Molecular and Cellular Biology, October 2001, p. 6796-6807, Vol. 21, No. 20
0270-7306/01/$04.00+0 DOI: 10.1128/MCB.21.20.6796-6807.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
First Department of Pathology,1 First Department of Medicine,2 and Department of Endoscopic & Photodynamic Medicine,3 Hamamatsu University School of Medicine, Hamamatsu 431-3192, Japan
Received 14 June 2001/Accepted 17 July 2001
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ABSTRACT |
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Benzo[a]pyrene [B(a)P], a potent procarcinogen
found in combustion products such as diesel exhaust and cigarette
smoke, has been recently shown to activate the c-Jun
NH2-terminal kinase 1 (JNK1) and induce caspase-3-mediated
apoptosis in Hepa1c1c7 cells. However, the molecules of the signaling
pathway that control the mitogen-activated protein kinase cascades
induced by B(a)P and the interaction between those and apoptosis by
B(a)P have not been well defined. We report here that B(a)P promoted
Cdc42/Rac1, p21-activated kinase 1 (PAK1), and JNK1 activities in 293T
and HeLa cells. Moreover, alpha-PAK-interacting exchange factor (
PIX) mRNA and its protein expression were upregulated by B(a)P. While
overexpression of an active mutant of
PIX (
CH) facilitated B(a)P-induced activation of Cdc42/Rac1, PAK1, and JNK1, overexpression of mutated
PIX (L383R, L384S), which lacks guanine nucleotide exchange factor activity, SH3 domain-deleted
PIX (
SH3), which lacks the ability to bind PAK, kinase-negative PAK1 (K299R), and kinase-negative SEK1 (K220A, K224L) inhibited B(a)P-triggered JNK1
activation. Interestingly, overexpression of
PIX (
CH) and a
catalytically active mutant PAK1 (T423E) accelerated B(a)P-induced apoptosis in HeLa cells, whereas
PIX (
SH3), PAK1 (K299R), and SEK 1 (K220A, K224L) inhibited B(a)P-initiated apoptosis. Finally, a
preferential caspase inhibitor, Z-Asp-CH2-DCB, strongly blocked the
PIX (
CH)-enhanced apoptosis in cells treated with B(a)P but did
not block PAK1/JNK1 activation. Taken together, these results indicate
that
PIX plays a crucial role in B(a)P-induced apoptosis through
activation of the JNK1 pathway kinases.
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INTRODUCTION |
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Benzo[a]pyrene [B(a)P] is a member of the polycyclic aromatic hydrocarbons (PAHs), compounds that are found in combustion products such as cigarette smoke and diesel exhaust and are thought to be carcinogens capable of tumor initiation, promotion, and progression (38). B(a)P binds to the aryl hydrocarbon receptor as a ligand and induces the expression of cytochrome P450-1A1 (CYP1A1) (40). Subsequently, B(a)P is metabolized by CYP1A1 to electrophilic species, such as anti-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene [B(a)PDE], which can eventually induce mutations or oxidative DNA damage (12, 20, 33). Via this mechanism, B(a)P and/or other PAHs can induce tumors in the mammary gland, liver, and lungs (15, 16, 18, 31). In addition, B(a)P and/or other PAHs have been shown to induce apoptosis, or programmed cell death, in vitro in Hepa1c1c7 hepatoma cells (27), Daudi human B cells (42), human ectocervical cells (39), and A20.1 murine B- cells (8). Recently, several studies have focused on the association between cellular signaling pathways and apoptosis induced by B(a)P. For example, B(a)P absorbed on carbon black induces the expression and release of tumor necrosis factor alpha, resulting in apoptosis of RAW 264.7 macrophage cells via mitogen-activated protein kinase (MAPK) (11). On the other hand, it has been demonstrated that B(a)P mimics signaling through the insulin-like growth factor-I receptor (IGF-IR) and increases cell survival through phosphatidylinositol 3-kinase (P13-K) activation in human mammary epithelial cells (48). It has also been suggested that c-Jun NH2-terminal kinase 1 (JNK1) activation might be independent of B(a)P-induced apoptosis in Hepa1c1c7 cells (27), although the interaction between the JNK1 signaling pathway and apoptosis induced by B(a)P has not been well elucidated.
Very recently, Kong et al. demonstrated that Rho-21 guanine nucleotide exchange factors (GEFs) are associated with apoptosis; the proto-oncogene Vav, which acts as a specific GEF on Rac and modulates cytoskeletal reorganization and cytokine production in T cells, has been reported to act upstream of caspase activation and regulate peptide-specific cell death in thymocytes (25). In addition, Tiam-1, a proto-oncogene and another GEF for Rac1, has been reported to regulate apoptosis induced by bufalin, a component of the Chinese medicine chan'su, via Rac1, p21-activated protein kinase (PAK) and JNK signaling pathways in HL60 and U937 cells (24). These findings together infer that GEFs in general may be closely involved in apoptosis.
Recently, novel GEFs for Rac1, termed the PAK-interacting exchange
factor (PIX) family, have been identified (29). PIX family proteins, which consist of two isoforms (
PIX and
PIX), bind tightly through the N-terminal SH3 domain to a conserved proline-rich PAK sequence located at the C terminus of the p21-binding domain (PBD)
and are colocalized with PAK to form activated Cdc42-and Rac1-driven
focal complexes (29, 37).
PIX is activated by signaling
cascades from the platelet-derived growth factor receptor and EphB2
receptor and from integrin-induced signaling through PI3-kinase,
leading to PAK activation (57). It has also been demonstrated that
PIX is involved in Rac-type morphological changes such as lamellipodia formation in PC12 cells and photoreceptor axon
guidance in Drosophila through interactions with PAK
(19, 36). In addition, PIX has been shown to bind the
G-protein-coupled receptor kinase-interacting protein, known as GIT1,
leading to focal complex disassembly and stimulation of cell motility
(59). Because PAK lies upstream of the JNK pathway
(3, 7, 58), we tried to elucidate the possible involvement
of
PIX in JNK signaling cascades and their various biological
effects including apoptosis on cells in response to B(a)P.
In this study, we show that
PIX mediates signals induced by B(a)P
leading to JNK1 kinase activation through Cdc42/Rac1, PAK1, and SEK1
kinases in 293T and HeLa cells. B(a)P also increases the levels of
PIX mRNA and its protein. While B(a)P induces
caspase-protease-mediated apoptosis,
PIX accelerates B(a)P-triggered
apoptosis through activation of JNK1 pathway kinases. These results
indicate that
PIX may play a pivotal role in B(a)P-induced apoptosis
through the JNK signaling pathway.
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MATERIALS AND METHODS |
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Materials. HeLa cells were kindly supplied by the Cell Resource Center of the Biomedical Research Institute of Development, Aging and Cancer, Tohoku University. A fluorogenic substrate for caspase-3 protease (Ac-DEVD-MCA) was purchased from the Peptide Institute (Osaka, Japan). B(a)P was from Sigma Chemical Co. (St. Louis, Mo.). All other chemicals were obtained through standard suppliers.
Plasmids and antibodies.
Plasmid carrying the full-length
cDNA of human
PIX was a gift from T. Nagase (KIAA0006). Deletion
mutants of
PIX tagged with a myc or hemagglutinin (HA) epitope at
the C terminus were generated using a PCR-based technique
(46). All constructs were subcloned into the pCS2+ vector
for transfection into 293T or HeLa cells and into pGEX2T (Phrmacia) to
make bacterial glutathione S-transferase (GST) fusion
constructs. pGEX fusion protein of the p21-binding domain (PBD) of PAK1
(pGEX-PBD) was generated by cloning a PCR-amplified fragment of
putative Cdc42 and the Rac binding domain of human PAK1 (amino acids 70 to 133) into pGEX2T for affinity precipitation. Amino-terminally
myc-tagged wild-type and mutant (K299R, T423E, and H83L H86L) human
PAK1 clones and wild-type Cdc42 and Rac1 were kindly provided by Bruce J. Mayer (45). Plasmid encoding wild-type human Bcl-2 was
kindly provided by Y. Eguchi and Y. Tsujimoto (44).
PIX, and the GST epitope tag were from
Santa Cruz.
Cell culture, DNA transfections, and in vitro kinase assay. 293T human embryonal kidney cells (expressing simian virus 40 T antigen) were transfected with a maximum of 20 µg of plasmid DNA per 10-cm-diameter dish by a calcium phosphate coprecipitation method with concurrent treatment with 25 µM chloroquine essentially as described previously (45). Plasmid DNA was transfected into HeLa cells using DMRIE-C reagent (Life Technologies, Gaithersburg, Md.). At 18 to 24 h after transfection, the cells were placed in medium with 0.5% fetal bovine serum (FBS) and incubated for a further 12 h. The cells were treated with B(a)P (Sigma), which was concentrated 1,000-fold in dimethyl sulfoxide (DMSO), prior to lysis or were left untreated. The cells were harvested for immunoprecipitation and kinase assays, which were performed as previously described (45). To purify myc-tagged PAK protein, monoclonal antibody against myc epitope (1 µg) was incubated with 500 µ 1 of cell lysate for 2 h at 4°C and precipitated with protein G-agarose (Boehringer, Mannheim, Germany). To purify GST-JNK protein, glutathione-Sepharose 4B (Amersham Life Science, Piscataway, N.J.) was incubated with 500 µ 1 of cell lysate for 30 min at 4°C. Immunoprecipitates were washed extensively before being used for immunoblotting or for an in vitro kinase assay with myelin basic protein or GST-c-Jun as substrate. The results were visualized with a Bio Imaging Analyzer (BAS1000; Fuji, Tokyo, Japan), and images were quantitated with NIH Image software. The relative activity of PAK/JNK was calculated by adjusting the densitometric value and standardization.
Drug treatment. Logarithmically growing 293T cells and HeLa cells were harvested by trypsinization and seeded in 10 ml of fresh medium in a 10-cm dish. After overnight incubation and subsequent 12-h prestarvation, B(a)P was added from 1,000-fold-concentrated stocks in DMSO with a final concentration of 0.1% DMSO in medium, which had no influence on the cells. After various periods of incubation, floating cells and/or trypsinized adhesive cells were combined and sedimented at 800 × g for 10 min, and then the following assays were performed.
Western blot analysis.
Cells were lysed in a lysis buffer
(25 mM Tris-HCl [pH 7.4], 10 mM
-glycerophosphate, 150 mM NaCl, 5 mM disodium EDTA, 10 mM sodium pyrophosphate, 1% Triton X-100, 1 mM
Na3VO4, 10% glycerol, 1 mM
phenylmethylsulfonyl fluoride (PMSF), 20 µg of aprotinin per ml). The
cell extracts were clarified by centrifugation, and the supernatants
were resolved by sodium dodecyl sulfate-polyacrylamide gel
electrophoresis. The proteins were then subjected to Western blot analysis.
Affinity precipitation. Affinity precipitation with GST-PBD was as described previously (4, 49), except that 293T and HeLa cells were lysed in lysis buffer (20 mM Tris-HCl [pH 7.5], 150 mM NaCl, 20 mM MgCl2, 1 mM Na3VO4, 0.5% Triton X-100, 5 µ g of aprotinin per ml, 1 mM PMSF), and precipitates were washed three times in the same buffer.
RT-PCR analysis.
Total RNA isolated from HeLa cells with or
without B(a)P treatment was prepared using ISOGEN (Wako Pure Chemical
Industries, Ltd., Osaka, Japan). The RNA samples were reverse
transcribed using Superscript reverse transcriptase (Life Technologies,
Inc.), oligo (dT) primers (Life Technologies, Inc.), and
deoxynucleostide triphosphate as specified by the manufacturer. The
synthesized cDNAs were amplified by PCR (30 and 25 cycles for
PIX
and
-actin, respectively) with Taq DNA polymerase
(Boehringer Mannheim) in the presence of deoxynucleostide triphosphate,
an appropriate pair of primers, and [
-32P]dCTP (ICN,
High Wycombe, United Kingdom). For PCR amplification of the
PIX gene, we used the forward primer
5'-AGTCCTACTCCCTGAGGAAGAGAAA-3' and the reverse primer
5'-TGAGGTCTTGCTACTGGACTCGCCT-3';
-actin primers (forward
primer 5'-GGTGAAGGTGTCAGCAGCAG-3' and the reverse primer
5'-GGCCAAGCAGCGCAGCACAG-3') were also used as a control. The
PCR products were subjected to electrophoresis in an 8% (wt/vol) acrylamide gel, and the results were visualized with a Bio Imaging Analyzer.
Measurement of CPP32-like caspase activity. After treatment with B(a)P, the cells were collected and lysed for 20 min on ice in lysis buffer containing 10 mM HEPES-KOH (pH 7.4), 2 mM EDTA, 0.1% 3-[(3-cholamidopropyl) dimethyl ammonio]-1-propanesulfonate (CHAPS), 1 mM PMSF, and 5 mM dithiothreitol. After centrifugation, the supernatants were collected as lysates. For measurement of caspase activity, 100 µg of lysate proteins in 50 µ l of lysis buffer were mixed with 50 µ l of 2× reaction buffer containing 40 mM HEPES KOH (pH 7.4), 20% glycerol, 1 mM PMSF, and 4 mM dithiothreitol DTT with 50 µ M fluorogenic substrate acetyl-Tyr-Val-Ala-Asp-4-methylcoumaryl-7-4,6-diamidino-2-phenylindole (Ac-DEVD-MCA) and incubated at 37°C for 90 min. The release of amino-4-methylcoumarin was monitored with a spectrofluorometer (Jasco model FP-777; Japan Spectroscopic Co., Ltd., Tokyo, Japan), using an excitation wavelength of 360 nm and an emission wavelength of 460 nm.
Cell death assays.
After a 12-h prestarvation, HeLa cells
were prepared with or without caspase inhibitor
(carbobenzoxy-L-Asp-
[(2,6-dichlorobenzoyl)oxy] methane
[Z-Asp-CH2-DCB] or carbobenzoxy-Tyr-Val-Ala-Asp-fluoromethyl ketone
[Z-DEVD-FMK]) for 90 min prior to addition of B(a)P. After treatment
with B(a)P, the cells were harvested by trypsinization and fixed in
phosphate-buffered saline with 4% paraformaldehyde for 30 min.
Following treatment with RNase (1 mg/ml in 0.1 M phosphate buffer [pH 7.0]), the cells were stained with 1 µ g of
4',6-diamidino-2-phenylindole (DAPI) per ml for 20 min. Cells were
placed on slides, and apoptotic cells with condensed or fragmented
nuclei were visualized and counted under a fluorescence microscope.
DNA fragmentation assay.
The DNA fragmentation assay was
performed as described previously (27). Briefly, HeLa
cells (106) were collected and lysed in buffer containing
40 mM Tris (pH 8.0), 150 mM NaCl, 25 mM EDTA, and 0.5% sodium dodecyl
sulfate, DNA was isolated with an equal volume of neutral
phenol-chloroform-isoamyl alcohol mixture and precipitated with 0.1 volume of 3 M sodium acetate (pH 5.2)-2 volumes of 100% ethanol at
20°C overnight. The precipitated DNA was dissolved in 50 µ l of
10 mM Tris (pH 8.0)-1 mM EDTA buffer. The DNA fragments were resolved
by electrophoresis in a 1.5% agarose gel and stained with ethidium bromide.
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RESULTS |
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B(a)P activates the PAK1/JNK1 signaling pathway.
We initially
investigated whether JNK1 was activated following B(a)P treatment in
293T cells as reported in Hepa1c1c7 cells (27). As shown
in Fig. 1A, JNK1 activation was observed
with 1 nM B(a)P, and this activity was maintained at least to 1 µ M B(a)P. In addition, JNK1 activation increased from 15 min after treatment with 0.1 µ M B(a)P in a time-dependent manner, and activity was maintained at least through 150 min (Fig. 1B). Since PAK lies upstream of the JNK1 pathway (3, 7, 58), we next examined whether PAK1 was activated in response to B(a)P. As shown in Fig. 1,
PAK1 was also activated by B(a)P in a time- and dose-dependent manner,
which coincided with the B(a)P-induced JNK1 activation. Strong
activation of PAK1 and JNK1 was also detected in 293T cells after
treatment with 10 µ M B(a)P as well as 0.1 and 1 µ M B(a)P (data
not shown). Western blot analysis confirmed equivalent amounts of the
corresponding kinase proteins (Fig. 1, lower panels). We further
examined Cdc42 or Rac1 activation induced by B(a)P in vivo by affinity
precipitation of GTP-bound Cdc42 or Rac1 with GST-PBD as previously
described (4), because Cdc42/Rac1 interacts with PAK and
stimulates PAK activity (3, 7, 58). B(a)P treatment
resulted in an increase in the amount of activated Cdc42 and Rac1 that
precipitated with GST-PBD in 293T cells in a time-dependent manner,
reaching a maximum at 3 h after treatment with 0.1 µM B(a)P
(Fig. 2B). B(a)P also increased the
amount of activated Cdc42 and Rac1 in a dose-dependent manner (data not
shown). Similar results were obtained with HeLa cells on treatment with
B(a)P under the same experimental conditions as in the experiments in Fig. 1 and 2 (data not shown). These results indicated that B(a)P induced the activation of Cdc42/Rac1 and PAK1 as well as JNK1 in a
time- and dose-dependent manner.
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PIX mediates the signal initiated by B(a)P, leading to the
activation of the JNK1 pathway kinases.
Human PIX family proteins
bind tightly to PAK and are colocalized with PAK to form activated
Cdc42- and Rac1-driven focal complexes (29, 37), leading
to PAK activation through Cdc42/Rac1 (2, 13, 57). We
therefore next studied the effect of
PIX on B(a)P-induced PAK1
activation. In our experiment, PAK1 kinase activity was measured after
B(a)P treatment in 293T cells coexpressing PAK1 with wild-type human
PIX [
PIX (WT)] (WT) or various mutants of human
PIX. We
performed the experiments under conditions ensuring that PAK1 or JNK1
was weakly activated by stimulation with B(a)P in the absence of
PIX
(Fig. 3A and B, lanes 1 and 3; Fig 3C, lanes 1 and 2). Without B(a)P stimulation,
PIX (WT) did not
adequately activate PAK1 (Fig. 3A, lane 2). However, in response to
B(a)P stimulation, wild-type as well as N-terminally deleted
PIX
(
CH), an active mutant of
PIX which has been reported to potently
activate PAK1 in response to PDGF or EphB2 stimulation
(57), enhanced the kinase activity of PAK1 (lanes 4 and
5). By contrast, mutated
PIX (L383R, L384S), which lacks GEF
activity, and SH3 domain-deleted
PIX (
SH3), which lacks the
ability to bind to PAK, suppressed B(a)P-induced PAK1 activation (lanes
7 and 8). These results indicate that
PIX mediates the signals
triggered by B(a)P stimulation, leading to PAK1 activation. It has also
been reported that Rac mediates signals to the MEKK-SEK-JNK pathway via
PAK (53). Therefore, we further examined whether
PIX
was involved in the activation of the JNK1 kinase induced by B(a)P. In
cells coexpressing GST-JNK1 with
PIX (WT) or mutated
PIX, the
kinase activity of JNK1 was measured after B(a)P stimulation. As shown
in Fig. 3B, while both
PIX (WT) and
PIX (
CH) enhanced the JNK1
activation by B(a)P (lanes 4, 6, and 7),
PIX (
SH3) and
PIX
(L383R, L384S) inhibited the kinase activity of JNK1 in cells treated
with B(a)P (lane 8 and data not shown). These results suggest that
PIX is involved in JNK1 activation induced by B(a)P. Moreover, we
evaluated whether the signals initiated by B(a)P were transduced
through PAK1 to JNK1. As shown in Fig. 3C, PAK1 (WT) modestly
accelerated the kinase activity of JNK1 in cells treated with B(a)P
compared with mock vector (lane 3). On the other hand, we detected much
higher JNK1 kinase activity with a constitutively active mutant, PAK1 (T423E), and PAK1 (H83L, H86L), which lacks the ability to bind Cdc42/Rac1, in cells treated with B(a)P than with a mock vector (Fig.
3C, lanes 4 and 5). In addition, we found that PAK1 (T423E) and PAK1
(H83L, H86L) effectively enhanced JNK1 kinase activity in the absence
of B(a)P (Fig. 3C, lane 8, and data not shown). In contrast,
kinase-negative PAK1 (K299R) inhibited JNK1 activation by B(a)P (lane
6). These findings show that PAK1 acts as an upstream mediator of JNK1
in response to B(a)P. In addition, coexpression of kinase-negative SEK1
(K220A, K224L), which is one of the MAPK kinases and lies upstream of
JNK1 (14), also strongly suppressed B(a)P-induced JNK1
activation (lane 10). We furthermore showed that PAK1 (T423E) could
also restore B(a)P-triggered JNK1 activation in cells expressing
PIX
(
SH3) (Fig. 3D, lane 3). Next, we examined the effect of
PIX on
Cdc42/Rac1 activation by B(a)P. As shown in Fig. 3E and F,
PIX
(
CH) and
PIX (WT) enhanced B(a)P-induced activation of Cdc42 and
Rac1 (lanes 4 and data not shown), which was detected by the increased
amount of GTP-bound Cdc42 or Rac1 in vivo as shown in Fig. 2. By
contrast,
PIX (
SH3) and
PIX (L383R, L384S) blocked the
activation of Cdc42/Rac1 by B(a)P (lanes 6 and data not shown). These
findings indicate that
PIX promotes Cdc42/Rac1 activity in response
to B(a)P. Similar results were obtained with HeLa cells under the same
experimental conditions as in the experiment in Fig. 3 (data not
shown). Taken together, these results strongly suggest that
PIX
plays a crucial role in the Cdc42/Rac1-PAK1-SEK1-JNK1 signaling pathway
initiated by B(a)P.
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B(a)P induces overexpression of
PIX.
B(a)P binds to AhR,
leading to the induction of CYP1A1 and other cytochrome P450 species,
which in turn metabolize B(a)P to nucleophilic species such as B(a)PDE
(12, 33, 40). It has also been reported that B(a)P causes
induction of the c-Ha-ras and c-myc
proto-oncogenes in rat aortic smooth muscle cells through a
transcriptional mechanism (6, 41). Therefore we next
investigated the effect of B(a)P on the induction of
PIX. To examine
the upregulation of
PIX due to B(a)P, we monitored the level of
expression of
PIX mRNA in HeLa cells. We used reverse
transcription-PCR (RT-PCR), with
-actin as an internal control for
semiquantitative analysis of mRNA expression. When HeLa cells were
treated with 0.1 µ M B(a)P, the level of expression of
PIX mRNA
was increased after B(a)P treatment, reaching a maximum at 1 h,
and decreased thereafter (Fig. 4A). We
also examined the effect of B(a)P on the expression of the
PIX
protein in HeLa cells. As shown in Fig. 4B, the level of
PIX protein
was also elevated 1 h after B(a)P treatment and the elevated
expression of
PIX protein was maintained at least through to 6 h.
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Cycloheximide inhibits B(a)P-induced JNK activation.
Next, we
examined the effect of an inhibitor of protein synthesis,
cycloheximide, on B(a)P-induced JNK1 activity to investigate a possible
mechanism of activation of JNK1 pathway kinases induced by B(a)P. As
shown in Fig. 5, cycloheximide, which
effectively inhibited an increase in the expression of
PIX protein
induced by B(a)P (data not shown), significantly blocked the
B(a)P-induced JNK1 activation (lane 4). These findings may suggest that
some other proteins, besides
PIX, overexpressed by B(a)P may be
involved in the JNK1 activation triggered by B(a)P.
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B(a)P induces apoptosis in HeLa cells.
Recent evidence
suggests that in addition to its carcinogenic effect, B(a)P may induce
apoptosis in vitro (27, 42). We therefore investigated the
effect of B(a)P on inducing apoptosis in HeLa cells. Since B(a)P by
itself is a poor inducer of cell death in HeLa cells (data not shown),
prestarvation was carried out, in which cells were placed in medium
with 0.5% FBS and incubated for a further 12 h before B(a)P
treatment, and then various apoptosis assays were performed. When HeLa
cells were treated with 10 µM B(a)P for 48 h, numerous cells
which showed morphological changes such as membrane blebbing, nuclear
condensation, and the formation of small apoptotic bodies
characteristic of apoptosis were observed (data not shown).
Furthermore, to confirm whether B(a)P-initiated cell death was indeed
apoptosis, a DNA fragmentation assay was performed and genomic DNA
digestion by B(a)P was detected in HeLa cells (data not shown). These
findings indicated that B(a)P could induce apoptosis in HeLa cells as
well as in Hepa1c1c7 and Daudi human B cells. Furthermore, to quantify
B(a)P-induced cell death, we counted apoptotic cells with condensed or
fragmented nuclei under a fluorescence microscope after staining them
with DAPI, as described in Materials and Methods. As shown in Fig.
6A, the percentage of apoptotic cells
increased in a dose-dependent manner at B(a)P concentrations ranging
from 0.1 to 100 µM. The percentage of apoptotic cells also began to
increase in a time-dependent manner from 24 h after 10 µM B(a)P
treatment (Fig. 6B). Moreover, the DNA fragmentation assay demonstrated
that B(a)P induced the increase in the extent of the typical DNA ladder
in a time- and dose-dependent manner, which coincided with the
percentage of apoptotic cells (data not shown). As reported previously,
caspase family proteases, especially caspase-3, play critical roles in the apoptotic process (30, 34, 50). It has also been
reported that caspase-3, but not caspase-1, plays a critical role in
B(a)P-induced apoptosis in Hepa1c1c7 cells (27). To
determine whether caspase-3 was involved in B(a)P-initiated apoptosis
in HeLa cells as well as in Hepa1c1c7 cells, a fluorogenic assay of
caspase-3 protease activity was performed. As shown in Fig. 6C, the
activation of caspase-3 induced by B(a)P was also detected in a
dose-dependent manner. In addition, the activation of caspase-3 began
to increase from 12 h to at least 24 h after B(a)P treatment
(Fig. 6D). This activation of caspase-3 followed the activation of JNK1
signaling pathway kinases (Fig. 1) and preceded the occurrence of
apoptosis induced by B(a)P (Fig. 6B).
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Effect of caspase inhibitors on B(a)P-induced PAK1/JNK1 activation
and apoptosis.
Since two pathways, the caspase protease cascade
and the JNK1 signaling pathway, were activated in response to B(a)P, we
next examined a possible regulatory association between these pathways, especially during apoptosis induced by B(a)P. In this study,
Z-Asp-CH2-DCB and Z-DEVD-FMK were used as a preferential caspase
inhibitor and caspase-3-specific inhibitor, respectively. HeLa cells
were pretreated for 90 min with the caspase inhibitors before being
subjected to B(a)P treatment, and then caspase-3 activation and the
percentage of apoptotic cells were measured as described above. Fig.
7A and B show that Z-Asp-CH2-DCB
completely inhibited the caspase-3 activation and apoptosis induced by
B(a)P. Interestingly, while Z-DEVD-FMK completely inhibited the
B(a)P-induced caspase-3 activation, Z-DEVD-FMK reduced the apoptotic
cell percentage by only approximately 20% (48% versus 39%),
suggesting that caspases other than caspase-3 might be predominantly
involved in apoptosis by B(a)P in HeLa cells. These results may explain
why the caspase-3 activation by B(a)P was mild in HeLa cells (Fig. 6C
and D). In contrast, Z-Asp-CH2-DCB did not prevent the B(a)P-initiated
PAK1 and JNK1 kinase activation in HeLa cells at all (Fig. 7C).
Interestingly, we found that treatment of cells with Z-Asp-CH2-DCB
induced PAK1 activation and, to a lesser extent JNK1 activation in the
absence of B(a)P. Although the detailed mechanism is unknown,
Z-Asp-CH2-DCB might activate the PAK1 and JNK1 kinases by acting as a
stress, because JNK1 pathway kinases are important mediators of stress signals. Z-DEVD-FMK also failed to inhibit PAK1 and JNK1 activation by
B(a)P (data not shown). These results indicate that caspases inhibited
by Z-Asp-CH2-DCB are important mediators of B(a)P-induced apoptosis and
suggest that PAK1/JNK1 kinases could lie upstream of caspase proteases
in a single cascade pathway or that PAK1/JNK1 and caspases could
participate in independent parallel pathways.
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Effects of
PIX on B(a)P-induced apoptosis.
Many reports
have demonstrated that JNK activation contributes to the induction of
apoptosis in response to various stimuli, such as UV, gamma, or
ionizing radiation; oxidative stress; heat; tumor necrosis factor;
withdrawal of nerve growth factor; and ceramide (9, 21, 22, 52,
54, 55). In addition, GEFs, such as Tiam-1 and Vav, regulate
apoptotic cell death (24, 25).
PIX and the
downstream JNK1 pathway kinases in B(a)P-triggered apoptosis. As
indicated in Fig. 8, in this study marker
plasmid expressing eGFP was transiently cotransfected into HeLa cells
together with expression plasmids for either mutated forms of
PIX or
other mutant JNK pathway kinases as well as a mock vector (negative control) or Bcl-2, a potent antiapoptotic protein (positive control). Expression of vector-encoded proteins was confirmed by immunoblotting (Fig. 8, bottom panels). HeLa cells were prestarved in medium with
0.5% FBS for a further 12 h after transfection and then treated with 10 µM B(a)P for 24 or 36 h or left untreated to evaluate apoptosis in eGFP-positive cells by selective apoptotic assay, as
described in Materials and Methods. When the cells were treated with 10 µM B(a)P for 24 h, overexpression of
PIX (
CH) led to a
significant increase in the number of apoptotic cells showing chromatin
condensation and nuclear fragmentation compared the number detected
after treatment with a mock vector (Fig. 8A, top). Overexpression of
PAK1 (T423E) also greatly promoted B(a)P-induced apoptosis. By
contrast, after treatment with B(a)P for 24 h, overexpression of
PIX (
SH3) and Bcl-2 slightly inhibited B(a)P-induced apoptosis compared with the effect of the empty vector. In addition,
overexpression of PAK1 (K299R) or SEK1 (K220A, K224L) also slightly
reduced apoptotic cell death by B(a)P. At 36 h after treatment
with 10 µM B(a)P, the inhibition of B(a)P-induced apoptosis was more
marked in cells overexpressing
PIX (
SH3), PAK1 (K299R), or SEK1
(K220A, K224L) as well as Bcl-2 (Fig. 8B, top). Likewise,
overexpression of mutated
PIX (L383R, L384S) also efficiently
reduced the percentage of apoptotic HeLa cells after B(a)P treatment
(data not shown). The effect of overexpressing mutant proteins on
B(a)P-induced cell death was not the result of differential
overexpression of mutant proteins, because similar levels of mutant
proteins were detected by immunoblotting (Fig. 8, bottom). Since
cleavage of genomic DNA into oligonucleosomal fragments is a hallmark
of apoptosis, we next examined the extent of B(a)P-induced DNA
fragmentation. As shown in Fig. 8A center, overexpression of
PIX
(
CH) or PAK1 (T423E) enhanced ladder formation by fragmented DNA.
By contrast, overexpression of
PIX (
SH3), PAK1 (K299R), or SEK1
(K220A, K224L), as well as Bcl-2, decreased the extent of DNA laddering
(Fig. 8B, center). SEK1 (K220A, K224L), Bcl-2, or the mock vector was transiently transfected into HeLa cells together with
PIX (
CH) and eGFP to determine whether SEK1 (K220A, K224L) could attenuate the
PIX (
CH)-enhanced apoptosis in cells treated with B(a)P. After
treatment with 10 µM B(a)P for 24 h, the percentage of apoptotic cells overexpressing SEK1 (K220A, K224L) as well as Bcl-2 was strongly
suppressed, compared with the percentage after treatment with the mock
vector (Fig. 8C).
|
PIX (
CH) in HeLa cells
exposed to 10 µ M B(a)P for 24 h (Fig. 8D). These results
verified that the death signal induced by B(a)P is mediated, at least
in part, via the
PIX-Cdc42/Rac1-PAK1-SEK1-JNK1 signaling pathway in
HeLa cells and that
PIX is involved in B(a)P-triggered apoptosis at
a step upstream of caspase activation.
| |
DISCUSSION |
|---|
|
|
|---|
B(a)P has been known as a carcinogen that affects cells by either a direct interaction with DNA or formation of hydroxy radicals and superoxides, leading to tumor initiation, promotion, and progression (12, 20, 33, 38, 40). On the other hand, a few recent studies have demonstrated that B(a)P induces apoptotic cell death and have focused on its association with cellular signaling pathways, especially the MAPK cascades and apoptosis (8, 27, 39, 42). For example, Lei et al. reported that B(a)P induces JNK kinase activation in Hepa1c1c7 cells during apoptosis (27), although the interaction between the JNK pathway and apoptosis induced by B(a)P has not been well elucidated.
The MAPK cascades occur in a variety of biological phenomena, including apoptotic cell death, cellular proliferation, differentiation, and transformation. Many reports have demonstrated that JNK activation contributes to the induction of apoptosis (9, 21, 22, 52, 54, 55). On the other hand, quite a few reports have demonstrated that the activation of JNK can be either nonapoptotic or antiapoptotic depending on the various cell biological settings (28, 35, 56). These findings appear to suggest that the JNK pathway kinases, such as SEK1 and JNK1, have bidirectional functions (cell proliferation and differentiation or programmed cell death) and that the actual function of the kinases depends on cosignals provided by different stimuli, cell types, and environmental conditions (10).
Therefore, in an attempt to identify the mediators of the JNK
activation induced by B(a)P on the JNK signaling pathway and the
interaction between the JNK pathway and the apoptotic cascade, we
especially focused on the commitment of
PIX.
PIX, a recently identified GEF for Cdc42/Rac1, has been reported to bind tightly to PAK
and to be colocalized with PAK to form activated Cdc42- and Rac1-driven
focal complexes, leading to PAK activation (29, 37), and
to be involved in various biological effects, including Rac-type
morphological changes such as lamellipodia formation and photoreceptor
axon guidance (19, 36).
In the present study, we found that Cdc42/Rac1, PAK1, and JNK1 activity
increased in a dose- and time-dependent manner in 293T and HeLa cells
treated with B(a)P (Fig. 1 and 2 and data not shown). We also confirmed
that the B(a)P-induced activation of Cdc42/Rac1, PAK1, and JNK1 kinases
was maintained for at least 6 h with parallel time kinetics (Fig.
2 and data not shown). To study the role of
PIX in the JNK pathway
triggered by B(a)P,
PIX was overexpressed in 293T and HeLa
cells.
PIX (
CH), an active form of
PIX, significantly
increased Cdc42/Rac1, PAK1, and JNK1 activation in cells treated with
B(a)P (Fig. 3A, B, E, and F). More importantly,
PIX (L383R, L384S),
which lacks GEF activity, and
PIX (
SH3), which abolishes the
ability to bind to PAK, clearly blocked B(a)P-initiated Cdc42/Rac1,
PAK1, and JNK1 activity (Fig. 3A, B, E, and F), demonstrating that
PIX lies on the B(a)P-triggered JNK1 signaling pathway and plays a pivotal role in the activation of JNK pathway kinases induced by B(a)P.
In addition, constitutively active mutants of PAK1, PAK1 (T423E) and
PAK1 (H83L, H86L), enhanced the B(a)P-initiated JNK1 activation,
whereas the kinase-negative mutant of PAK1, PAK1 (K299R), or of SEK1,
SEK1 (K220A, K224L), significantly inhibited the JNK1 activity induced
by B(a)P (Fig. 3C), suggesting that PAK1 and SEK1 may also lie on the
B(a)P-induced JNK1 signaling cascade. Moreover, we found evidence that
B(a)P triggers an increase in both
PIX mRNA and protein expression
(Fig. 4). With respect to the mechanism of activation of JNK1 pathway
kinases induced by B(a)P, the following possibilities can be
considered. One possibility is that B(a)P may primarily cause an
increase in expression of
PIX protein itself, a key regulatory
factor in this pathway, thus activating the Cdc42/Rac1-PAK1 signaling
pathway. In fact, we found that an inhibitor of protein synthesis,
cycloheximide, which effectively inhibited an increase in the
expression of
PIX protein (data not shown), significantly blocked
the B(a)P-induced JNK1 activation (Fig. 5). We can deduce from these
findings that some other proteins besides
PIX that are upregulated
by B(a)P may be involved in the JNK1 activation by B(a)P. This
hypothetical mechanism may explain why the maximal activation of
B(a)P-induced JNK1 pathway kinases requires 3 to 6 h. Our
observation that the overexpression of
PIX protein induced by B(a)P
required 1 h to appear and it maintained at least for another
5 h (Fig. 4B) is also consistent. Another possibility is that
B(a)P may activate the JNK1 pathway through the mitogen receptors, such
as IGF-IR, because some reports have demonstrated that PAHs mimic
antigen and mitogen receptor signaling by protein tyrosine kinases
(1, 26, 32). In addition, it has been reported that B(a)P
can mimic signaling through the IGF-IR and significantly increase the
activity of PI3-K via insulin receptor substrate 1 and Shc (48). Via this mechanism, B(a)P may be able to activate
the Cdc42/Rac1-PAK1-SEK1-JNK1 signaling pathway, since Cdc42/Rac are activated by PI3-K (17, 47). Since we previously reported that
PIX is activated by direct association with the p85 regulatory subunit of PI3-K, leading to the activation of Cdc42/Rac1-PAK1 pathway
(57), B(a)P may activate the Cdc42/Rac1-JNK1 pathway through
PIX. However, the possibility that B(a)P may induce the overexpression of some proteins, which enhances
PIX activity, is not
excluded. It is more likely that other unknown mechanisms, together
with those mentioned above, may synergistically cause the B(a)P-induced
activation of the JNK1 pathway. This complicated mechanism could
explain why exogenous overexpression of
PIX does not simply mimic
the actions of B(a)P and give rise to a marked stimulation of the JNK1
pathway kinases. Again, it is possible that the
Cdc42/Rac1-PAK1-SEK1-JNK1 pathway might also have been activated
through another pathway that does not involve
PIX. Furthermore, with
respect to the mechanism of upregulation of
PIX protein expression
by B(a)P, the following possibilities can be considered. One is that
B(a)P activates the Cdc42/Rac1-PAK1-SEK1-JNK1 signaling pathway almost
instantly, and thus
PIX would probably be among the genes controlled
by transcription factors phosphorylated by activated JNK
(23). Positive feedback, including the activated
PIX,
would further enhance signals through Cdc42/Rac1 to JNK1. Another
possible mechanism is that expression of
PIX might be increased by
some unknown signaling pathway(s) other than the PAK1-SEK1-JNK1-transcription factor pathway. Taken together, these findings demonstrate that
PIX is, at least in part, functionally involved in JNK1 activation via activation of the downstream kinase cascade Cdc42/Rac1-PAK1-SEK1 pathway.
Next, we examined the role of
PIX in B(a)P-induced apoptotic cell
death. In the present study, while we found that B(a)P induced
caspase-3 activation and subsequent apoptosis in HeLa cells treated
with B(a)P in a dose- and time-dependent manner (Fig. 6), other
caspases may be predominantly involved in B(a)P-induced apoptosis (Fig.
7A and B). Interestingly, overexpression of
PIX (
CH) induced a
significant increase in B(a)P-induced apoptotic cell death (Fig. 8A)
while overexpression of
PIX (
SH3) and
PIX (L383R, L384S)
greatly inhibited B(a)P-triggered apoptosis (Fig. 8B and data not
shown), demonstrating that
PIX plays a crucial role in apoptosis
triggered by B(a)P. In addition, overexpression of PAK1 (T423E)
promoted B(a)P-induced apoptosis (Fig. 8A) whereas overexpression of
PAK1 (K299R) and SEK1 (K220A, K224L) greatly suppressed B(a)P-triggered
apoptosis. Likewise, overexpression of dominant negative Cdc42 or Rac1
(Cdc42- or Rac1-T17N, respectively), which inhibited B(a)P-induced
PAK1/JNK1 activation, also suppressed apoptotic cell death by B(a)P
(data not shown). Moreover, overexpression of SEK1 (K220A, K224L)
significantly blocked the apoptotic cell death enhanced by coexpressing
PIX (
CH) in HeLa cells treated with B(a)P (Fig. 8C). These
observations strongly indicate that
PIX plays a crucial role in
B(a)P-initiated apoptosis via regulation of the JNK pathway kinases in
HeLa cells. Our findings showed that B(a)P-induced apoptosis followed
the activation of the JNK pathway kinases and subsequent caspase
proteases and that Z-Asp-CH2-DCB could completely prevent the apoptotic
cell death facilitated by
PIX (
CH) in HeLa cells treated with
B(a)P, but not by the JNK pathway kinases (Fig. 7C and 8D),
demonstrating that the JNK pathway lies upstream of the caspase
proteases. Figure 9 illustrates our model
of the B(a)P-induced apoptotic cascade via activation of JNK pathway
kinases in HeLa cells. The targets for JNK in induction of apoptosis
most probably involve transcriptionally regulated gene products, such
as c-Jun, whose activation has been reported to be sufficient to
initiate apoptotic cell death (5). This possibility
remains to be confirmed. By contrast, it has recently been reported
that PAK1 protects against apoptosis induced by interleukin-3
deprivation in FL5.12 lymphoid progenitor cells (43).
However, our observations clearly indicate that PAK1 contributes to
apoptosis, but not to cell survival, induced by B(a)P stimulation. Consistent with our observations, Thomas et al. recently reported that
PAK1 and JNK1 might play a role downstream of Cdc42 in transducing the
p53-dependent apoptotic signal via phosphorylation of Bcl-2 (51). PAK1 may very well have bidirectional functions for
the regulation of cell survival and programmed cell death as well as
some other JNK pathway kinases, including SEK1 and JNK1
(10).
|
In summary, our findings suggest that
PIX plays an important role in
B(a)P-induced apoptotic cell death via the activation of the
Cdc42/Rac1-PAK1-SEK1-JNK1 signaling pathway.
| |
ACKNOWLEDGMENTS |
|---|
We thank Bruce J. Mayer, Department of Genetics and Developmental Biology, University of Connecticut Health Center, Farmington, Yutaka Eguchi and Yoshihide Tsujimoto, Osaka University Graduate School of Medicine, and Takahiro Nagase, Kazusa DNA Research Institute, for providing us with plasmids used in these experiments. Technical support by Research Equipment Center, Hamamatsu University School of Medicine, is acknowledged.
This work is supported by grant-in-aids from the Ministry of Education, Science, Culture, Sports, and Technology of Japan (B,C-2:12218215), the Ministry of Health and Welfare for Comprehensive 10-Year Strategy for Cancer Control, and the Smoking Research Foundation.
| |
FOOTNOTES |
|---|
* Corresponding author. Mailing address: First MCB 92-01 Department of Pathology, Hamamatsu University School of Medicine, 3600 Handa-cho, Hamamatsu 431-3192, Japan. Phone: 81-53-435-2220. Fax: 81-53-435-2225. E-mail: hsugimur{at}hama-med.ac.jp.
| |
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