase (ERK). MAPKs are
proline-directed serine/threonine kinases that are activated by
dual phosphorylation on threonine and tyrosine residues in
response to a wide variety of stress stimuli (Pearson et al.,
2001). They mediate signal transduction from the cell surface
to the nucleus and are involved in the expression of a variety
of genes (Karin, 1995). Pharmacological inhibitors of p38
enhanced the ability of proteasome inhibitors to induce apo-
ptosis and seemed also to help overcome resistance to protea-
some inhibitors in models of lymphoma (Meriin et al., 1998;
Chauhan et al., 2003; Hideshima et al., 2004), indicating that
manipulation of these signalling pathways might influence
the anti-tumour effectiveness of proteasome inhibitors.
In the present study, we characterized the role of MAPK
signalling pathways in the induction of BAG3, following
exposure to proteasome inhibitor. Proteasome inhibition acti-
vated several sequential protein kinase pathways, leading to
increased activity of ERK, JNK and p38 MAPK in kidney
cancer cells. We demonstrated that the JNK inhibitor
SP600125 significantly suppressed the induction of BAG3 by
MG132, whereas the ERK inhibitor PD98059 and the p38
MAPK inhibitor SB203580 had little effect on BAG3 induc-
tion. We also demonstrated that the JNK inhibitor SP600125
or dominant-negative JNK1 (DN-JNK1) significantly sup-
pressed the induction of BAG3 by MG132. We further
observed that inhibition of the JNK pathway significantly
increased levels of apoptosis induced by MG132. In addition,
ectopic expression of BAG3 reversed the enhancing effects of
the JNK inhibitor, suggesting that, in kidney cancer cells, the
JNK pathway plays anti-apoptotic roles after proteasome inhi-
bition, mediated, at least in part, by induction of BAG3.
Methods
Cell culture
The A498, Caki-1 and Caki-2 kidney cancer cell lines were
maintained in DMEM supplemented with 10% FBS (Sigma-
Aldrich, Saint Louis, MO).
20S proteasome activity assay
Cytosolic extracts (without protease inhibitors) were used to
measure proteasome activity using a 20S proteasome assay kit
(Chemicon International, Temecula, CA) following the manu-
facturer’s instructions. The assay is based on detection of the
fluorophore 7-amino-4-methylcoumarin (AMC) after cleavage
from the labelled substrate LLVY-AMC. Levels of released
AMC were measured using an excitation wavelength of
380 nm and an emission wavelength of 460 nm with an
automatic multi-well plate reader. The relative activity was
standardized by protein concentration, determined using
Coomassie Protein Assay Reagent (Pierce, Rockford, IL).
Western blot analysis
Cells were lysed in lysis buffer (20 mM Tris-HCl, 150 mM
NaCl, 2 mM EDTA, 1% Triton-X100) containing a protease
inhibitor cocktail (Sigma-Aldrich, Saint Louis, MO). Cell
extract protein amounts were quantified using the BCA
protein assay kit. Equivalent amounts of protein (20 mg) were
separated using 12% SDS-PAGE and transferred to PVDF
membranes (Millipore Corporation, Billerica, MA).
Luciferase assay
The 5-flanking region of human BAG3 genomic DNA
between -1556 and +5(+1 represents the translation start site)
was amplified by polymerase chain reaction (PCR) from HeLa
genomic DNA and subcloned into the reporter plasmid pGL4
(Promega, Madison, WI). The luciferase activity was deter-
mined using the Dual-Luciferase
®
Reporter Assay System
(Promega, Madison, WI), according to the manufacturer’s
instructions. All transfection experiments were repeated for
three times, each in triplicate. Firefly (Photinus pyralis)
luciferase activities normalized by Renilla (Renilla reniformis)
activities are presented as fold induction relative to the nor-
malized firefly luciferase activity in cells transfected with the
pGL4 empty vector only, which was taken as 1.0.
Construction of plasmids and transfection
A cDNA encoding human BAG3 or JNK1 was generated by
PCR from human brain cDNA library (Invitrogen, Carlsbad,
CA) and subcloned into the eukaryotic expression plasmid
pcDNA3 tagged with Flag epitope. The DN-JNK1 cDNA was
produced by amino acid substitution of threonine (ACG) 183
for alanine (GCG) and tyrosine (TAT) 185 for phenylalanine
(TTT) in the dual-activating phosphorylation sites of wild-
type JNK1 and subcloned into pcDNA3 tagged with Flag
epitope. A498 cells were transfected with indicated vector
using Lipofectamine 2000 reagent according to the protocol
of the manufacturer.
RNA isolation and real-time reverse transcription PCR
RNA isolation and real-time reverse transcription PCR was
performed as previously reported (Wang et al., 2007). For
BAG3, the forward primer was 5-ATGCAGCGATTCCGAA
CTGAG-3 and the reverse was 5-AGGATGAGCAGTCAG
AGGCAG-3, the amplicon size is 191 base pair (bp). For
b-actin, the forward primer was 5-GAGACCTTCAAC
ACCCCAGCC-3 and the reverse was 5-GGATCTTCAT
GAGGTAGTCAG-3, the amplicon size is 205 bp. Results were
normalized against those of b-actin.
Chromosomal immunoprecipitation assay
Chromosomal immunoprecipitation (IP) assays were per-
formed using a kit from Upstate Biotechonology Inc. (Lake
Induction of BAG3 by MG132 via JNK
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British Journal of Pharmacology (2009) 158 1405–1412
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Placid, NY) according to the supplied protocol. In brief, cells
were exposed to 2 mM MG132 for 8 h and fixed with 1%
formaldehyde in phosphate-buffered saline to cross-link chro-
matin. Cell lysates were prepared and sonicated on ice to
break chromatin DNA to an average length of 400 bp. After a
preclearing step, IP was carried out at 4°C overnight with
anti-HSF1 antibody or normal goat IgG (negative control anti-
body). Immune complexes were collected with salmon sperm
DNA saturated protein A-agarose beads. After extensive
washing the immunoprecipitated complexes were eluted with
0.1 M NaHCO
3
and 1% SDS, and then protein-DNA cross-
links were reversed by incubating at 65°C for 5 h. DNA was
purified using proteinase K digestion, phenol: chloroform
extraction and ethanol precipitation. Real-time PCR was per-
formed using primers specific for the BAG3 promoter
sequence between -327 and -174 (forward: 5-GAT
TATAGCCGATGACTCAGGGCG-3 and reverse: 5-AGTGT
CTGGAAATAGCCTCC-3) to generate a 154 bp amplification
product containing the HSE sequence. A standard curve was
prepared using serial dilutions of the pBAG3/-825 promoter
construct. The amount of BAG3 promoter that was present in
the IP and input fractions was calculated from the standard
curve. The input represents 1% of the material used in the IP
assay. The results were expressed as the IP/input ratios of the
PCR products,which were used for comparison.
Detection of cell death
For cell death assays, cells were washed twice in phosphate-
buffered saline and then stained with Annexin V-FITC
(Biovision, Mountainview, CA) and propidium iodide
(Sigma-Aldrich) according to the manufacturer’s instructions.
After staining with annexin V-FITC and propidium iodide,
samples were analysed by fluorescence-activated cell scanner
(FACScan) flow cytometer (Becton Dickinson, Franklin
Lakes, NJ).
Statistics
The statistical significance of the difference was analysed by
ANOVA and post hoc Dunnett’s test. Statistical significance was
defined as P < 0.05. All experiments were repeated three times,
and data were expressed as the mean SD (standard devia-
tion) from a representative experiment.
Materials
MG132, PD98059, SB203580 and SP600125 were purchased
from Calbiochem (La Jolla, CA). The following antibodies
were used in this study: mouse anti-p44/42 MAPK (ERK1/2)
monoclonal antibody (Cell Signaling Technology, Danvers,
MA), rabbit anti-phospho-p44/42 MAPK (ERK1/2) (Thr202/
Tyr204) monoclonal antibody (Cell Signaling Technology,
Danvers, MA), rabbit anti-c-Jun polyclonal antibody (Abcam,
Cambridge, MA), rabbit anti-JNK monoclonal antibody (Cell
Signaling Technology, Danvers, MA),mouse anti-phospho-
JNK (Thr183/Tyr185) monoclonal antibody (Cell Signaling
Technology, Danvers, MA), rabbit anti-p38 MAPK mono-
clonal antibody (Cell Signaling Technology, Danvers, MA),
rabbit anti-phospho-p38 (Thr180/Tyr182) monoclonal anti-
body (Cell Signaling Technology, Danvers, MA) and rabbit
anti-BAG3 polyclonal antibody (Abcam, Cambridge, MA).
Results
Sequential activation of protein kinase pathways by MG132
MG132 quickly suppressed proteasome activity in A498,
Caki1 and Caki2 renal cancer cells, and more than 40% sup-
pression was observed upon exposure to 2 mM MG132 for
30 min (Figure 1A). The suppression peaked at 2 h and was
maintained for 24 h (Figure 1A), suggesting that the dose of
MG132 used in this study effectively suppressed proteasome
activity in kidney cancer cells. A498 cells were then treated
with MG132 and the activation state of mitogenic signalling
pathways (ERK, p38 MAPK and JNK) was measured over 24 h
using antibodies against the phosphorylated active forms of
the proteins and Western blot analysis (Figure 1B). ERK, JNK
and p38 activities are all activated by MG132 exposure.
However, the regulation of each pathway by MG132 is differ-
ent as characterized below. ERK activity increased rapidly to a
maximal level within 1 h of treatment. Levels of phospho-p38
Figure 1 Proteasome inhibition activates several mitogenic signal-
ling pathways. (A) A498, Caki1 and Caki2 kidney cancer cells were
treated with 2 mM MG132 for the indicated times and 20S protea-
some activity was analysed. (B) A498 cells were incubated in the
presence of vehicle or 2 mM MG132 for the indicated time. Total
cytosolic proteins were isolated and subjected to Western blotting to
assess levels of three mitogen-activated protein kinases, extracellular
signal-regulated kinase (ERK)1/2, c-Jun N-terminal kinase (JNK)1/2
and p-38. We used antibodies for the active, phosphorylated protein
and for the total level of protein. Blots are representative of three
individual experiments.
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British Journal of Pharmacology (2009) 158 1405–1412
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increased time-dependently to a maximum at 8–12 h. JNK
and its major substrate c-Jun activation were seen 4 h after
addition of MG132 and peaked at 8–12 h (Figure 1B).
Effects of MAPK inhibitors on MG132-induced BAG3 expression
Because studies have shown that the MAPK pathway is critical
for the activation of gene expression upon various stimuli, we
next sought to determine if the activation of these pathways
by MG132 exposure influences BAG3 induction. To evaluate
the roles of MAPKs in BAG3 induction by MG132, the small
molecule inhibitors, PD98059, SB203580 and SP600125, were
used as inhibitors for ERK, p38 kinase and JNK respectively. To
confirm that the inhibitors were functional in our model,
cells were treated with vehicle or MG132, with or without the
specific mitogenic inhibitors. The activation state of the path-
ways was then measured by Western blot analysis (Figure 2A).
All these inhibitors specifically inhibited the activity of their
respective target proteins and had no cross-reactivity with the
other pathways examined (Figure 2A). As reported in other
types of cancer cells (Wang et al., 2008), real-time PCR con-
firmed that MG132 significantly induced BAG3 expression in
A498 kidney cancer cells (Figure 2B). BAG3 induction by
MG132 was significantly inhibited by pre-treatment with
SP600125, but not in A498 cells pre-treated with PD98059 or
SB203580 (Figure 2B). To assess whether regulation of BAG3
induction by these pathways was a cell line-specific response,
these experiments were repeated using two other kidney
cancer cell lines, Caki1 and Caki2 (Figure 2C). Both cell lines
displayed induction of BAG3 in response to MG132 treatment
Figure 2 Effect of pharmacological inhibitors on BAG3 induction by MG132. (A) A498 cells were pre-treated with vehicle, PD98059 (10 mM),
SB203580 (10 mM) or SP600125 (20 mM) for 1 h. The cells were then incubated with MG132 for 8 h, and Western blot was performed using
the indicated antibodies. (B) A493 cells were pre-treated with various inhibitors for 1 h, subsequently treated with MG132 for additional 8 h,
and real-time polymerase chain reaction was performed. (C) Caki1 and Caki2 cells were treated as (B) and BAG3 mRNA was measured using
real-time polymerase chain reaction. *P < 0.001.
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British Journal of Pharmacology (2009) 158 1405–1412
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that was significantly suppressed by SP600125 pre-treatment,
whereas PD98059 and SB203580 had little effect on the levels
of BAG3 mRNA (Figure 2C). These results indicate that JNK
pathways are implicated in MG132-induced BAG3 expression
in kidney cancer cells.
Role of JNK pathway in activation of BAG3 transcription
by MG132
To confirm that JNK activity was involved in the MG132-
induced BAG3 expression in A498 cells, we investigated
whether administration of a JNK inhibitor or expression of
dominant-negative JNK could affect MG132-induced activa-
tion of BAG3 gene. Pre-treatment of cells with SP600125
inhibited BAG3 luciferase reporter activity in a dose-
dependent manner (Figure 3A). In addition, overexpression of
JNK1 increased BAG3 luciferase reporter activity induced by
MG132, whereas a DN-JNK1 dramatically suppressed BAG3
luciferase reporter activity (Figure 3B). The data thus confirm
that the JNK signalling pathway was involved in activation of
BAG3 transcription by MG132. Earlier, we had shown that
MG132 induced BAG3 expression via activation of heat shock
factor 1 (HSF1: Du et al., 2009). We further performed chro-
mosomal IP assay to investigate whether the JNK pathway
affected the binding of HSF1 to MG132-responsive elements
on the BAG3 promoter (Figure 3C). Treatment with MG132
recruited HSF1 proteins to the MG132-responsive sites located
on the BAG3 promoter, whereas the signal was significantly
suppressed by pre-treatment of the cells with SP600125
(Figure 3C). These results suggested that SP600125 inhibited
BAG3 induction by MG132 via suppressing the interaction of
HSF1 with MG132-responsive sites of the BAG3 promoter.
Effects of JNK inhibition or BAG3 overexpression on
MG132 cytotoxicity
We then investigated whether the activation of the JNK
pathway affects the apoptotic response to proteasome inhibi-
tion. Whereas treatment with the JNK inhibitor SP600125
alone had little effect, when used in combination with
MG132, it significantly increased levels of apoptosis induced
by MG132 in A498 cells (Figure 4A). The sensitizing effects of
Figure 3 c-Jun N-terminal kinase (JNK) pathways are involved in activation of the BAG3 promoter. (A) A498 cells were transfected with
reporter plasmid containing the BAG3 promoter. At 24 h after transfection, the cells were pre-treated with the indicated concentration of
SP600125 for 1 h, and then treated with 2 mM MG132 for an additional 4 h. Cells were harvested and luciferase and Renilla assays were
performed. *P < 0.05; **P < 0.001 versus treatment with MG132 alone. (B) Cells were co-transfected with the reporter plasmid containing the
BAG3 promoter with mock, JNK1 or dominant-negative JNK1 (DN-JNK1) expression plasmid for 24 h, then treated with 2 mM MG132 for
additional 4 h and luciferase activity was measured. The data are expressed as fold effect from three independent assays where luciferase
activity was normalized with Renilla expression. *P < 0.05; **P < 0.001. (C) Cells were pre-treated with vehicle or SP600125 for 1 h, then
exposed to 2 mM MG132 for 8 h; cross-linked chromatin was extracted and immunoprecipitated with an anti-HSF1 antibody. Immunopre-
cipitated DNA was amplified by real-time polymerase chain reaction. *P < 0.05.
Induction of BAG3 by MG132 via JNK
H-Q Wang et al
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British Journal of Pharmacology (2009) 158 1405–1412
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this inhibitor on the apoptotic responses of Caki1 and Caki2
cells to MG132 were broadly similar to the responses observed
in A498 cells (Figure 4B), suggesting that the JNK signal
pathway might possess anti-apoptotic roles in kidney cancer
cells treated with MG132.
Our previous studies indicated that proteasome inhibitors
induced BAG3 expression (Wang et al., 2008). Importantly, we
have also shown that blocking BAG3 up-regulation in thyroid
cancer cells using RNAi strategy can enhance cytotoxicity of
proteasome inhibitors (Wang et al., 2008). In the current
study, we found that activation of JNK was involved in induc-
tion of BAG3 by MG132. To confirm the potential role of
BAG3 in sensitizing effect of the JNK inhibitor, BAG3 was
ectopically overexpressed in A498 cells (Figure 4C) and the
cells were then treated with a combination of MG132 and
SP600125. The results (Figure 4D) showed that, in the pres-
ence of BAG3 overexpression, the pro-apoptotic effects of
MG132 were slightly, though significantly, less, than in the
mock-transfected cells. However, the addition of SP600125 to
MG132 now decreased this apoptotic effect in BAG3-
overexpressing cells, instead of potentiating it, as in normal
cells (see Figure 4A).
Discussion
Proteasome inhibitors represent a class of drugs that have
anti-cancer activity through a variety of cellular mechanisms,
including induction of apoptosis, interference with cell cycle
progression, inhibition of angiogenesis, and the suppression
of NF-kB (Voorhees and Orlowski, 2006). However, we have
previously shown that proteasome inhibitors also induced
BAG3 expression at the transcriptional level, and its induc-
tion suppressed anti-tumour effects of proteasome inhibitors
(Wang et al., 2008). Similar to our findings, several other dis-
tinct anti-apoptotic responses to proteasome inhibitor treat-
ments have been described. Induction of heat shock proteins
[Hsp27, Hsp70 (Hsp72) and Hsp90] has been implicated in
proteasome inhibitor-induced anti-apoptotic signalling
(Meriin et al., 1998; Robertson et al., 1999; Mitsiades et al.,
2002; Chauhan et al., 2003; Hideshima et al., 2004). Orlowski
et al. (2002) found that proteasome inhibitors increased the
cellular levels of mitogen-activated kinase phosphatase-1, a
specific deactivator of pro-apoptotic JNK activity in breast
cancer cells (Small et al., 2004; Shi et al., 2006). The suppres-
sion of these various responses by small molecule inhibitors
Figure 4 Effects of the c-Jun N-terminal kinase pathway or of overexpression of BAG3 on the apoptotic response of kidney cancer cells to
MG132. (A) A498 cells were pre-treated with vehicle, SP600125 (20 mM) for 1 h, subsequently treated with 2 mM MG132 for additional 24 h
and apoptotic cells were analysed. (B) Caki1 or Caki2 cells were treated as (A) and apoptotic cells were analysed. (C) A498 cells were transfected
with mock or BAG3-expressing plasmid; total cellular protein was analysed by Western blot. (D) 24 h after transfection with mock or
BAG3-expressing plasmid, A498 cells were pre-treated with vehicle or SP600125 for 1 h, subsequently treated with MG132 for additional 24 h
and apoptotic cells were analysed. *P < 0.05; **P < 0.001.
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British Journal of Pharmacology (2009) 158 1405–1412
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or small interfering RNA significantly enhanced the apoptotic
response of various cell types to proteasome inhibition
(Meriin et al., 1998; Robertson et al., 1999; Mitsiades et al.,
2002; Chauhan et al., 2003; Hideshima et al., 2004; Small
et al., 2004; Wang et al., 2008). Evaluation of the mechanisms
underlying induction of these anti-apoptotic molecules may
contribute to the development of improved therapies using
proteasome inhibitors.
In the current study, we investigated the role of MAPKs in the
induction of BAG3 by MG132. Our data showed that p38 and
ERK inhibitors had little effect on the induction of BAG3 by
MG132 but that inhibition of JNK significantly reduced BAG3
expression by MG132. Several earlier studies have looked at the
effects of bortezomib or MG132 treatment on molecular sig-
nalling in different cancer types. In liver, breast and pancreatic
cancer cells treated with a proteasome inhibitor, JNK and ERK
showed very similar patterns of activation to those described
here in kidney cancer cells (Yang et al., 2004; Codony-Servat
et al., 2006; Lauricella et al., 2006; Sloss et al., 2008).
The JNK pathway has been associated with the induction of
apoptosis in response to various cellular stresses such as treat-
ment with anti-cancer drugs. We did not expect JNK activa-
tion to be involved in BAG3 induction and that inhibition of
JNK activation would enhance apoptotic responses of kidney
cancer cells to proteasome inhibition. Meriin et al. (1998) had
suggested that the proteasome inhibitor-induced JNK activa-
tion would be pro-apoptotic in lymphoid tumours. However,
a growing body of evidence suggests that JNK activation is
also central to anti-apoptotic and growth-promoting effects.
For instance, JNK has been shown to be activated and/or
overexpressed in an anti-apoptotic manner in various cancers,
and JNK inhibition has been shown to increase anti-cancer
effects of proteasome inhibitors in pancreatic cancer cells,
suggesting an anti-apoptotic role for JNK in this cell type
(Malicet et al., 2003; Li et al., 2007; Sloss et al., 2008). The JNK
pathway is also reported to be involved in the activation of
Hsp70 (Kim et al., 2005), one of whose most important func-
tions is to prevent apoptosis. Although the reported results to
date may seem contradictory, they may more likely represent
tissue and cell type-specific differences in the response to
different stimuli.
In the current study, our data showed that the JNK pathway
was related to the activation of BAG3 expression in kidney
cancer cells. These data indicate that the JNK pathway might
play opposing roles in the induction of cellular apoptosis and
maintenance of cellular homeostasis, suggesting that JNK
triggers and signals to cellular apoptosis, as well as to the
pathways leading to the cell survival.
Acknowledgements
This work was supported by National Natural Science Foun-
dation of China (30870522) and Shenyang Outstanding
Talent Foundation to H-Q.W.
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  • Source
    • "Therefore, the expression profile of Bis in GI tract, showing the specific localization in the cells of less self-renewing capacity, suggest the possible role for Bis in the maintenance of cell survival probably by suppressing cell death. The death suppressor function of Bis has been demonstrated by previous experiments performed in vitro, as well as in vivo data, showing higher expression of Bis in the various kinds of cancers [1, 2,91011. Thus, our results presenting the expression of Bis in the normal cells lacking proliferation potential, but with long life span, support the pro-survival role of Bis in the physiological condition, in addition to a pathological condition. "
    [Show abstract] [Hide abstract] ABSTRACT: The Bcl-2 interacting death suppressor (Bis) protein is known to be involved in a variety of pathophysiological conditions. We recently generated bis-deficient mice, which exhibited early lethality with typical nutritional deprivation status. To further investigate the molecular basis for the malnutrition phenotype of bis deficient mice, we explored Bis expression in the digestive system of normal mice. Western blot analysis and quantitative real time reverse transcription polymerase chain reaction analysis indicated that Bis expression is highest in the esophagus, followed by the stomach, colon, jejunum and ileum. Immunohistochemical data indicated that Bis expression is restricted to the stratified squamous epitheliums in the esophagus and forestomach, and was not notable in the columnar epitheliums in the stomach, small intestine and colon. In addition, strong Bis immunoreactivity was detected in the striated muscles surrounding the esophagus and smooth muscles at a lesser intensity throughout the gastrointestinal (GI) tract. Ganglionated plexuses, located in submucous layers, as well as intermuscular layers, were specifically immunoreactive for Bis. Immunofluorescence studies revealed that Bis is co-localized in glial fibrillary acidic protein-expressing enteric glial cells. Immunostaining with neuron specific esterase antibodies indicate that Bis is also present in the cell bodies of ganglions in the enteric nervous system (ENS). Our findings indicate that Bis plays a role in regulating GI functions, such as motility and absorption, through modulating signal transmission between the ENS and smooth muscles or the intestinal epitheliums.
    Full-text · Article · Sep 2012 · Anatomy & cell biology
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    • "Hsp90 was previously reported to cooperate with Hsp70 to stabilize HIF-1α under hypoxia or heat-shock circumstance [39]. Moreover, it has been reported that MG132 could induce cellular apoptosis through the JNK1 pathway [68]. Our most recent studies have also demonstrated that JNK1 activation is crucial for HIF-1α protein accumulation due to hypoxia and nickel exposure through induction of both Hsp90/Hsp70 [34]. "
    [Show abstract] [Hide abstract] ABSTRACT: Hypoxia-inducible factor-1α (HIF-1α) has been reported to regulate over 100 gene expressions in response to hypoxia and other stress conditions. In the present study, we found that arsenite could induce HIF-1α protein accumulation in both mouse epidermal Cl41 cells and mouse embryonic fibroblasts (MEFs). Knockout of p85α, a regulatory subunit of PI-3K, in MEFs (p85α(-/-)) dramatically decreased the arsenite-induced HIF-1α accumulation, indicating that p85α is crucial for arsenite effects on the stabilization of HIF-1α protein. Our further studies suggest that arsenite could induce inducible Hsp70 expression, and transfection of inducible Hsp70 into p85α(-/-) MEFs could restore HIF-1α protein accumulation. Moreover, the results using EMSA and Supershift assays indicate that p85α is crucial for arsenite-induced activation of the heat-shock transcription factor 1 (HSF-1), which is responsible for transcription of inducible Hsp70. Taken together, p85α-mediated HIF-1α stabilization upon arsenite exposure is specifically through HSF-1 activation and subsequent up-regulation of the inducible Hsp70 expression.
    Full-text · Article · Feb 2011 · Cellular and Molecular Life Sciences CMLS
  • [Show abstract] [Hide abstract] ABSTRACT: In this work, we report an extensive characterisation of fully silicided gate (FUSI) devices with oxynitride (SiON) and Hf-silicate gate dielectrics. Enhanced drive current is obtained, in comparison with poly gate devices, together with an increase in electron/hole mobility and reduction in CET values. We show that the work function (WF) can be engineered by doping of the poly gates prior to FUSI for SiON devices but not for Hf-silicate devices. With reference to the poly gate, Hf-silicate/FUSI devices exhibit improved TDDB reliability behavior, having higher acceleration factor (γ) values. NBTI gives a maximum operating voltage above 1.2 V for ΔV<sub>T</sub> = 10% or 30 mV, as extrapolated for a 10 years-lifetime.
    No preview · Conference Paper · Jan 2005
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