From the Guthrie Research Institute, Laboratory of Molecular Immunology, Guthrie Medical Center, Sayre, Pennsylvania 18840
Received for publication, August 16, 2002, and in revised form, December 31, 2002
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ABSTRACT |
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Transient activation of c-Jun N-terminal kinase
(JNK) promotes cell survival, whereas persistent JNK activation induces
apoptosis. Bovine testicular hyaluronidase PH-20 activates JNK1 and
protects L929 fibroblasts from staurosporine-mediated cell death. PH-20 also induces the expression of a p53-interacting WW domain-containing oxidoreductase (WOX1, also known as WWOX or FOR) in these cells. WOX1
enhances the cytotoxic function of tumor necrosis factor and mediates
apoptosis synergistically with p53. Thus, the activated JNK1 is likely
to counteract WOX1 in mediating apoptosis. Here it is demonstrated that
ectopic JNK1 inhibited WOX1-mediated apoptosis of L929 fibroblasts,
monocytic U937 cells, and other cell types. Also, JNK1 blocked WOX1
prevention of cell cycle progression. By stimulating cells with
anisomycin or UV light, JNK1 became activated, and WOX1 was
phosphorylated at Tyr33. The activated JNK1
physically interacted with the phosphorylated WOX1, as determined by
co-immunoprecipitation. Alteration of Tyr33 to
Arg33 in WOX1 abrogated its binding interaction with JNK1
and its activity in mediating cell death, indicating that
Tyr33 phosphorylation is needed to activate WOX1. A
dominant negative WOX1 was developed and shown to block p53-mediated
apoptosis and anisomycin-mediated WOX1 phosphorylation but could not
inhibit JNK1 activation. This mutant protein bound p53 but could not
interact with JNK1, as determined in yeast two-hybrid analysis. Taken
together, phosphorylation of JNK1 and WOX1 is necessary for their
physical interaction and functional antagonism.
WW domain-containing oxidoreductase
(WOX1,1 also known as WWOX or
FOR) is a proapoptotic protein and is known to enhance the cytotoxic
function of tumor necrosis factor (TNF) as well as overexpressed TNF
receptor-associated death domain protein (1-3). Enhancement of
TNF cytotoxicity by WOX1 is due, in part, to its significant down-regulation of the apoptosis inhibitors Bcl-2 and Bcl-xL but up-regulation of proapoptotic p53 (1, 2). WOX1 physically interacts
with p53, and these proteins mediate apoptosis synergistically (1, 2).
Blocking of WOX1 expression by antisense mRNA abolishes p53
apoptosis, suggesting that WOX1 is a potential partner of p53 in
apoptosis (1). Interestingly, hyaluronidases PH-20, Hyal-1, and Hyal-2
induce WOX1 expression and enhance TNF cytotoxicity (4, 5).
The WWOX gene coding for human WOX1 is mapped to a fragile
site on the chromosome ch16q (for reviews, see Refs. 2 and 3). High
frequency of loss of heterozygosity of this chromosomal region has been
shown in breast (6) and prostate cancers (7, 8). This chromosome region
appears to confer suppression of the metastatic activity in a prostatic
cancer cell line (9). Both in vitro and in vivo
experiments showed that WOX1 could act as a tumor suppressor (10).
WOX1 (46 kDa) possesses two N-terminal WW domains (containing
conserved tryptophan residues), a nuclear localization sequence (NLS) and a C-terminal short-chain alcohol dehydrogenase (ADH) domain (1). The human WWOX gene has nine exons. At least
seven alternatively spliced human WOX protein variants have been found. The ADH domain region, which is encoded by exons 4-8, is frequently deleted or alternatively spliced in the variants. In contrast, the WW
domain region is rarely deleted, and its amino acid sequence is highly
conserved among humans, mice, and rats. However, in Drosophila, the sequence of the N-terminal WW
domain region is altered (11).
A portion of cytosolic WOX1 is located in the mitochondria, and the
mitochondrial targeting sequence was mapped within the ADH domain (1).
Induction of mitochondrial permeability transition by TNF,
staurosporine, and atractyloside results in WOX1 release from the
mitochondria and subsequent nuclear translocation (1, 2).
c-Jun N-terminal kinase (JNK) is involved in cellular stress response
and has been implicated in both cell growth and apoptosis (for a
review, see Ref. 12). JNK1 is involved in signaling pathways that
initiate cell cycle checkpoints and cell cycle progression (13). In
contrast, inhibition of the JNK signaling pathway prevents death of
trophic factor-deprived neurons (14). Persistent activation of JNK1
causes apoptosis (15). However, early activation of JNK1 is essential
for protecting cells against TNF-mediated apoptosis (16, 17). Transient
activation of JNK by growth factors promotes survival of human
thyrocytes (18). Notably, hyaluronidase activation of JNK1 and JNK2
protects L929 fibroblasts from staurosporine-mediated cell death
(19).
In this study, we demonstrated that anisomycin and UV light activated
JNK1 and induced WOX1 phosphorylation in Tyr33, and both
proteins physically interacted. Ectopic JNK1 inhibited WOX1-mediated
apoptosis of L929 fibroblasts, monocytic U937 cells, and other tested
cells. Also, WOX1 prevention of cell cycle progression was abolished by
JNK1. The mechanisms of JNK1 and WOX1 binding interaction and the
functional significance of this interaction were investigated.
Cell Lines--
Cell lines used in these studies were murine
L929 fibroblasts, human monocytic U937 cells, human prostate Du145
cells, and human neuroblastoma SK-N-SH cells. These cells were grown
according to the instructions of the ATCC (Manassas, VA).
cDNA Expression Constructs--
Expression constructs for
the murine wild type Wox1 cDNA (sense and antisense
directions) and a nuclear localization mutant Wox1mtNLS were
established as previously described (1). These constructs were made in
pEGFP-C1 vector (Clontech, Palo Alto, CA). The
expressed proteins were tagged with an N-terminal enhanced green
fluorescent protein.
The FLAG-tagged wild type JNK1 (pcDNA3-Flag-JNK1) and dominant
negative JNK1 (DN-JNK1 or pcDNA3-Flag-JNK1apf) expression
constructs were kind gifts of Dr. Roger Davis (University of
Massachusetts Medical Center, Worcester, MA). The HA-tagged wild type
JNK2 and JNK3 (peVRFO-HA-JNK2 and peVRFO-HA-SAPK Cell Cycle Analysis and Cell Death Assays--
Where indicated,
L929 cells were electroporated with the wild type or DN-JNK constructs
(20 µg of DNA/3 × 106 cells; 200 V and 50 ms; BTX
ECM 830 Square Wave Electroporator; Genetronics, San Diego, CA).
Electroporation caused immediate cell death (or necrosis) by
~20-40%. These electroporated cells were seeded and cultured on
60-mm Petri dishes (Corning Glass) overnight. The culture supernatants
were discarded to remove dead cells. The cells were fed with fresh
medium (RPMI 1640 plus 10% fetal bovine serum) and then treated with
or without bovine testicular hyaluronidase (100 units/ml; Sigma) for
24 h. The cell layers, reaching 80-90% confluence in 48 h,
were gently washed once with phosphate-buffered saline, treated with
70% ethanol, harvested by repeat pipetting, precipitated by
centrifugation (500 × g), and then stained with
propidium iodide. The cellular DNA content was analyzed by flow
cytometry (Becton Dickinson, Franklin Lakes, NJ).
In some experiments, L929 and other cells were electroporated with WOX1
and/or JNK1 constructs and cultured for 24 h (in eight replicates). The extent of cell death was examined by crystal violet
staining (1) or by Promega's MTS proliferation assay (Promega,
Madison, WI).
Antibodies, Western Blotting, and
Co-immunoprecipitation--
Antibody against the N-terminal
amino acid sequence of WOX1 was generated as previously described (1).
Commercial antibodies used in the Western blotting were against JNK1
and a phospho-JNK1 peptide (Santa Cruz Biotechnology, Inc., Santa Cruz,
CA). Co-immunoprecipitation was performed as previously described (1,
20). Briefly, U937, L929, or SK-N-SH cells were treated with anisomycin
(100 µM) for various indicated times to activate JNK1.
The cytosolic and nuclear fractions of these cells were prepared using
the NE-PER nuclear and cytoplasmic extraction reagents (Pierce).
Endogenous JNK1 was precipitated by the specific anti-JNK1 antibody
(Santa Cruz Biotechnology) and protein A-agarose beads (Pierce),
followed by separation in SDS-PAGE and detection by immunostaining.
Co-presence of WOX1 in the precipitates was examined similarly by
immunostaining. Alternatively, immunoprecipitation was performed using
anti-WOX1 or p53 (Santa Cruz Biotechnology) antibody.
Generation of Antibody against a Phospho-Tyr33 WOX1
Peptide--
Generation of antibody against synthetic peptides in
rabbits was performed as described (1). A synthetic peptide
NH2-CKDGWVpYYANHTEEKT-COOH, with tyrosine 33 phosphorylation (pY), was made (Genemed Synthesis, South San Francisco,
CA). A control peptide without phosphorylation was made also. The
N-terminal cysteine was added to allow the peptide to covalently
conjugate to keyhole limpet hemocyanin for immunization or to SulfoLink
beads for antibody purification (Pierce). The peptide sequence is
located at the first WW domain of murine WOX1 (amino acids 28-38).
Antibody generation was performed using the EZ antibody production and
purification kit (Pierce). The antiserum was preadsorbed with a column
covalently conjugated with the control peptide, and then the antibody
was purified by affinity chromatography using a column immobilized with
the synthetic phosphopeptide.
Yeast Two-hybrid Interactions and Constructs--
The Ras
rescue-based CytoTrap yeast two-hybrid system (Stratagene, La Jolla,
CA) for protein binding interaction was used (1, 20). Briefly, binding
of a cytosolic Sos-tagged bait protein to a cell membrane-anchored
target protein (tagged with a myristoylation signal) results in
activation of the Ras signaling pathway in yeast. Activation of the Ras
signaling pathway allows mutant yeast Cdc25H to grow in 37 °C
using a selective agarose plate containing galactose. Without binding,
yeast cells fail to grow at 37 °C. The human JNK1 cDNA was
constructed in a pMyr vector and utilized as target. The bait
constructs in a pSos vector for binding interactions with JNK1 were
murine Wox1 (1), human WOX3 (1), and the first WW
domain of murine Wox1 (1). Additional constructs for control
experiments were human p53 in pMyr, MafB in both pMyr and pSos,
collagenase in pSos, and lamin C in pMyr (1, 20).
Site-directed Mutagenesis--
Site-directed mutagenesis was
performed using the QuikChange site-directed mutagenesis kit
(Stratagene). Tyr33 in WOX3 was mutated to
Arg33 (designated Y33R) using the following primers
by PCR: forward, 5'-AAGGACGGCTGGGTTCGATACGCCAATCACACC; reverse,
5'-GGTGTGATTGGCGTATCGAACCCAGCCGTCCTT. Lys28 and
Asp29 in Wox1 were mutated to Thr28
and Val29 (designated K28T/D29V): forward,
5'-AGAGAACCACCACGGTCGGCTGGGTGTACT; reverse,
5'-AGTACACCCAGCCGACCGTGGTGGTTCTCT.
Statistical Analysis and Data Presentation--
Where indicated,
Student's t tests were performed to analyze the
experimental data (using the Microsoft Excel program). All experiments
indicated above were performed 2-5 times. In most cases, a
representative set of data is shown.
Dominant Negative JNK1 Induces Apoptosis in Murine L929
Fibroblasts--
Bovine testicular hyaluronidase PH-20 activates JNK1
in L929 fibroblasts (19). PH-20 promotes the proliferation of L929 cells (4). By measuring cellular DNA content using flow cytometry, PH-20 was shown to increase cell cycle progression toward the S and
G2/M phases during 24-h exposure (Fig.
1). No PH-20-mediated apoptosis or cell
death was observed (Sub G0/G1) (Fig. 1).
To examine the possible role of JNK1 in the PH-20-mediated growth
regulation, L929 cells were electroporated with a wild type human JNK1
or a dominant negative JNK1 construct. The cells were cultured
overnight, exposed to PH-20 for 24 h, and then subjected to cell
cycle analysis by flow cytometry. In controls, the cells were
electroporated with nothing (or medium only) and treated similarly. The
results showed that ectopic wild type JNK1 did not induce apoptosis or
death of L929 cells (Fig. 2). Exposure of
these JNK1-expressing cells to PH-20 could not induce apoptosis (Fig.
2). In contrast, dominant-negative JNK1 significantly induced L929 cell
death (56.4%), and PH-20 did not significantly increase the extent of
cell death (60.9%) (Fig. 2).
In comparison, ectopic human wild-type JNK2 and JNK3 mediated apoptosis
or death of L929 cells by ~30%, and PH-20 limitedly increased the
cell death (~40%) (Fig. 2). In contrast, dominant negative JNK2 and
JNK3 significantly mediated cell death up to 60-70%, and PH-20
slightly increased the cell death (Fig. 2).
JNK1 Inhibition of WOX1-mediated Cell Death--
PH-20 induces the
expression of proapoptotic p53 and WOX1 in L929 cells, thereby
increasing cellular sensitivity to the cytotoxic effect of TNF (1).
Nonetheless, PH-20 also activates JNK1 in these cells (19). Here, we
examined whether JNK1 inhibited WOX1-mediated apoptosis of L929 cells.
In agreement with our previous observations (1), electroporation of
L929 cells with a wild-type WOX1 expression construct (tagged with
enhanced green fluorescent protein) resulted in cell death (Fig.
3). The extent of cell death was
determined by the MTS assay. Similar results were observed by staining
the cells with crystal violet (data not shown). The extent of WOX1 protein expression was ~60-75% in L929 cells, as determined by counting cells under fluorescence microscopy.
When L929 cells were cotransfected with both wild type JNK1 and WOX1
constructs, and JNK1 blocked WOX1-mediated cell death (Fig. 3). In
contrast, WOX1mtNLS, which possesses a mutated sequence at the NLS, had
a significantly reduced activity in mediating cell death (Fig. 3). When
in combination, JNK1 and WOX1mtNLS could not induce cell death (Fig.
3). The mutation in WOX1mtNLS prevents nuclear translocation of this
protein (1). In agreement with our previous observations (1),
expression of antisense WOX1 mRNA in L929 cells failed to result in
cell death, and JNK1 had no effect in mediating cell death during
cotransfection (Fig. 3). Together, WOX1 mediates apoptosis in the
nucleus, and JNK1 blocks the WOX1-mediated cell death at the
nuclear level.
JNK1 Blocks WOX1-mediated Inhibition of Cell Cycle Progression
in U937 Cells--
To further verify the above observations, we
examined the ability of wild type JNK1 in blocking apoptosis of
monocytic U937 cells by WOX1. U937 cells were electroporated with
various amounts of WOX1 in the presence or absence of wild type JNK1.
The cells were cultured 48 h and subjected to cell cycle analysis
by flow cytometry. The results showed that WOX1-mediated apoptosis was suppressed by JNK1 (see the sub-G0/G1 phase)
(Fig. 4). Additionally, WOX1-mediated
reduction of cellular distribution in the
G0/G1, S, and G2/M phases was
reversed by JNK1 (Fig. 4). Thus, our data strongly support the
protective role of JNK1 in blocking WOX1-mediated cell death.
WOX1 Physically Interacts with JNK1--
We investigated whether
JNK1 physically interacts with WOX1, thereby blocking WOX1-mediated
apoptosis. As determined by yeast two-hybrid analysis (1, 20), the wild
type human JNK1 interacted with murine WOX1 and human WOX3
in vivo, as evidenced by the growth of mutant Cdc25H yeast
at 37 °C (Fig. 5). Interestingly, the
WOX3/JNK1 binding interaction promoted a strong yeast cell growth,
compared with that of the WOX1/JNK1 interaction (Fig. 5). Human WOX3
has a deletion of its ADH domain at the C-terminus, whereas its
N-terminal WW domain region is highly homologous to that of
murine WOX1 (Fig. 5) (1). The first WW domain also interacted with JNK1
(Fig. 5). These results suggest that the first WW domain is responsible for binding to JNK1. A conserved phosphorylation site at the first WW
domain, Tyr33, was then altered to arginine in WOX3. This
mutant, designated WOX3(Y33R), could not interact with JNK1 (Fig. 5).
The observations further suggest that phosphorylation at
Tyr33 is essential for WOX1 and JNK1 binding
interaction.
In positive control experiments, the binding interactions between p53
and WOX1 (1) and the self-binding interaction of MafB were demonstrated
(Fig. 5). In negative controls, no binding interactions were observed
when testing empty pSos vector versus empty pMyr vector, or
collagenase versus lamin C (Fig. 5).
Anisomycin Activates JNK1 and Mediates Tyr33
Phosphorylation in WOX1 and Their Binding Interactions--
To further
verify the above observations, U937 cells were treated with anisomycin
to activate JNK1. Anisomycin is a potent activator of JNK1 (17). In
resting cells, no JNK1 and WOX1 binding interaction was observed, as
determined by co-immunoprecipitation using specific anti-JNK1 antibody
(Fig. 6A). Upon stimulation of
cells with anisomycin for 30 min, expression of JNK1 was increased, and
a portion of JNK1 interacted with WOX1 (Fig. 6A).
Similarly, exposure of SK-N-SH cells to anisomycin for 30 min also
resulted in an increased binding interaction between JNK1 and WOX1, as
determined using anti-WOX1 antibody in co-immunoprecipitation (Fig.
6B). Similar results were observed when L929 cells were treated with anisomycin, or the above cells were exposed to UV light
(data not shown).
To further verify Tyr33 phosphorylation in WOX1, we
synthesized a peptide containing phospho-Tyr33 for
immunization in rabbits. Anisomycin rapidly increased Tyr33
phosphorylation in WOX1, as well as JNK1 phosphorylation, in SK-N-SH
cells (Fig. 7A). Similar
results were observed using L929 and U937 cells (data not shown).
Exposure of L929 cells to UV light, followed by growing for 1 h,
resulted in increased p53 expression (Fig. 7B). p53 is known to interact with WOX1 (1) and JNK1 (21). Co-immunoprecipitation of p53
with a specific antibody resulted in the presence of p53, JNK1, and
phosphorylated WOX1 in the precipitates (Fig. 7B).
Dominant Negative WOX1 Abolishes WOX1 Phosphorylation and p53
Apoptosis but Not JNK1 Activation--
We developed a
dominant-negative WOX1. Lys28 and Asp29 in
Wox1 were mutated to Thr28 and
Val29, respectively. These residues are located at the
first WW domain. In agreement with our previous observations (1), p53
and WOX1 mediated U937 cell death in a synergistic manner, and
antisense WOX1 abolished p53-mediated cell death (Fig.
8A). The mutated full-length
WOX1 (WOX1mt) abolished p53 apoptosis (Fig. 8A). Similarly, the mutated WW domain region (WOX1wwmt) also abolished p53 apoptosis (Fig. 8A). These data support the dominant negative effect
of the generated WOX1 mutant.
Anisomycin-mediated WOX1 phosphorylation in Tyr33 was
abolished by the dominant negative WOX1 (Fig. 8B). SK-N-SH
cells were transfected with a GFP-tagged WOX1wwmt or a control GFP
construct by electroporation. The cells were cultured for 48 h and
then treated with anisomycin for 30 min. The dominant negative WOX1wwmt abolished anisomycin-mediated WOX1 phosphorylation in Tyr33
(Fig. 8B). However, WOX1wwmt failed to inhibit
anisomycin-induced JNK1 activation (Fig. 8B).
By yeast two-hybrid analysis, both WOX1mt and WOX1wwmt physically
interacted with p53, whereas WOX1wwmt could not bind JNK1 (Fig.
8C). Apparently, these dominant negative WOX1 proteins have an altered conformation, and inhibition of p53 apoptosis by the dominant negatives is due to a direct binding interaction. In contrast,
these dominant negatives failed to bind and prevent JNK1 activation.
Tyr33 Phosphorylation Is Necessary for WOX1 Apoptotic
Function--
Overexpression of the WW domain region of WOX1,
designated WOX1ww, induces cell death (1). Alteration of
Tyr33 to Arg33 was performed in WOX1ww, and the
resulting mutant, WOX1ww(Y33R), had a significantly reduced activity
(~50% decrease) in mediating death of L929 and Du145 cells (Fig.
9). The findings were also observed using
U937, SK-N-SH, and other types of cells (data not shown). JNK1
significantly blocked WOX1ww-mediated death of L929 and Du145 cells by
30-50% (Fig. 9). Interestingly, JNK1 increased the apoptotic function
of WOX1ww(Y33R) mutant in L929 but not in Du145 cells (Fig. 9).
In this study, we demonstrated that stress stimuli such as
anisomycin and UV light mediated WOX1 phosphorylation at
Tyr4. The phosphorylation is essential for functional
activation of WOX1 in mediating apoptosis. Alteration of
Tyr33 to Arg33 reduced the WOX1 activity. In
agreement with our previous observations (1), WOX1-mediated apoptosis
occurs at the nuclear level. Mutation of the nuclear localization
signal reduced the WOX1 apoptotic function.
We determined that ectopic JNK1 counteracted WOX1-mediated apoptosis,
as well as cell cycle progression. By stimulating cells with
anisomycin, JNK1 became activated and physically interacted with the
Tyr33-phosphorylated WOX1, as determined by
co-immunoprecipitation. A dominant negative WOX1 blocked p53-mediated
apoptosis and anisomycin-mediated WOX1 phosphorylation but failed to
inhibit JNK1 activation. This dominant negative WOX1 bound p53 but
could not interact with JNK1, as determined in yeast two-hybrid
analysis. Together, these observations indicate that a physical contact
between activated or phosphorylated WOX1 and JNK1 is essential for
their functional antagonism. How JNK1 inhibits WOX1 function remains to
be determined.
The upstream kinase that phosphorylates WOX1 is unknown. Nonetheless,
we do not exclude the possibility that activated JNK1 binds WOX1 and
then phosphorylates WOX1. Alternatively, an unknown kinase
phosphorylates WOX1 in response to stress stimuli. The phosphorylated
WOX1 then interacts with JNK1.
The dominant negative WOX1 bound p53 and blocked p53 apoptosis. Also,
this mutant protein inhibited WOX1 phosphorylation, suggesting its
possible interaction with the wild type WOX1. This potential binding
interaction remains to be determined by co-immunoprecipitation and
yeast two-hybrid analysis.
An additional conserved phosphorylation site in WOX1 is
Tyr61. Alteration of this residue to Arg61
results in abrogation of the binding interaction between WOX1 and p53
in yeast.2 Thus, it is likely
that both Tyr33 and Tyr61 in WOX1 are
phosphorylated when cells are exposed to stress stimuli. Functional
significance of Tyr61 phosphorylation in WOX1 is being
determined in this laboratory.
The functional property of WOX3 in vivo and in
vitro is unknown. Whether WOX3 expression is increased during
stress response is unknown. By RT-PCR and Northern blot, Driouch
demonstrated that about 50% of breast tumors have overexpressed WOX1
and WOX3 (22). WOX1 appears to have a tumor suppressor function (10, 11). Presumably, the overexpressed WOX3 enhances cancer growth by
neutralizing the WOX1 function.
In addition to activate JNK1, anisomycin inhibits protein synthesis and
mediates apoptosis (23, 24). Anisomycin-mediated apoptosis of leukemia
HL-60 cells depends upon JNK1 (24). Most interestingly,
ansiomycin-activated JNK1 phosphorylates Bcl-2, thereby increasing cell
survival (25). Previously, we found that when L929 cells were exposed
to anisomycin for less than 1 h, these cells became resistant to
TNF cytotoxicity (17). We determined that I Hyaluronidase PH-20 activates JNK1, and the activation is necessary to
prevent L929 cell death by various anticancer drugs such as
staurosporine, daunorubicin, and doxorubicin (19). In contrast, PH-20
increases L929 cell sensitivity to TNF cytotoxicity by increasing the
expression of WOX1 (1). Apparently, different mechanisms are involved
in the regulation of cellular sensitivity to TNF and anticancer drugs.
Indeed, we determined that ectopic JNK1 and exogenous PH-20 act
synergistically in blocking L929 cell death by WOX1 in the presence of
staurosporine, daunorubicin, or doxorubicin.2 We believe
that PH-20 activates both endogenous and ectopic JNK1 that block the
cell death. Whether JNK1 mediates Bcl-2 phosphorylation in these tested
cells is unknown.
The hyaluronidase HYAL1 gene, also known as
LUCA1, is a candidate tumor suppressor (for a review, see
Ref. 26). Inactivation of the HYAL1 gene by aberrant
splicing of pre-mRNA has been shown in head and neck squamous cell
carcinomas (27). Nonetheless, overexpressed hyaluronidases are
frequently associated with metastatic cancers (28-30). Also, plasma
HYAL1 is capable of promoting tumor cell cycling (31). PH-20 enhances
cell proliferation (4). Here, we demonstrated that PH-20 increased cell
cycle progression in L929 cells. PH-20-activated JNK1 is essential for
cell survival, since dominant negative JNK1 mediated apoptosis in L929 cells.
UV light up-regulates and activates p53 (32) and also activates JNK1
(12). Co-immunoprecipitation of the anisomycin-treated cells with
anti-p53 resulted in the presence of p53, JNK1, and WOX1 in the
precipitate. The observation suggests the presence of a
p53·WOX1·JNK1 complex during stress response. Most recently, we
determined that a portion of cytosolic I Although transient JNK1 activation provides a signal for cell survival
(13, 16-19), a persistent JNK1 activation can mediate cell death (14,
15). How the antiapoptotic JNK1 turns into apoptotic is not known. It
may be related to the status of JNK1 phosphorylation, or it may be
associated with JNK1-regulated gene expression at different stages of
cell differentiation or stress. We believe that at a certain stage of
apoptosis, the apoptotic JNK1 may act synergistically with WOX1 in
facilitating cell death.
In transient expression, both wild type JNK2 and JNK3 mediated L929
cell death, yet the dominant negatives greatly increased the cell
death. The likely explanation is that the kinetics of activation of JNK
isoforms is different, and their functional roles may be different
also. For example, JNK1 and JNK2 have distinct roles in antiviral
immunity, in which JNK1 is involved in survival of activated T cells
during immune responses, and JNK2 plays a role in control of CD8(+) T
cell expansion in vivo (33). Similarly, JNK1 and JNK2 play
distinct roles in controlling the activation of CD8(+) T cells (34).
Also, JNK1-deficient cells resist UV-mediated apoptosis, whereas
JNK2-deficient cells have increased sensitivity to UV irradiation (35).
JNKs are widely distributed in the nervous system (12), and JNK3 is
involved not only in neuronal development but also apoptosis of
neuronal cells (36).
L929 cells express both JNK1 and JNK2 (17, 19). Whether JNK3 is
expressed in L929 cells is unknown. Dominant negative JNK1 mediated
L929 cell apoptosis, suggesting that JNK1 is essential for the survival
of these cells. Inhibition of JNK2 and JNK3 functions by dominant
negatives induced cell death, suggesting that L929 cells also require a
certain level of JNK2 and JNK3 for survival. In contrast to the wild
type JNK1, transiently expressed wild type JNK2 and JNK3 induced L929
cell death, indicating that the kinetics of activation of JNK isoforms
is different.
Taken together, phosphorylation of Tyr33 and probably
Tyr61 is important for activating WOX1. Our data support
the possibility that WOX1 is a signaling protein in the p53 and JNK1
stress pathways. Whether activated JNK1 phosphorylates WOX1 remains to
be determined.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
) and dominant
negative JNK2 and JNK3 (peVRFO-HA-JNK2-K55R and peVRFO-HA-SAPK
-K55R
or DN-JNK2 and DN-JNK3) were kind gifts of Dr. Michael Kracht (Medical School Hannover, Hannover, Germany).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Hyaluronidase PH-20 promoted L929 fibroblast
progression toward S and G2/M phases in the cell
cycle. Freshly harvested L929 cells were grown overnight and
treated with bovine testicular hyaluronidase PH-20 for 24 h,
followed by determining cellular DNA content using flow cytometry (see
"Experimental Procedures"). PH-20 increased cellular distribution
in the S and G2/M phases and a reduced distribution in the
G0/G1 phase. No PH-20-mediated cell death or
apoptosis was observed (sub-G0/G1 phase). A
representative set of data from two experiments is shown.
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Fig. 2.
Dominant-negative JNK1 mediated apoptosis of
L929 cells. L929 cells were electroporated with a wild type or a
dominant-negative (dn) JNK1 (B), JNK2
(C), or JNK3 (D) construct and cultured
overnight. The cells were treated with or without hyaluronidase PH-20
(100 units/ml) for 24 h and then subjected to cell cycle analysis
by flow cytometry. In controls, the cells were electroporated with
nothing or medium (mock) and treated similarly (A). The wild
type JNK1 did not induce apoptosis of L929 cells. In contrast,
dominant-negative JNK1 significantly induced L929 cell death, and
hyaluronidase (HAase) slightly increased the extent of cell
death. In comparison, wild type JNK2 and JNK3 mediated L929 cell death
by ~30%, and the dominant negatives of these proteins significantly
caused cell death (~60-70%). A representative set of data from two
experiments is shown.
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Fig. 3.
JNK1 inhibited WOX1-mediated death of L929
cells. L929 cells were transfected with JNK1 and/or various WOX1
constructs by electroporation and cultured for 24 h. The extent of
cell death was measured by the MTS assay. Wild type WOX1 mediated L929
cell death (~40%), which was blocked by JNK1 (~20% death)
(p < 0.0001, n = 8, Student's
t test). JNK1 alone slightly increased cell proliferation
(0.001 < p < 0.05, n = 8, Student's t test). In contrast, WOX1mtNLS, which has a
mutated sequence at the NLS, had a significantly reduced activity in
inducing cell death (~70% reduction). This mutant failed to undergo
nuclear translocation (1). When in combination, JNK1 and WOX1mtNLS
could not significantly mediate cell death (p > 0.1, n = 8, Student's t test). Expression of
antisense WOX1 mRNA in L929 cells could not mediate cell death.
JNK1 and the antisense WOX1, in combination, could not induce cell
death either (p > 0.1, n = 8, Student's t test). Experiments were done in eight
replicates (mean ± S.D.). A representative set of data from two
experiments is shown.
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Fig. 4.
JNK1 blocked WOX1-mediated apoptosis and
inhibition of cell cycle progression in U973 cells. U937 cells
were transfected with wild type JNK1 (JNK1wt; 20 µg) and/or WOX1
(GFP-tagged WOX1 or WOX1gfp) by electroporation. Following 16-24 h in
culture, the cells were subjected to cell cycle analysis. WOX1 mediated
apoptosis (Sub G0/G1) and inhibited cell cycle progression
to the G0/G1, S, and G2/M phases.
JNK1 alone had litter or no effect on the cell cycle progression,
whereas it prevented WOX1-mediated apoptosis and inhibition of the cell
cycle progression. A representative set of data from two experiments is
shown.
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Fig. 5.
WOX1 physically interacts with JNK1. The
Ras rescue-based yeast two-hybrid analysis was performed (1, 20). In
positive controls, the binding interactions between p53 and WOX1 (1)
and the self-binding interaction of MafB are shown, as evidenced by the
growth of mutant Cdc25H yeast at 37 °C in the presence of
galactose. In negative controls, no binding interactions were observed
when testing empty pSos vector versus empty pMyr vector or
collagenase versus lamin C. The wild type human JNK1
physically interacted with murine WOX1 and human WOX3. Human WOX3 has a
deletion of its ADH domain at the C-terminus, whereas its
N-terminal WW domain region is highly homologous to that of
murine WOX1 (1). The first WW domain interacted with JNK1. Alteration
of a conserved phosphorylation site at Tyr33 to
Arg33 at the first WW domain in WOX3 was performed. This
mutant, designated WOX3(Y33R), could not bind JNK1. A representative
set of data from three experiments is shown.
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Fig. 6.
JNK1 and WOX1 binding interaction in
anisomycin-treated cells. A, monocytic U937 cells were
treated with anisomycin (100 µM) to activate JNK1 (17).
The cells were then processed for immunoprecipitation (IP)
using specific anti-JNK1 antibody. The presence of a cytosolic JNK1 and
WOX1 complex was observed in cells treated with anisomycin for 30 min.
In negative controls, no JNK1 was observed when protein A-agarose beads
alone were used in the precipitation. IgH, IgG heavy chain;
Pre-IP, one-tenth of the total cytosolic and nuclear
proteins (~50 µg) were loaded onto SDS-PAGE for Western blotting
analysis. B, similar results were observed by exposure of
SK-N-SH neuroblastoma cells to anisomycin (100 µM) for 30 min, followed by co-immunoprecipitation using anti-WOX1 antibody.
Representative data from three experiments are shown.
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Fig. 7.
Anisomycin and UV light mediated Tyr33
phosphorylation in WOX1. A, SK-N-SH cells were
stimulated with anisomycin (100 µM) for various indicated
times. Activation (or phosphorylation) of JNK1 and phosphorylation of
WOX1 were observed. B, L929 cells were exposed to UV light
and then cultured for 1 h, followed by processing
co-immunoprecipitation using specific anti-p53 antibody. UV induced p53
expression and its binding interaction with JNK1 and the
Tyr33-phosphorylated WOX1. IgH, IgG heavy chain.
Representative data from three experiments are shown.
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Fig. 8.
Dominant negative WOX1 abolishes WOX1
phosphorylation and p53 apoptosis but not JNK1 activation.
A, a dominant-negative WOX1 was developed by mutating
Lys28 and Asp29 to Thr28 and
Val29, respectively. U937 cells were electroporated with
WOX1 expression constructs or nothing (null) in the presence or absence
of a p53 expression construct. The cells were harvested 48 h
postelectroporation, and DNA fragmentation was analyzed by agarose gel
electrophoresis. p53-mediated cell death was blocked by the mutated
full-length WOX1 (WOX1mt), the mutated WW domain region (WOX1wwmt), and
antisense WOX1 (WOX1as). The relative increase in DNA fragmentation
(versus controls) was analyzed by the NIH Image program. The
data represent average from two experiments. B, SK-N-SH
cells were transfected with a GFP-tagged WOX1wwmt or a control GFP
construct by electroporation, cultured for 48 h, and then treated
with anisomycin for 30 min. WOX1wwmt abolished anisomycin-mediated WOX1
phosphorylation in Tyr33. However, WOX1wwmt failed to
inhibit anisomycin-induced JNK1 activation. C, both WOX1mt
and WOX1wwmt physically interacted with p53, as determined in yeast.
WOX1ww interacted with JNK1, whereas WOX1wwmt could not bind JNK1. A
representative set of data from 2-4 experiments is shown in
B and C.
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Fig. 9.
Tyr33 phosphorylation is
necessary for WOX1 apoptotic function. Alteration of
Tyr33 to Arg33 reduced WOX1ww-mediated death of
L929 and Du145 death by ~50% (p < 0.0001, Student's t test). JNK1 significantly blocked
WOX1ww-mediated death of L929 cells (p < 0.01, Student's t test). Similar results were observed using
Du145 cells (p < 0.01, Student's t test;
see the representative crystal violet stain). Interestingly, JNK1
increased the apoptotic function of WOX1ww(Y33R) mutant in L929 but not
in Du145 cells. Experiments were done in eight replicates for L929
cells and four replicates for Du145 cells (mean ± S.D.). A
representative set of data from two experiments is shown. The extent of
cell death was determined by crystal violet stain.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B
, the inhibitor of
NF-
B, maintains the basal level of JNK1 activation and regulates
JNK1-mediated TNF resistance (17). However, whether this protective
event is related to JNK1-mediated Bcl-2 phosphorylation remains to be determined.
B
binds p53 in resting cells and the complex dissociates in response to apoptotic stress, hypoxia, DNA damage, and tumor growth factor-
1-mediated growth suppression (20). The dissociation allows p53 nuclear translocation. Alternatively, when dissociated from I
B
, p53 may complex with WOX1 and JNK1.
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ACKNOWLEDGEMENTS |
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We thank Terri Zimmer for antibody production in rabbits, John Heath and Lori Schultz for technical assistance, and Drs. Roger Davis and Michael Kracht for JNK constructs.
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FOOTNOTES |
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* This work was supported by the American Heart Association and the Guthrie Foundation for Education and Research.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Guthrie Research
Institute, Sayre, PA 18840. Fax: 570-882-4643; E-mail:
nschang@ inet.guthrie.org.
§ Summer research student from the University of Rochester (Rochester, NY) in 2001.
¶ Summer research student from Roberts Wesleyan College (Rochester, NY) in 2002.
Published, JBC Papers in Press, January 6, 2003, DOI 10.1074/jbc.M208373200
2 N.-S. Chang, J. Doherty, and A. Ensign, unpublished results.
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ABBREVIATIONS |
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The abbreviations used are: WOX1, WW domain-containing oxidoreductase; JNK, c-Jun N-terminal kinase; TNF, tumor necrosis factor; NLS, nuclear localization sequence; ADH, short-chain alcohol dehydrogenase; GFP, green fluorescent protein.
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