Distinctive Functions of Syk and Lyn in Mediating Osmotic Stress- and Ultraviolet C Irradiation-induced Apoptosis in Chicken B Cells*

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 apopto-sis. 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 radiationinduced 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)(22)(23)(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 * This study was supported by the Grants-in-Aid provided by the Ministry of Education, Science and Culture, Japan for General Scientific Research and Scientific Research on Priority Areas; by the Uehara Foundation, Daiichi Phamaceutical Co., LTD; by the Yamanouchi Foundation for Research on Metabolic Disorders; the Naito Foundation; and a Research Grant from the Princess Takamatsu Cancer Research Fund, Osaka Cancer Research Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
ʈ To whom correspondence should be addressed: Tel.: 81-78-341-7451, ext. 3250; Fax: 81-78-371-8734. 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 stressinduced 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 Sykdeficient 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.

EXPERIMENTAL PROCEDURES
Materials-The generation of DT40/Lyn Ϫ , DT40/Syk Ϫ , DT40/Syk Ϫ / Syk, and DT40/Syk Ϫ /Syk(K Ϫ ) 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 ϳ79) of c-Jun was a gift from Dr. Hibi (Osaka University, Japan).
Cell Culture and Harvest-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 g/ml streptomycin in a humidified 95% air, 5% CO 2 atmosphere. The parent culture was maintained in continuous logarithmic growth between (5-10) ϫ 10 5 cells/ml. For experiment use, cells were collected by centrifugation, washed once in NaCl/P i buffer (136. 8  Preparation of GST Fusion Protein-pGEX3X-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.
Immunoblot Analysis-Cell extracts were immunoprecipitated with 0.3 g of anti-Syk antibody, 1 g of anti-JNK1 antibody, or 3 l of anti-Lyn antisera with 40 l 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 Activity-Cell extracts were immunoprecipitated with 1 g of anti-JNK1 with 40 l 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 MgCl 2 , pH 7.6), and once with kinase assay buffer (10 mM Hepes, 10 mM MgCl 2 , 10 M cold ATP, and 10 M vanadate, pH 7.6). Immune complex kinase assays were performed in 30 l of kinase assay buffer containing 1 Ci of [␥-32 P]ATP and 5 g 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).
DNA Fragmentation Analysis-Cells (5 ϫ 10 5 /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 ϫ 10 5 /ml) in RPMI 1640 media were irradiated in an open tissue culture dish and then cultured for the indicated time. 5 ϫ 10 6 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 g/ml ethidium bromide.

RESULTS
Activation of Syk by NaCl, but Not by UVC Irradiation, in DT40 Cells-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 antiphosphotyrosine 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/m 2 , 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 Ϫ 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 Ϫ 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 Ϫ cells, and the selected clone was designated as DT40/Syk Ϫ /Syk cells. It is highly conceivable that DT40/Syk Ϫ /Syk cells would be resistant to osmotic stress if Syk, in fact, negatively regulates osmotic stressinduced apoptosis. As shown in Fig. 2A, expression of wild-type porcine Syk into DT40/Syk Ϫ 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).
Requirement for the Kinase Activity of Syk in the Protection of Cells from Osmotic Stress-induced Apoptosis-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 Ϫ cells, we transfected kinase-inactive porcine syk cDNA into DT40/Syk Ϫ 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 Ϫ cells largely failed to block cell death (Fig. 3B, right) while expression of wild-type porcine Syk in DT40/Syk Ϫ cells was sufficient to elicit an antiapoptotic 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-CD19genistein 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 Ϫ ) 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 stressinduced apoptosis when compared with that observed in wildtype cells. The extent of DNA fragmentation was comparable in DT40 and DT40/Lyn Ϫ 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/m 2 (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 Irradiation-Relaying 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 Ϫ , and DT40/Lyn Ϫ 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 ϳ1000 J/m 2 ) failed to induce a significant increase in JNK activity in DT40, DT40/ Syk Ϫ , and DT40/Lyn Ϫ cells under the experimental conditions employed although DT40/Lyn Ϫ cells displayed an ϳ2-fold higher basal activity compared with wild-type cells (Fig. 5B, and data not shown). DISCUSSION 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

FIG. 3. Requirement of the kinase activity of Syk for protecting cells from osmotic stress-induced apoptosis.
A, tyrosine kinase activity and protein expression of porcine wild-type and kinaseinactive Syk in DT40/Syk Ϫ cells. Lysates from DT40/Syk Ϫ , DT40/Syk Ϫ / Syk, and DT40/Syk Ϫ /Syk(K Ϫ ) cells were immunoprecipitated with anti-Syk antibody and divided into two portions. One was used for the Syk immunocomplex kinase assay (top), and the other was used for immunoblot analysis using an anti-Syk antibody (bottom). B, DNA fragmentation assay. Extracted DNA was analyzed on a 2% agarose gel, containing ethidium bromide, to detect DNA laddering. IP, immunoprecipitation; IB, immunoblot. 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 stressinduced 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 Ϫ cells leads to an apoptotic response very similar to that observed in wild-type DT40 cells. Enhanced apoptosis in DT40/Syk Ϫ 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)(24)(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 Sykdeficient 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 irradiationinduced 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 func- tions 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 Ϫ cells. Second, the enhanced DNA fragmentation in DT40/Syk Ϫ cells does not occur in DT40/Lyn Ϫ cells under the same condition. Third, overexpression of porcine Syk in DT40/Syk Ϫ 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.