(Received for publication, January 29, 1997, and in revised form, May 12, 1997)
From the Departments of Biochemistry, Kobe University School of Medicine, Kobe 650, Japan, the § Fukui Medical School, Fukui 910-11, Japan, and the ¶ Kansai Medical University, Moriguchi 570, Japan
By taking advantage of the established chicken B cell line, DT40 cells, which do not express tyrosine kinase Syk or Lyn, functional roles of Syk and Lyn in apoptotic response elicited by cellular stress were investigated. DT40 cells underwent apoptosis after hyperosmotic stress. In Syk-deficient DT40 cells, this apoptotic process was significantly enhanced. Ectopic expression of wild type, but not kinase-inactive, porcine Syk in Syk-deficient cells rescued cells from osmotic stress-induced apoptosis, demonstrating that the presence of functionally active Syk is necessary to protect cells from osmotic stress-induced apoptosis. In comparison, there was no effect on osmotic stress-induced apoptosis in Lyn-deficient DT40 cells. Interestingly, while Syk was not involved in ultraviolet C (UVC)-induced apoptosis, a deficiency of Lyn rendered cells resistant to UVC irradiation. These observations defined Syk and Lyn as important mediators of apoptosis in DT40 cells in response to osmotic stress and UVC irradiation, respectively. Furthermore, osmotic stress, but not UVC irradiation, could activate c-Jun N-terminal kinase (JNK) in DT40 cells. A deficiency in either Syk or Lyn did not affect the osmotic stress-induced activation of JNK. We, therefore, concluded that Syk and Lyn regulate the apoptotic responses to osmotic stress and UVC irradiation independently of the JNK pathway in DT40 cells.
Apoptosis, which is widely observed in different cells of various organisms, is the unique morphological pattern of cell death characterized by chromatin condensation and membrane blebbing. The most prominent event in the early stages of apoptosis is internucleosomal DNA cleavage by undefined endonuclease activities, which is widely used as a biochemical marker of apoptosis. It is generally believed that apoptosis plays important roles in developmental processes, maintenance of homeostasis, and elimination of damaged cells (1, 2). Cells usually undergo apoptosis when they suffer from cellular stress. The molecular mechanisms by which cellular stress regulates cell apoptosis are still poorly understood.
A growing body of evidence demonstrates that several protein kinases
participate in regulating stress-triggered apoptosis. Abl, a
nonreceptor protein-tyrosine kinase (PTK),1
which is localized to the nucleus and the cytoplasm and shares structural features with Src family PTKs (3, 4), has been identified as
a negative regulator of apoptosis. Constitutive expression of the p210
Bcr-Abl proteins in chronic myelogenous leukemia progenitor cells
confers resistance to apoptosis upon interleukin-3 withdrawal (5).
Moreover, the down-regulation of Bcr-Abl protein levels by antisense
oligonucleotides has been shown to render K562 cells susceptible to
apoptosis.(6). More recently, Btk has also been demonstrated as a
mediator of radiation-induced apoptosis of DT40 cells (7). In addition
to the PTKs, c-Jun N-terminal kinases (JNKs) have an unusually high
affinity for their substrate, c-Jun, and phosphorylate it on specific
N-teminal serine residues at positions 63 and 73, leading to enhanced
c-Jun transactivation potential (8). JNKs are strongly activated by
stimuli other than growth factors, including signals as diverse as UV
irradiation (9), osmotic shock (10, 11), protein synthesis inhibitors
(12), and tumor necrosis factor (TNF-
) stimulation (13). The
pathways of JNK activation have been partially delineated and
Rac/Cdc42-MEKK1-JNKK protein kinases have been shown to be upstream of
JNK (14, 15). Furthermore, Pyk2 and c-Abl have recently been identified
as the upstream regulators of JNK activation in response to certain
cellular stresses although the convergence point of both these PTKs
into the JNK pathway is unclear (16-18). The growing evidence, which
indicates various cellular stresses as the potential activators of JNK,
suggest that JNK activation may play an important role in mediating
cell death or cell survival in cells exposed to various stresses. In fact, JNK activation has been demonstrated to correlate with cell apoptosis triggered by cellular stress (19, 20). Ectopic expression of
a dominant-negative c-Jun mutant lacking the N terminus or a
dominant-negative kinase-inactive JNKK abolishes cellular
stress-induced cell apoptosis (19). In PC12 cells, ectopic expression
of various mutants, which either activate or inhibit the JNK signaling
pathway, also enhance or inhibit nerve growth factor withdrawal-induced apoptosis (20). In comparison, expression of human wild-type, but not
kinase-negative, JNK in yeast lacking the protein kinase Hog1 is able
to promote growth on hyperosmolar media (10), which under normal
conditions inhibits growth of these cells, suggesting that JNK
activation delivers a signal for cell survival.
We and others have demonstrated that certain cellular stresses such as
oxidative stress are potent activators of the Syk family PTKs, Syk and
ZAP70, in lymphocytes (21-24). Recently, we found that osmotic stress
can also activate Syk in human and chicken B cells (25). Thus, it is of
interest to further investigate the functional roles of PTKs in the
stress response and the possible mechanisms by which PTKs execute their
function in stress signaling. Mammalian cells are exposed to
hyperosmotic conditions in the distal tubule of the kidney, during hemo
or peritoneal dialysis, when the concentration of serum sodium rises as
a consequence of dehydration or due to an infusion of hypertonic saline
(26). Oxidative stress may occur in response to inflammation due to the
production of superoxide anion and hydrogen peroxide by neutrophils and
monocytes. Inflammatory cytokines such as TNF- and interleukin-1 can
also stimulate the production of hydrogen peroxide and reactive oxygen
intermediates, thus leading to cells being exposed to oxidant stress
(27). Under physiological conditions, lymphocytes are rarely exposed to
these types of stresses, yet elucidating the functional roles of PTKs
in stress-induced responses in vitro would provide a better
understanding of the pathogenesis in response to these
pathophysiological stresses in vivo. By taking advantage of
established Syk- or Lyn-deficient cells, we therefore investigated the
functional roles of Syk and Lyn (in particular Syk) in the apoptotic
response triggered by osmotic stress or ultraviolet C (UVC)
irradiation. Here, we report that a deficiency of Syk, but not Lyn,
results in a drastically enhanced apoptotic response to osmotic stress
when compared with wild type DT40. Ectopic expression of wild type, but
not the kinase-inactive, porcine Syk in Syk-deficient cells
significantly promoted cell survival in response to osmotic stress. In
contrast, Lyn is a positive mediator of the apoptotic response elicited
by UVC irradiation, whereas Syk does not appear to participate in this
apoptotic process. These results demonstrate that in DT40 cells, Syk
may function as a specific inhibitor of osmotic stress-induced
apoptosis while Lyn acts as a positive mediator of UVC
irradiation-induced apoptosis.
The generation of DT40/Lyn,
DT40/Syk
, DT40/Syk
/Syk, and
DT40/Syk
/Syk(K
) cells and antisera against
Lyn or Syk was carried out as described previously (28). RPMI 1640 was
purchased from ICN Biomedicals Inc. Fetal bovine serum was from Life
Technologies, Inc. Protein A was from Calbiochem Corp.
Anti-phosphotyrosine antibody (4G10) was from Upstate Biotechnology
Inc. Mouse anti-human JNK1 monoclonal antibody was purchased from
Pharmingen (San Diego, CA). Enhanced chemiluminescence reagents were
from Amersham Corp.. Glutathione-Sepharose 4B was from Pharmacia
Biotech Inc. GST expression vector containing the N-terminal fragment
(amino acids 1 to ~79) of c-Jun was a gift from Dr. Hibi (Osaka
University, Japan).
DT40 (chicken B cells) and Raji (human B cells) cells were maintained in RPMI 1640 medium supplemented with 10% (v/v) fetal bovine serum, 100 units/ml penicillin, and 100 µg/ml streptomycin in a humidified 95% air, 5% CO2 atmosphere. The parent culture was maintained in continuous logarithmic growth between (5-10) × 105 cells/ml. For experiment use, cells were collected by centrifugation, washed once in NaCl/Pi buffer (136.8 mM NaCl, 2.68 mM KCl, 8.04 mM Na2HPO4, 1.47 mM KH2PO4, pH 7.4), and then suspended at a density of 1 × 107 cells/ml in Hanks' balanced salt solution (136.7 mM NaCl, 5.4 mM KCl, 0.81 mM MgSO4, 1.3 mM CaCl2, 0.33 mM Na2HPO4, 0.44 mM KH2PO4, 5.6 mM dextrose, 4.2 mM NaHCO3, pH7.4). Osmotic stress was achieved by the addition of a concentrated sodium chloride solution. Cells were stimulated at 37 °C under gentle aggitation.
Preparation of GST Fusion ProteinpGEX3X-c-Jun (amino acids 1 to 79) encodes a GST-fusion protein containing the JNK binding domain and the serine residues (at positions 63 and 73), the phosphorylation of which correlates well with the increased transcriptional activity of c-jun. Escherichia coli XL1Blue were transfected with this glutathione S-transferase fusion protein expression vector. Proteins were purified following the protocol recommended by the manufacturer (Pharmacia). The amounts of purified proteins were estimated by SDS-polyacrylamide gel electrophoresis and subsequent staining with Coomassie Blue.
Preparation of Cell ExtractsStimulated cells (1 × 107 cells) were lysed in ice-cold lysis buffer (5 mM EDTA, 150 mM NaCl, 2% Triton X-100, 100 µM vanadate, 2 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, 50 mM Tris, pH 7.4). Lysates were clarified by centrifugation at 16,000 × g for 15 min at 4 °C.
Immunoblot AnalysisCell extracts were immunoprecipitated with 0.3 µg of anti-Syk antibody, 1 µg of anti-JNK1 antibody, or 3 µl of anti-Lyn antisera with 40 µl of protein A-Sepharose 4B (50% slurry) for 1 h at 4 °C. Immunoprecipitates were washed three times with lysis buffer, once with 10 mM Hepes, pH 8.0, buffer containing 500 mM NaCl, and once with the same Hepes buffer without NaCl. The washed immunoprecipitates were boiled with SDS sample buffer for 3 min, resolved on a 10% SDS-polyacrylamide gel electrophoresis, transferred electrically to polyvinylidene difluoride membranes, and then immunoprobed with 4G10 to detect tyrosine phosphorylation. The corresponding antibody was used to detect the protein levels of Syk, JNK, or Lyn. Immunoreactive proteins were visualized using enhanced chemiluminescence.
Assays for JNK ActivityCell extracts were
immunoprecipitated with 1 µg of anti-JNK1 with 40 µl of protein
A-Sepharose 4B (50% slurry) for 1 h at 4 °C. Anti-JNK
immunoprecipitates were washed three times with lysis buffer, once with
washing buffer (50 mM Hepes and 10 mM MgCl2, pH 7.6), and once with kinase assay buffer (10 mM Hepes, 10 mM MgCl2, 10 µM cold ATP, and 10 µM vanadate, pH 7.6).
Immune complex kinase assays were performed in 30 µl of kinase assay buffer containing 1 µCi of [-32P]ATP and 5 µg of
GST-c-Jun as a substrate. After a 20-min incubation at 30 °C, the
reaction was terminated by the addition of SDS sample buffer followed
by boiling for 5 min. The samples were separated by SDS-polyacrylamide
gel electrophoresis. Autoradiography was carried out utilizing a
phosphoimager (Fuji BAS 2,000).
Cells (5 × 105/ml) were treated for the time stated with the indicated concentration of sodium chloride dissolved in RPMI 1640 media. UV irradiation was performed using a model 1800 Stratalinker UV cross-linker (Stratagene, La Jolla, CA) with 254-nm lamps. Cells (5 × 105/ml) in RPMI 1640 media were irradiated in an open tissue culture dish and then cultured for the indicated time. 5 × 106 cells were lysed in 0.5 ml of lysis buffer (10 mM Tris-HCl, pH 7.5, 10 mM EDTA, 200 mM NaCl, 0.4% Triton X-100, and 0.1 mg/ml proteinase K) for 20 min at room temperature followed by a 30-min incubation with 0.1 mg/ml RNase A at 50 °C. DNA fragmentation was analyzed on a 2% agarose gel in the presence of 0.5 µg/ml ethidium bromide.
To investigate the activation of Syk in response to low
levels of hyperosmotic stress (0.2 M NaCl), anti-Syk
immunoprecipitates from DT40 cell lysates, treated with or without
sodium chloride, were subject to immunoblotting with an
anti-phosphotyrosine antibody. As shown in Fig.
1A, top, exposure of DT40 cells to
0.2 M sodium chloride stimulated a rapid and sustained
tyrosine phosphorylation of Syk. This increase in tyrosine
phosphorylation reached a maximum at 1 min of exposure and remained
elevated over a 15-min incubation time. The observed tyrosine
phosphorylation of Syk was dependent on the concentration of sodium
chloride used. Exposure of cells to 0.1 M sodium chloride
for 5 min induced a significant increase in Syk tyrosine
phosphorylation. Immunoblot analysis with an anti-Syk antibody revealed
that the amounts of Syk immunoprecipitated from treated or untreated
DT40 cells were comparable (Fig. 1A, bottom). Therefore, the elevated tyrosine phosphorylation was a specific response to sodium chloride treatment. Activation of Syk by osmotic stress was also observed in the human B cell line, Raji (Fig. 1B), indicating that osmotic stress-induced Syk activation
was not unique to DT40 cells. In contrast, no detectable tyrosine phosphorylation of Syk was observed following UVC irradiation, up to
1,000 J/m2, under our experimental conditions (Fig.
1C, top; data not shown) although the amounts of
Syk immunoprecipitated from untreated and treated DT40 cells were
comparable (Fig. 1C, bottom). Thus, Syk was not
activated by UVC irradiation in DT40 cells.
Inhibition of Osmotic Stress- but Not UVC-induced Apoptosis by Syk
To explore whether Syk plays a role in regulating osmotic
stress-induced cell apoptosis, DT40 and DT40/Syk cells
were treated with 0.2 M sodium chloride. Cell apoptosis was
assessed by DNA fragmentation, a typical biochemical marker of
apoptosis, by running extracted DNA on a 2% ethidium
bromide-containing agarose gel. As presented in Fig.
2A, in DT40 cells, the typical nucleosomal
DNA ladders appeared 16 h after sodium chloride treatment. In
comparison, in DT40/Syk
cells, DNA fragmentation occurred
4 h after sodium chloride exposure (Fig. 2A). The
intensity of fragmented DNA was continuously increased as a function of
exposure time, indicating the progression of massive fragmentation of
chromosomal DNA.
To support this observation that Syk may have a role in regulating
apoptotic response triggered by osmotic stress, we made use of genetic
approaches in which wild-type porcine syk cDNA was
transfected into DT40/Syk cells, and the selected clone
was designated as DT40/Syk
/Syk cells. It is highly
conceivable that DT40/Syk
/Syk cells would be resistant to
osmotic stress if Syk, in fact, negatively regulates osmotic
stress-induced apoptosis. As shown in Fig. 2A, expression of
wild-type porcine Syk into DT40/Syk
cells protected
Syk-deficient cells from osmotic stress-induced cell death,
demonstrating that Syk may be an inhibitor of osmotic stress-induced
apoptosis in DT40 cells. Interestingly, UVC irradiation, which was
unable to activate Syk in DT40 cells (Fig. 1B), triggered rapid DNA fragmentation independently of Syk (Fig. 2B).
To evaluate an
important functional role of the kinase activity of Syk in mediating
the enhanced DNA fragmentation induced by osmotic stress in
DT40/Syk cells, we transfected kinase-inactive porcine
syk cDNA into DT40/Syk
cells. The lack of
Syk kinase activity was demonstrated by an in vitro kinase
assay that showed there was no detectable autophosphorylation, which is
seen under normal conditions (Fig. 3A,
top). Expression levels of wild-type and kinase-inactive
porcine Syk were comparable, as revealed by immunoblotting (Fig.
3A, bottom). Expression of kinase-inactive
porcine Syk in DT40/Syk
cells largely failed to block
cell death (Fig. 3B, right) while expression of
wild-type porcine Syk in DT40/Syk
cells was sufficient to
elicit an anti-apoptotic response to osmotic stress (Fig.
3B, middle). This finding indicated that the
kinase activity of Syk is required for the anti-apoptotic effect
observed.
Lyn, though Not Involved in Osmotic Stress-induced Apoptosis, Mediates UVC-induced Apoptosis
Lyn is another major
nonreceptor-type PTK predominantly expressed in B-lineage cells (28,
29). Lyn is physically and functionally associated with CD19 (29), and
inhibition of Lyn activity by an anti-CD19-genistein immunoconjugate
triggers rapid apoptotic cell death in Ramos Burkitt lymphoma cells,
suggesting that Lyn in association with CD19 is an important regulator
of apoptosis (30). To investigate the specificity or the functional
redundancy of PTKs in mediating osmotic stress-induced apoptotic
process in DT40 cells, the roles of Lyn in cell apoptosis were examined using established Lyn-deficient (DT40/Lyn) cells. After a
12-h exposure to the indicated concentration of sodium chloride, the
extracted DNA from untreated and treated cells was separated on 2%
agarose gels. As presented in Fig. 4A, exposure to sodium chloride induced apoptosis in DT40 cells, which was
significant at a concentration of 0.2 M sodium chloride. A deficiency of Syk produced a drastically enhanced,
dose-dependent apoptotic response based on DNA
fragmentation (Fig. 4A). In contrast, a deficiency of Lyn
did not have any effect on osmotic stress-induced apoptosis when
compared with that observed in wild-type cells. The extent of DNA
fragmentation was comparable in DT40 and DT40/Lyn
cells.
Therefore, in DT40 cells, Syk, but not Lyn, appears to be a specific
negative regulator of apoptosis in response to osmotic stress. To
examine the role of Lyn in UVC-induced apoptosis, cells were irradiated
by the indicated doses of UVC. Cells were harvested 12 h after
irradiation, and apoptosis was analyzed by DNA laddering. Intriguingly,
although a Syk deficiency did not affect UVC-induced apoptosis (Figs.
2B and 4B), a deficiency of Lyn rendered cells resistant to UVC-induced apoptosis. The resistance of Lyn-deficient cells to UVC-induced apoptosis was observed from 100 up to 1,000 J/m2 (Fig. 4B).
Thus, it has become apparent that Lyn has distinct roles in the mediation of osmotic stress- or UVC irradiation-induced apoptosis. To further elucidate the possible functional role of Lyn in cell death signaling, the extent and kinetics of Lyn activation by these two different stresses were examined. As revealed by anti-phosphotyrosine immunoblotting analysis (Fig. 4C), both osmotic stress and UVC irradiation were able to activate Lyn to different degrees. Activation of Lyn by osmotic stress and UVC irradiation was rapid and sustained within the time examined.
Differential Responses of JNK to Osmotic Stress and UVC IrradiationRelaying stress signals to the JNK pathway remains
poorly understood. However, Pyk2 and c-Abl have recently been shown to positively regulate the activation of JNKs in response to osmotic stress (16) and genotoxic stress (UVC irradiation or ara-C treatment) (17, 18). Further, JNK activation has been shown to correlate with
apoptosis induced by certain forms of extracellular stress (19). These
observations lead us to analyze whether Syk and/or Lyn regulate
apoptosis in DT40 cells via the JNK pathway. As revealed by
phosphorylation of an exogenous substrate, GST-c-Jun-(1-79), exposure
of DT40 cells to 0.2 M sodium chloride induced a 7-9-fold increase in JNK activity over the control (Fig.
5A, top). A deficiency in either
Syk or Lyn had a marginal effect, if any, on JNK activation since JNK
activity in DT40, DT40/Syk, and DT40/Lyn
cells was comparable following exposure to osmotic stress. Immunoblot analysis with an anti-JNK antibody showed that the amounts of JNK in
each sample were comparable (Fig. 5A, bottom).
These results indicate that both Syk and Lyn are not involved in
osmotic stress-induced JNK activation. In contrast, UVC irradiation
(100 to ~1000 J/m2) failed to induce a significant
increase in JNK activity in DT40, DT40/Syk
, and
DT40/Lyn
cells under the experimental conditions employed
although DT40/Lyn
cells displayed an ~2-fold higher
basal activity compared with wild-type cells (Fig. 5B, and
data not shown).
Cellular stress, including ionizing irradiation, hydrogen peroxide, sodium chloride, and low energy electromagnetic fields, activates several nonreceptor PTKs such as Btk, Syk, Lyn, and ZAP70 (7, 21, 22, 24, 25, 31, 32), which are predominantly expressed in lymphocytes. In addition, cellular stress usually damages cells, thereby resulting in elimination of injured cells by apoptosis (19, 20). The mechanisms by which extracellular stimuli trigger cell apoptosis are not well understood, yet PTKs have been indicated to play an important role in mediating cell apoptosis in response to extracellular stimuli. Constitutive expression of Bcr-Abl confers resistance to interleukin-3 withdrawal-induced apoptosis in leukemia progenitor cells while down-regulation of Bcr-Abl renders K562 cells susceptible to apoptosis (5, 6). Immature B cells undergo apoptosis when activated through the B cell receptor, and a Syk deficiency blocks this apoptotic response (33).
DT40 cells that lack the expression of either Syk or Lyn provide a
powerful tool to study the exact role of the respective PTKs in stress
signaling. In this study, we have focused on the functions of Syk and
Lyn (in particular Syk) in the osmotic stress- and UVC
irradiation-triggered apoptotic response in DT40 cells. We have
observed in wild-type DT40 cells that, after a 16-h exposure to sodium
chloride (osmotic stress), there is a significant induction of cell
apoptosis. However, a deficiency in Syk results in a drastic enhancement of cell apoptosis, indicating that the presence of Syk
inhibits osmotic stress-induced apoptosis in DT40 cells. The negative
regulatory role of Syk in the osmotic stress-induced apoptotic response
is further emphasized by the fact that ectopic expression of the
wild-type porcine syk gene into DT40/Syk cells
leads to an apoptotic response very similar to that observed in
wild-type DT40 cells. Enhanced apoptosis in DT40/Syk
cells is also observed, but to a much lesser extent, when cells are
subject to oxidative stress using 1 mM hydrogen peroxide
(data not shown), which is a stronger activator of Syk (21, 23-25). In
addition, experiments utilizing kinase-inactive Syk mutant highlight
that the kinase activity of Syk is required to render Syk-deficient
cells resistant to osmotic stress since expression of the
kinase-inactive form of Syk largely fails to protect Syk-deficient cells from osmotic stress-induced apoptosis. Consistent with the requirement for kinase activity, ultraviolet C irradiation, which fails
to activate Syk in DT40 cells (Fig. 1B), induces a very similar DNA fragmentation pattern in both wild type and Syk-deficient cells (Fig. 2B).
Lyn is expressed predominantly in B-lineage cells (28, 29), and Lyn in
association with CD19 is an important mediator of apoptosis in Ramos
Burkitt lymphoma cells (30). These findings led us to examine whether
Lyn was involved in mediating the apoptotic response induced by osmotic
stress. Unlike Syk-deficient cells, a deficiency of Lyn does not alter
the sensitivity of cells to osmotic stress, when compared with wild
type cells, although Lyn is activated when cells are exposed to osmotic
stress.(Fig. 4, A and C). These results clearly
indicate that Syk, but not Lyn, is an inhibitor of osmotic
stress-induced apoptosis in DT40 cells. On the other hand, the
functional roles of Syk and Lyn in UVC irradiation-induced apoptosis
are quite different from those in osmotic stress-induced apoptosis.
Upon UVC irradiation, Lyn acts as a positive mediator of apoptosis. In
fact, abolishment of Lyn, but not Syk, blocks UVC irradiation-induced
apoptosis (Fig. 4B). In B cells, Btk, Syk, and Lyn are
abundantly expressed (28, 34, 35). All of them are activated following
B-cell receptor engagement (28, 34, 35) and -ray irradiation (7). An interesting issue to be addressed is the functional roles of each PTK
activated by a specific agonist. In the case of the apoptotic response,
induction of apoptosis by B-cell receptor engagement is mediated by
both Syk and Btk but not Lyn (7, 33). Btk, but not Syk and Lyn, is
involved in
irradiation-triggered apoptosis (7). Although Btk
participates in both B-cell receptor engagement- and
irradiation-induced apoptosis, phospholipase C
2, which is downstream
of BTK, is only used in B-cell receptor signaling as a putative signal
transducer to relay the death signal to the nucleus (7, 33). Both Syk
and Btk positively mediate apoptosis induced by B-cell receptor
engagement and
irradiation (7, 33). Similarly, Lyn, but not Syk,
functions as a positive mediator of UVC-induced apoptosis (Fig.
4B). In contrast, Syk, but not Lyn, functions as a negative
mediator of osmotic stress-induced apoptosis (Figs. 2 and 4). In the
same cell system, the apoptotic responses induced by these types of
extracellular stress require the participitation of different members
of these three nonreceptor PTKs to relay the death signals to
downstream effectors. Although the critical factors that determine the
specificity of the PTK and the signaling pathways responsible for
apoptosis are poorly defined at present, one can assume that both the
docking sites provided by PTKs and the various sets of downstream
signaling molecules utilized by them may be critical in determining the fate of the cell.
Recently, reseachers have paid much attention to the roles of the JNK
pathway in the apoptotic response induced by cellular stress (19, 20).
Blocking the JNK pathway abolishes apoptosis induced by extracellular
stress, including hydrogen peroxide, heat shock, UVC irradiation, and
radiation (19). Furthermore, it has been shown, that c-Abl and Pyk2
work upstream of JNK activation in response to certain extracellular
stresses (16-18). We therefore analyzed the activation of JNK in wild
type and Syk- and Lyn-deficient DT40 cells after exposure to osmotic
stress or UVC irradiation. The results show that JNK activation in
these cells was comparable in response to osmotic stress (Fig.
5A), excluding the possibility that the susceptibility of
Syk-deficient cells to osmotic stress-induced apoptosis is due to the
altered JNK activity. The signaling pathways involved in cell death are
not well understood. There are several pathways that may work either
independently or interactively to execute signal transmission. For
example, nerve growth factor withdrawal activates JNK and induces
apoptosis in PC12 cells. Following nerve growth factor withdrawal, some
survival-promoting agents, such as Bcl2 and
N-acetylcysteine, promote cell survival and block JNK
activation simultaneously, whereas others significantly promote cell
survival without affecting JNK activity (36). The idea that JNK
activation and c-Jun are crucial mediators of apoptosis in response to
tumor necrosis factor (19, 37) has been challenged by recent findings
that demonstrate that cell apoptosis, elicited by tumor necrosis
factor, is not linked to JNK activation (38). With respect to cell
death signaling in response to various types of extracellular stress,
many questions remain to be answered. What determines Btk as the
mediator of
radiation-induced apoptosis among activated nonreceptor
PTKs? Why is it that Syk functions as an inhibitor of apoptosis in
response to osmotic stress but Lyn positively mediates UVC
irradiation-induced apoptosis? To fully address these issues further
investigation is warranted.
In summary, our present studies demonstrate that a deficiency in Syk
selectively confers a strong susceptibility to osmotic stress from the
combined results of three different experimental approaches. First, DNA
laddering, a hallmark of lymphocyte apoptosis, is enhanced in
DT40/Syk cells. Second, the enhanced DNA fragmentation in
DT40/Syk
cells does not occur in DT40/Lyn
cells under the same condition. Third, overexpression of porcine Syk in
DT40/Syk
cells renders cells more resistant to apoptosis.
In contrast, a deficiency of Lyn, but not Syk, blocks the apoptotic
response induced by UVC irradiation. These results indicate that, in
DT40 lymphoma cells, Syk is a negative mediator of osmotic
stress-induced apoptosis while Lyn is a positive mediator of UVC
irradiation-induced apoptosis.
We thank Dr. Allison Stewart for help in manuscript preparation.