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The Journal of Immunology, 2001, 166: 1641-1649.
Copyright © 2001 by The American Association of Immunologists

Prevention of Anti-IgM-Induced Apoptosis Accompanying G1 Arrest in B Lymphoma Cells Overexpressing Dominant-Negative Mutant Form of c-Jun N-Terminal Kinase 11

Eiko Takada, Hiroko Toyota, Jun Suzuki and Junichiro Mizuguchi2

Department of Immunology and Intractable Disease Research Center, Tokyo Medical University, Tokyo, Japan


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
A family of mitogen-activated protein (MAP) kinases comprising the extracellular signal-regulated kinases (ERKs), c-Jun N-terminal kinases (JNKs), and p38 MAP kinases are involved in proliferation and apoptosis. However, there are some arguments concerning the role of these kinases in Ag-induced B cell apoptosis. Two of the B lymphoma cell lines (CH31 and WEHI-231) susceptible to anti-IgM-induced apoptosis were used as a model. To address these issues, we examined the kinetics of anti-IgM-induced activation of MAP kinases and established cell lines overexpressing a dominant-negative (dn) mutant form of JNK1 (dnJNK1). Anti-IgM induced a sustained JNK1 activation with a peak at 8 h, with a marginal activation of ERK1/ERK2 in CH31 cells. The sustained JNK1 activation was not a secondary event through a caspase activation. The peak point of the JNK1 activation was just before the onset of a decline in mitochondrial membrane potential, which preceded anti-IgM-induced cell death. Following anti-IgM stimulation, dnJNK1 prevented a decline in mitochondrial membrane potential at 24 h, with a prolonged inhibition up to 72 h in WEHI-231, although it did so only partially during a later time period in CH31. The dnJNK1 cells also demonstrated diminished procaspase-3 activation and a decreased rate of apoptosis upon anti-IgM stimulation, with a concomitant increased arrest in G1 phase, which could be explained by enhanced levels of cyclin-dependent kinase inhibitor p27Kip1 protein. Thus, anti-IgM-induced JNK activation might be implicated in cell cycle progression as well as in apoptosis regulation, probably involving p27Kip1 protein.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Interaction of membrane Ig (mIg)3 with self-Ag contributes to an elimination of self-reactive B cells at several developmental stages including immature and germinal center B cells (1, 2), probably through induction of apoptosis. B lymphoma cell lines CH31 and WEHI-231, representing an immature B cell, have been widely used as a model for analysis of Ag-induced B cell unresponsiveness (3, 4, 5, 6, 7, 8) because cross-linking of mIg results in an accumulation of cells in late G1, followed by apoptotic cell death.

Mitogen-activated protein (MAP) kinases play a crucial role in various cellular responses including apoptosis, proliferation, and differentiation (9, 10, 11). The MAP kinase family comprises three distinct species of kinases: extracellular signal-regulated kinases (ERKs), c-Jun N-terminal kinases (JNKs), and p38 MAP kinases. Although these kinases are differentially activated in response to a variety of stimuli, signal transduction pathway(s) responsible for induction of apoptosis in B lymphocytes as well as in other cell types still remain controversial (11, 12, 13, 14, 15, 16, 17). For example, Sutherland et al. (15) showed that activation of ERK, occurring within 30 min after anti-IgM stimulation, paralleled anti-IgM-induced apoptosis in WEHI-231 cells. The correlation of ERK2 activation with cell death was also supported by the observation that MAP phosphatase-1 (MKP-1) interfered with both anti-IgM-induced ERK2 activation and apoptosis (14). In contrast, a slow and sustained increase in JNK activity correlated with anti-IgM-induced apoptosis in the human B lymphoma cell line B104 (12, 13), suggesting that JNK activation is involved in mIg-mediated apoptosis. Such a close association of JNK activation with induction of apoptosis has also been reported in response to certain stimuli, such as irradiation (17), growth factor withdrawal (11), and a chemotherapeutic agent (16).

Several pieces of evidence suggest the notion that mitochondria function as an integrator of a variety of proapoptotic stimuli (18). A decline in mitochondrial membrane potential ({Delta}{psi}m) is accompanied by an early phase of apoptosis in many situations (19, 20, 21) and is considered to be mediated by opening of the mitochondrial permeability transition pore, which is composed of a multiprotein complex located at the contact site between the inner and outer mitochondrial membranes (22). The decline in {Delta}{psi}m is preceded by a release of cytochrome c from mitochondria into the cytosol. The released cytochrome c, in conjunction with Apaf-1 and ATP, causes proteolytic activation of procaspase-9, resulting in activation of effector caspases such as caspase-3 and caspase-7 (23). The activated caspase-3 is considered to be involved in morphological changes and DNA degradation typical of apoptosis (24), probably through the cleavage of several substrates such as DNA fragmentation factor (25) and poly(ADP-ribose) polymerase (26).

There is growing evidence to suggest that induction of apoptosis is associated with the late G1 cell cycle checkpoint (27, 28). The G1 arrest can potentiate or prevent apoptosis (29, 30, 31). Progression from the G1 to the S phase is regulated by G1 cyclins that bind to cyclin-dependent kinases (CDKs), resulting in the activated kinases that phosphorylate substrates required for the progression (32). The cyclin/CDK activity is markedly affected by CDK inhibitor proteins such as p27Kip and p21Cip1 (33). Thus, anti-IgM-induced arrest in late G1 accompanies increased amounts of p27Kip1 protein, leading to the prevention of cyclin A- or cyclin E-dependent CDK2 activity required for G1/S transition (34, 35).

To address whether mIg-induced JNK activation leads to apoptosis, we used cell lines overexpressing the dominant-negative (dn) mutant form of JNK1 (dnJNK1). Our results support the notion that JNK activation is implicated in mIg-induced apoptosis as well as in cell cycle progression. Possible mechanisms underlying mIg-induced apoptosis will be discussed.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell lines

B lymphoma cells CH31 and WEHI-231 were maintained in RPMI 1640 medium supplemented with 10% (v/v) FBS, 50 µM 2-ME, 2 mM glutamine, and 100 µg/ml kanamycin at 37°C in humidified air with 5% CO2. CH31 cells were obtained from Dr. Geoffrey Haughton (University of North Carolina, Chapel Hill, NC) (36).

Cells were stimulated with anti-IgM for various time periods, as described below. Preliminary dose-response analysis showed that 10 µg/ml Bet-1 and 1 µg/ml Bet-2 were optimal for induction of apoptosis in WEHI-231 and CH31, respectively (data not shown). The hybridoma cell lines Bet-1 and Bet-2 were a gift from Dr. William E. Paul (National Institutes of Health, Rockville, MD) (37), and Ab was purified from ascites of scid mice by ammonium sulfate precipitation. The other reagents are of standard grade unless otherwise stated.

Flow cytometric analysis of apoptosis, {Delta}{psi}m, and membrane IgM

To evaluate apoptosis, hypodiploid DNA was analyzed using a flow cytometer (FACScalibur, Nippon Becton Dickinson Company, Tokyo, Japan) and CellQuest software (Becton Dickinson Immunocytometry Systems, San Jose, CA) as previously described (38). Apoptotic cell nuclei containing subdiploid DNA were enumerated as a percentage of the total population. To determine {Delta}{psi}m, fluorochrome 3,3'dihexyloxycarbocyanine iodide (DiOC6) (Molecular Probes, Eugene, OR), which incorporates into mitochondria and emits the spectrum of green light (525 nm), was used as previously described (21). Briefly, the cells (1 x 106), stimulated with or without anti-IgM in six-well plates (152795; Nunc, Naperville, IL) for various times, were loaded with DiOC6 (40 nM in RPMI 1640 medium) for 30 min at 37°C, followed by analysis on a flow cytometer. As a positive control for dissipation of {Delta}{psi}m, cells were incubated for 30 min in RPMI 1640 medium at 37°C and 5% CO2 with the uncoupling reagent carbonyl cyanide m-chlorophenylhydrazone (mCICCP; 50 µM) (Sigma, St. Louis, MO), a protonophore that disrupts {Delta}{psi}m. In some experiments, the DiOC6-labeled cells were washed twice and then labeled simultaneously with propidium iodide (PI; 50 µM) (Sigma) for 15 min at room temperature, followed by analysis on a flow cytometer. The PI fluorescence, detected at 620 nm, served as a measure for the percentage of loss of membrane integrity. To evaluate membrane IgM density, cells were stained with anti-IgM (Bet-2), followed by incubation with fluorescein-labeled mouse anti-rat Ab (39), and analyzed using the flow cytometer.

In vitro immune complex kinase assay

In vitro immune complex kinase assay was performed as previously described (39). Briefly, cells cultured with or without anti-IgM for various times were solubilized in a lysis buffer (10 mM Tris-HCl (pH 7.4)/0.1% SDS/1% Triton X-100/1% sodium deoxycholate/1 mM Na3VO4/4 mM EDTA/10 µg/ml leupeptin/1 mM PMSF/10 µg/ml aprotinin). The lysates, incubated with primary Ab (anti-JNK1/JNK2, anti-ERK1/ERK2) overnight at 4°C, were immunoprecipitated with protein G-agarose (Life Technologies, Rockville, MD): rabbit anti-JNK1(C17)/anti-JNK2(N18) Abs (Santa Cruz Biotechnology, Santa Cruz, CA); mouse anti-human ERK1 mAb and anti-rat ERK2 mAb (Transduction Laboratories, Lexington, KY). The immunoprecipitates for each kinase were suspended in kinase buffer (20 mM HEPES (pH 7.4)/100 mM NaCl/5 mM MnCl2/5 mM MgCl2) containing 10 µCi of [{gamma}-32P]ATP (3000 Ci/mmol; NEN Life Science Products, Boston, MA) in the presence of substrates (myelin basic protein, GST-c-Jun, respectively) and then incubated for 30 min at 30°C. Myelin basic protein was obtained from Upstate Biotechnology (Lake Placid, NY), and GST-c-Jun 1–79(1–79) cDNA was obtained from Dr. Roger J. Davis (University of Massachusetts Medical Center, Howard Hughes Medical Institute, Worcester, MA). The samples were separated on SDS-PAGE, followed by autoradiography. Levels of phosphates incorporated into proteins were measured by BAS 2000 (Fuji Photo Film, Tokyo, Japan).

Western blotting

Western blotting was done as previously described by Yanase et al. (38). Briefly, samples were separated by 12.5% SDS-PAGE. Proteins were transferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA), blocked with 5% nonfat dry milk, and then washed with PBST buffer (PBS/0.5% nonfat dry milk/0.05% Tween 20). The membranes were blotted with primary Abs: anti-ERK1/ERK2, anti-JNK1, mouse anti-p27Kip1 mAb (Transduction Laboratories, Lexington, KY), and rabbit anti-actin (Sigma). The bound primary Abs were then incubated with secondary Abs: HRP-labeled goat anti-rabbit IgG Fc Ab (EY Laboratories, San Mateo, CA) or HRP-goat anti-mouse IgG Ab (Cappel Research Products, Durham, NC). After washing, membrane-bound HRP-conjugated Ab was visualized with ECL (Amersham Life Science, Buckinghamshire, U.K.).

Caspase-3 assay

Caspase-3 activity was assayed by colorimetric protease assay kit according to the manufacturer’s instructions (MBL, Nagoya, Japan). Briefly, anti-IgM-stimulated cells were suspended in a lysis buffer, kept on ice for 10 min, and then centrifuged. The supernatants were collected for the assay. Enzyme reaction was performed in a buffer containing supernatant proteins (100 µg/sample) and caspase substrate DEVD-p-nitroanilide (pNA) at 37°C for 2 h, followed by colorimetric detection of pNA at a wavelength of 405 nm using a microplate reader (Bio-Rad, Hercules, CA). In some experiments, the cells were pretreated with a caspase inhibitor peptide, benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (Z-VAD-fmk) (Enzyme Systems Products, Livermore, CA) for 1 h before the anti-IgM stimulation.

Construction of a JNK expression plasmid

Flag-JNK1 cDNA was kindly provided by Dr. Roger Davis (University of Massachusetts Medical Center, Howard Hughes Medical Institute) (40). The dnJNK1 mutant was created by replacing Thr183 and Tyr185 with Ala and Phe, respectively, using a PCR-based Quickchange site-directed mutagenesis kit (Stratagene, La Jolla, CA) according to the manufacturer’s instructions. Both wild-type (wt) Flag-JNK1 and Flag-dnJNK1 cDNA were subcloned at XhoI and NotI into the expression vector pMKIT-Neo, which was generously provided by Dr. K. Maruyama (Tokyo Medical and Dental University, Tokyo, Japan). Recombinant DNA was sequenced to confirm the fidelity of each construct.

Transfections and generations of stable cell lines overexpressing dnJNK1

CH31 and WEHI-231 cells were transfected by electroporation using gene pulser (270 V, 960 µF; Bio-Rad) with 30 µg of either pMKIT-Neo-Flag-JNK1, pMKIT-Neo-Flag-dnJNK1, or vector-alone control (Neo) plasmids. Transfectants (2.5 x 104 cells/well) were cultured in 96-well plates for 48 h and then selected in the RPMI 1640 medium with Geneticin (G418, 500 µg/ml) (Life Technologies). Several individual clones were obtained by limiting dilution. The level of Flag-JNK1 protein was determined by Western blotting, as described below.

Statistical analysis

Data were expressed as means ± SD for each group. Statistical significance was determined by Student’s t test, and a difference of p < 0.05 was considered to indicate statistical significance.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Stimulation of CH31 B lymphoma cells with anti-IgM induced apoptosis and reduced {Delta}{psi}m

CH31 B lymphoma cells were cultured with 1 µg/ml of anti-IgM (Bet-2) for various times and were assayed for apoptosis and cell cycle analysis by the PI staining method. A careful analysis of the cell cycle revealed that a slightly increased proportion of cells in the G1 phase was obtained at 14 h (53.3 ± 4.5% vs 38.3 ± 1.8% in medium alone), with a subsequent decline through a concomitant increase in apoptotic cells after anti-IgM stimulation (Fig. 1Go). These findings confirmed the previous observations that CH31 cells are similar to WEHI-231 in terms of an occurrence of G1 arrest, accompanied by induction of apoptosis (4, 5, 6, 7).



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  		FIGURE 1. Kinetics of anti-IgM-induced G1 arrest and apoptosis in CH31 cells. The CH31 cells were stimulated with 1 µg/ml anti-IgM for various time periods, fixed with ethanol, and then stained with PI. The labeled cells were analyzed using a FACScan flow cytometer. The percentage of apoptosis or G1 phase was determined: (number of cells in G1 or apoptosis/number of total cells) x 100. Each value is the mean ± SD of two experiments done in triplicate. *, Significantly different from medium control.

 
Apoptosis has been demonstrated to be accompanied by a decline in {Delta}{psi}m in several cell types (18). Therefore, we examined the {Delta}{psi}m using amphophilic cationic fluorochrome DiOC6. Cells were loaded with DiOC6, and retention of fluorescence was then monitored in a flow cytometer. To confirm that DiOC6 dye was sensitive to {Delta}{psi}m, unstimulated cells were treated with mitochondrial uncoupling agent mCICCP (50 µM) for 30 min. The mCICCP-treated cells showed a shift to weaker fluorescence than control cells (Fig. 2GoA, left), suggestive of a decline in {Delta}{psi}m, as is usually observed in apoptotic cells (41). A decrease in {Delta}{psi}m was detected in a significant proportion of cells at 20 h (36.9% vs 6.1% in medium alone) after anti-IgM stimulation (Fig. 2GoA, right). To examine more carefully the sequential relationship between a decrease in {Delta}{psi}m and cell death, the stimulated cells were loaded with DiOC6, followed by incubation with PI. The cells that failed to exclude the PI were considered dead (PI+), reflecting loss of plasma membrane integrity. More than 90% of the unstimulated cells were PI-negative/{Delta}{psi}m-high (PI-/{Delta}{psi}mhigh) (Fig. 2GoB). Following anti-IgM stimulation, the cells displayed a decreased {Delta}{psi}m with intact plasma membrane integrity (PI-/{Delta}{psi}mlow) at 9 h (4.0 ± 1.0% vs 2.1 ± 0.6% in medium alone), with the percentage reaching 11.5 ± 1.6% at 12 h, accompanied by a concomitant decrease in PI-/{Delta}{psi}mhigh cells. The percentage of both PI+/{Delta}{psi}mlow and PI+/{Delta}{psi}mhigh cells was almost unaltered by anti-IgM during the observed periods. These findings suggest that the anti-IgM-induced decline in {Delta}{psi}m precedes cell death.



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  		FIGURE 2. Anti-IgM-induced decline in {Delta}{psi}m. The cells stimulated with anti-IgM for various time periods were loaded with DiOC6, and fluorescence was monitored using a flow cytometer. A, Histogram of DiOC6. Unstimulated cells were treated with mCICCP for 30 min to ascertain that DiOC6 dye was sensitive to {Delta}{psi}m (left). The cells stimulated with anti-IgM for 20 h showed a shift to weaker fluorescence, representing a decline in {Delta}{psi}m (right). B, Simultaneous detection of {Delta}{psi}m and loss of plasma membrane integrity (cell death) in anti-IgM-stimulated cells. The anti-IgM-stimulated cells were loaded with DiOC6 and then PI, followed by flow cytometric analysis. The percentage of cells was determined as described in Fig. 1Go. The experiments were done two to three times, with identical results.

 
Anti-IgM induces a sustained JNK1 activation, but only a meager ERK1/ERK2, in CH31 cells

Activation of a family of MAP kinases following mIg engagement has been reported in WEHI-231 and B104 cells (12, 13, 14, 15, 42). To assess the role of these kinases in anti-IgM-induced apoptosis, we examined the activity of both JNK1 and ERKs 1/2 at various time points following anti-IgM stimulation. JNK1 activity was assayed by phosphorylation of a GST-c-Jun fusion protein as in vitro substrate. Anti-IgM substantially enhanced JNK1 activity at 15 min, with a peak at 8 h followed by a decline to levels of unstimulated cells (Fig. 3GoA, left). These data suggest that anti-IgM induces a prolonged JNK1 activation, with the end point being just before the onset of the decline in {Delta}{psi}m in CH31 cells (Figs. 2GoB and 3A). In contrast, ERK1 and ERK2 activities in the anti-IgM-stimulated CH31 cells were only slightly elevated compared with those in the WEHI-231 cells (Fig. 3GoA). Such a slightly increased activity of the ERKs in the stimulated cells was confirmed by Western blotting using anti-pERK mAbs (our unpublished observation). WEHI-231 cells also displayed a prolonged elevation of JNK1 activity, with a peak level at 12 h as described previously (43). The levels of JNK1 and ERKs 1/2 proteins were unaltered during the observed periods in both cell lines, as assessed by Western blotting (Fig. 3GoB, and Ref. 43). These results indicate that a sustained increase in JNK1 activity is obtained in the anti-IgM-stimulated cells, whereas ERK activation is only transient or meager in its extent.



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  		FIGURE 3. Kinetics of anti-IgM-induced activation of ERK1/ERK2 and JNK1. The CH31 and WEHI-231 cells were stimulated with anti-IgM for various time periods and lysed with a Triton X-100 lysis buffer. The lysates were immunoprecipitated with anti-ERK1, anti-ERK2, or anti-JNK1 Abs. A, Time course of kinase activity of ERK1/ERK2 and JNK1 in response to anti-IgM. Kinase assay was done as described in Materials and Methods. The data were expressed as fold increase in enzymatic activities compared with unstimulated controls. B, Levels of ERK1/ERK2 and JNK1 proteins. Protein levels were determined by Western blotting. Experiments were done three times, with identical results.

 
Pretreatment with Z-VAD-fmk does not affect anti-IgM-induced JNK activation

To address whether the anti-IgM-induced sustained JNK1 activation is secondary to apoptotic stress such as activation of caspases (44), we examined the effect of pan-caspase inhibitor Z-VAD-fmk on the anti-IgM-induced JNK1 activation. The cells pretreated with 20 µM Z-VAD for 1 h were stimulated with anti-IgM for 15 min or 8 h and were assayed for JNK1 activity using an in vitro kinase assay. The Z-VAD pretreatment did not affect the increase in JNK1 activity (Fig. 4GoA), suggesting that JNK activation is not mediated through caspase activation. As expected, such pretreatment prevented caspase-3 activation and induction of apoptosis after anti-IgM stimulation (Fig. 4Go, B and C, and Refs. 45, 46, 47). These findings suggest that caspase activation is not required for the mIg-induced JNK1 activation.



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  		FIGURE 4. Pretreatment with Z-VAD-fmk fails to prevent anti-IgM-induced JNK1 activation, whereas the inducible apoptosis is blocked in CH31 cells. The CH31 cells pretreated with Z-VAD for 1 h were stimulated with anti-IgM for 15 min plus 8, 20, or 24 h and then assayed for JNK1 activation (A), caspase-3 activation (B), and apoptosis (C), respectively. The caspase-3 activity was expressed as absorbance of pNA light at a wavelength of 405 nm. The experiments were done three times, with identical results.

 
Expression of dnJNK1 in both CH31 and WEHI-231

To address whether an anti-IgM-induced increase in JNK activity is involved in the induction of apoptosis and a reduction in {Delta}{psi}m, we used dnJNK1 because levels of JNK1 expression were greater than those of JNK2 in the cell lines used, as assessed by Western blotting (our unpublished observation). CH31 cells transfected with a Flag-dnJNK1 expression vector pMKIT-Neo-Flag-dnJNK1(T183A/Y185F) or control Neo were selected by incubation with G418. Several independent transformants were isolated and assayed for Flag-dnJNK1 level by Western blotting. Flag-dnJNK1 protein was detected at almost the same, or higher, levels than endogenous JNK1 in three clones (CH #5, CH #8, and CH #12), but not controls (wt, Neo) (Fig. 5GoA, left). The membrane IgM densities of the three dnJNK cells were almost identical with that of control cells (data not shown). WEHI-231 clones overexpressing Flag-dnJNK1 (WEHI #9, WEHI #10) were also established using the same procedure, with the IgM density almost identical with that of control cells (Fig. 5GoA, right, and data not shown). The transformants overexpressing wt Flag-JNK1 have not yet been isolated, possibly through an elimination of cells with overexpressed wt JNK1 during a selection procedure because of a possible proapoptotic nature of JNK (our unpublished observation). These five clones overexpressing Flag-dnJNK1 were used in the following experiments.



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  		FIGURE 5. CH31 and WEHI-231 cell lines overexpressing Flag-dnJNK1 (T183A/Y185F). CH31 and WEHI-231 cells were transfected with pMKIT-Neo-Flag-dnJNK1 or control Neo. Transfectants were selected with RPMI 1640 medium containing G418, followed by limiting dilution to obtain an individual clone. A, Cell lines with overexpressed Flag-dnJNK1. Levels of Flag-dnJNK1 and endogenous JNK1 proteins in individual clones (CH #5, CH #8, CH #12 (left); WEHI #9, WEHI #10 (right)) and control Neo were determined by Western blotting. B, Anti-IgM-induced endogenous JNK1 activity is severely impaired in the CH-dnJNK1 (left) and WEHI-dnJNK1 cells (right). JNK1 precipitates from the cells stimulated with anti-IgM for the optimal time were used for in vitro immune complex kinase assay using a substrate GST-c-Jun. The experiments were performed twice, with identical results.

 
dnJNK1 prevents anti-IgM-induced endogenous JNK activation, reduced {Delta}{psi}m, caspase-3 activation, and apoptosis in both CH31 and WEHI-231 cells

Following anti-IgM stimulation, all the clones (CH #5, CH #8, CH #12, WEHI #9, and WEHI #10) overexpressing Flag-dnJNK1 demonstrated a decreased JNK1 activity compared with controls (Neo, wt), as assessed by in vitro kinase assay (Fig. 5GoB), indicating that dnJNK1 prevents mIg-induced endogenous JNK1 activation.

Therefore, we tested whether dnJNK1 affects anti-IgM-induced {Delta}{psi}m. The decline in {Delta}{psi}m upon anti-IgM stimulation was substantially inhibited at 24 h in all five dnJNK1 cells compared with the controls (Fig. 6GoA). Interestingly, the WEHI-dnJNK1 cells exhibited marked inhibition of the anti-IgM-induced decline in {Delta}{psi}m even during later time points (48–72 h) (Fig. 6GoA, right), whereas the CH-dnJNK1 cells showed only a partial inhibition during 36–48 h (Fig. 6GoA, left).



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  		FIGURE 6. Prevention of anti-IgM-induced decline in {Delta}{psi}m, caspase-3 activation, and apoptosis, accompanied by enhanced G1 arrest, in both CH-dnJNK1 (left) and WEHI-dnJNK1 cells (right). The dnJNK1 and control cells were assayed for {Delta}{psi}m (A), caspase-3 activity (B), apoptosis (C), and G1 phase (D) at various time points after anti-IgM stimulation. The percentage was determined as described in Fig. 1Go. Each point was performed in triplicate, and the results were represented as the mean ± SD. Essentially similar results were obtained from two other experiments.

 
Because dysfunction of {Delta}{psi}m provides caspase activation (18, 45, 48, 49), dnJNK1 and control cells stimulated with anti-IgM were lysed and assayed for proteolytic caspase-3 activity using a substrate DEVD-pNA. The anti-IgM-induced caspase-3 activation was substantially prevented in all the dnJNK cells (Fig. 6GoB).

Because caspase activation is implicated in anti-IgM-induced apoptosis, the anti-IgM-induced apoptosis was examined in both the dnJNK and control cells. The dnJNK1 markedly prevented the anti-IgM-induced apoptosis in both dnJNK1 cell lines (Fig. 6GoC), with the kinetics similar to that of the decline in {Delta}{psi}m. Altogether, dnJNK1 inhibits anti-IgM-induced decline in {Delta}{psi}m, procaspase-3 activation, and apoptosis in B lymphoma cells.

Increase in G1 arrest in dnJNK1 cells following anti-IgM stimulation, accompanied by elevation of p27Kip1

Induction of apoptosis has been shown to be related to the cell cycle phase (27, 28). Therefore, we determined the cell cycle position in the dnJNK1 cells after anti-IgM stimulation. Anti-IgM-stimulated CH31 and WEHI-231 cells demonstrated G1 arrest around 14 and 24 h, respectively, followed by a decreased proportion in G1 phase as a result of a concomitant rise in a fraction of apoptosis (Figs. 1Go and 6GoD). Interestingly, a marked increase in the proportion of phase G1 was obtained during 24–36 h in all the dnJNK1 cells after anti-IgM stimulation. The increased proportion was maintained up to 72 h in the WEHI-dnJNK1 cells, whereas it started to decline at 48 h in CH-dnJNK1 cells, reflecting a concomitant rise in apoptotic cells (Fig. 6Go, C and D).

Because anti-IgM-induced G1 arrest in WEHI-231 cells has been shown to be accompanied by an increase in levels of p27Kip1 protein (34, 35), p27Kip1 levels in the dnJNK1 cells upon mIg engagement were examined. An increase in the levels of p27Kip1 was confirmed in both the wt CH31 (around 12–18 h) and WEHI-231 cells (at 24 h) after anti-IgM stimulation (Fig. 7Go, upper and lower panels), and then the levels declined to baseline levels at 24 h (CH31) or 48 h (WEHI-231). Interestingly, the enhanced levels of p27Kip1 were maintained up to later time points (24 and 48 h) in the CH- and WEHI-dnJNK1 cells, respectively (Fig. 7Go). Altogether, dnJNK1 might promote the anti-IgM-induced growth arrest in G1 phase, at least in part through the maintenance of a heightened level of p27Kip1 protein.



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  		FIGURE 7. Enhancement of p27Kip1 protein levels in the anti-IgM-stimulated dnJNK1 cells. The dnJNK1 and control cells stimulated with anti-IgM for various time periods were assayed for p27Kip1 level by Western blotting. Upper panel, CH31 cells and their derivatives; lower panel, WEHI-231 cells and their derivatives. The p27Kip1 levels were normalized to actin levels and expressed as a fold of the p27Kip1 level from unstimulated cells. The experiments were performed three times, with identical results.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Engagement of mIg in B cells has been implicated in the induction of self-tolerance as well as activation (50, 51). B cell tolerance is thought to be mediated by deletion (apoptosis) or anergy (1, 2), and the molecular mechanisms underlying mIg-induced apoptosis remain elusive. Because mIg engagement results in apoptosis in CH31 and WEHI-231 mouse B lymphoma cell lines representing immature cells (5, 6, 7, 8), they would be suitable for analysis of B cell tolerance on a molecular level.

A family of MAP kinases has recently been proposed to be involved in mIg-induced apoptosis in B cells as well as other cell types. However, there are some arguments as to which component(s) of the kinases are implicated in the induction of apoptosis (9, 10, 11, 12, 13, 14, 17, 42, 52, 53). Thus, anti-IgM-induced enhancement of ERK2 activation correlated with the induction of apoptosis in WEHI-231 cells (14). In contrast, Graves et al. (12, 13) have observed that anti-IgM-induced sustained JNK activation paralleled the induction of apoptosis. To address whether JNK activation is required for anti-IgM-induced apoptosis, we employed a genetic approach in using dnJNK1. Both the CH31 and WEHI-231 cells overexpressing dnJNK1 failed to display a decline in {Delta}{psi}m and procaspase-3 activation by anti-IgM, compared with controls (Fig. 6Go, A and B). The dnJNK1 cells also demonstrated a decreased rate of apoptosis, with a concomitant arrest in G1 phase, which might be provided by elevation of p27Kip1 level (Figs. 6Go, C and D, and 7).

The duration of MAP kinase activity is thought to be crucial for cell fate, whether proliferation, apoptosis, or differentiation (11, 54). Anti-IgM stimulation markedly induced a transient activation of ERK2 in WEHI-231 cells (Fig. 3GoA, right, and Refs. 14, 15 , and 42). The transient ERK2 activation was suggested to affect anti-IgM-induced apoptosis because the MKP1-mediated blockade of ERK2 activation prevented the anti-IgM-induced apoptosis (14). However, it should be noted that MKP-1 recognizes ERK2, JNK, and p38 MAP kinase, not a specific inhibitor of ERK2 (55). Unlike WEHI-231 cells, only a meager increase in ERK2 activation was observed in CH31 cells after anti-IgM stimulation (Fig. 3GoA, left). Moreover, in our preliminary experiments, the addition of specific MKK (an upstream activator of ERK) inhibitor PD98059 did not prevent the anti-IgM-mediated apoptosis (data not shown) in CH31 cells, suggesting that ERK2 activation does not appear to result in induction of apoptosis. Additional experiments are required to verify the involvement of ERK2 activation in mIg-induced apoptosis in B lymphoma cells.

Instead of a transient activation of ERK2, a prolonged activation of JNK might be involved in mIg-mediated apoptosis, as claimed by Graves et al. (13) using human B lymphoma cell line B104. Consistent with this notion, engagement of mIg in both cell lines CH31 and WEHI-231 induced a sustained increase in JNK1 activity, with a peak time point before the onset of a decreased {Delta}{psi}m and apoptosis in CH31 (Figs. 1Go, 2GoB, and 3A) and WEHI-231 cells (43). Furthermore, dnJNK1 prevented the anti-IgM-induced apoptosis (Fig. 6GoC), suggesting that a sustained JNK activation is involved in the apoptosis in response to mIg engagement. It is also conceivable that JNK activation relative to that of ERK is critical for the apoptosis in B cells, as proposed by Xia in an apoptosis model of PC12 cells following withdrawal of growth factor (11).

The induction of apoptosis is thought to be triggered by mitochondria; the opening of the mitochondrial transition pore, as revealed by a reduction in {Delta}{psi}m, occurs in various cell lines undergoing apoptosis (18, 19, 20, 41). The present study confirmed the previous observation that anti-IgM stimulation induces a decline in {Delta}{psi}m in B lymphoma cells (21). The decline in {Delta}{psi}m in response to anti-IgM occurred before the induction of cell death (PI-positive) (Fig. 2GoB), reflecting a loss of plasma membrane integrity, in terms of kinetics, as reported in staurosporin-mediated apoptosis in CEM cells (56). Moreover, dnJNK1 blocked the anti-IgM-induced {Delta}{psi}m (Fig. 6GoA), suggesting that JNK1 activation affects the {Delta}{psi}m in B cells.

A decline in {Delta}{psi}m is preceded by the release of cytochrome c from mitochondria into the cytosol in a variety of cells after apoptotic stimuli (18). The cytosolic cytochrome c, in conjunction with Apaf-1, has been clearly demonstrated to activate procaspase-9 in the presence of ATP (23), leading to an activation of effector caspases such as caspase-3 in B cells as well as other cell types (24, 46, 47, 48). The anti-IgM-induced procaspase-3 activation was considerably prevented in all the dnJNK1 cells (Fig. 6GoB). Moreover, the pretreatment with pan-caspase inhibitor ZVAD-fmk inhibited anti-IgM-induced apoptosis, but not the decline in {Delta}{psi}m (Fig. 4GoC, our unpublished observation, and Refs. 45 and 47), suggesting that caspase activation is required for anti-IgM-induced apoptosis, but not mitochondrial membrane dissipation. Altogether, the following sequence of events might be suggested in the anti-IgM-stimulated B cells: JNK1 activation -> mitochondrial dysfunction -> caspase activation -> apoptosis.

The prolonged activation of JNK1 is not due to the stress of the cell death that can activate JNK, because JNK activation was still observed in the presence of caspase inhibitor Z-VAD-fmk in CH31 (Fig. 4GoA) and WEHI-231 cells (our unpublished observation). In contrast to our findings, Graves et al. (12) reported the Z-VAD-sensitive JNK activation in human B104 cells after anti-IgM stimulation. The difference between their observations and ours might be explained by the following: the JNK activation occurred before mitochondrial membrane dissipation and apoptosis in our system, whereas they observed Z-VAD-sensitive JNK activation in cells undergoing apoptosis, which may represent a secondary feedback amplification due to apoptotic stress such as caspase activation (57). Such a late induction of JNK activation was also observed in Fas (CD95)-induced cell death (44), where large amounts of caspase-8 activation is initiated (58). Although the activated caspase(s) can result in JNK activation through MAP/ERK kinase-1 activation (59), mIg-induced apoptosis does not involve CD95/CD95 ligand (60, 61).

It is thought that the induction of apoptosis is closely linked with cell cycle control because several proteins that can induce apoptosis are in fact implicated in the regulation of the cell cycle progression (27, 62, 63). Although it remains unclear whether JNK activation plays a role in cell cycle progression, it is possible that dnJNK1 somehow enhances anti-IgM-induced accumulation of G1 phase with a concomitant increase in p27Kip1 levels, which may delay the anti-IgM-induced apoptosis. Alternatively, the dnJNK1-mediated prevention or delay of anti-IgM-induced apoptosis may be responsible for the observed accumulation of G1 involving enhanced p27Kip1. The precise role of JNK activation in the different consequences is currently unknown.

The dnJNK1 provided a partial inhibition of anti-IgM-induced apoptosis in CH31 compared with WEHI-231 cells (Fig. 6GoC). Although both cell lines behaved similarly in the sequence of events (G1 arrest and then apoptosis) after anti-IgM stimulation, there are some quantitative differences: WEHI-231 cells showed slightly less sensitivity to anti-IgM (64), a more pronounced G1 arrest, and somewhat higher levels of p27Kip1 protein (around 2-fold) (Fig. 7Go, and our unpublished observation) than CH31 cells. The levels of p27Kip1 protein may at least partly contribute to the differential features among the two cell lines and their derivatives because cells lacking p27Kip1 protein displayed marked sensitivity to apoptosis (65), whereas those overexpressing p27Kip1 protein delayed it (29, 31).

Taken together, we show here that JNK activation might be implicated in anti-IgM-induced apoptosis in mouse B lymphoma cell lines CH31 and WEHI-231. Although the present study does not clarify how JNK activation regulates the cell cycle progression and/or mitochondrial function, our observations would provide some understanding of mIg-mediated apoptosis, and hopefully also have implications for the understanding of self-tolerance of B cells.


   Acknowledgments
 
We thank Masae Furuhata and Prof. J. Patrick Barron (International Medical Communications Center, Tokyo Medical University, Tokyo, Japan) for technical assistance and reading of the manuscript, respectively. We also thank Dr. Geoffrey Haughton (University of North Carolina, Chapel Hill, NC), Dr. William E. Paul (National Institutes of Health, Rockville, MD), Dr. Roger Davis (University of Massachusetts Medical Center, Howard Hughes Medical Institute, Worcester, MA), and Dr. K. Maruyama (Tokyo Medical and Dental University) for providing CH31 line, hybridoma cell lines Bet-1/Bet-2, Flag-JNK1 cDNA and GST-c-Jun (1–79) cDNA, and expression vector pMKIT-Neo, respectively.


   Footnotes
 
1 This work was supported in part by a grant from the Intractable Disease Center, Tokyo Medical University, Tokyo, Japan. Back

2 Address correspondence and reprint requests to Dr. Junichiro Mizuguchi, Department of Immunology and Intractable Disease Research Center, Tokyo Medical University, 6-1-1 Shinjuku, Shinjuku-ku, 160-0022, Tokyo, Japan. Back

3 Abbreviations used in this paper: mIg, membrane Ig; MAP, mitogen-activated protein; ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; MKP-1, MAP phosphatase-1; {Delta}{psi}m, mitochondrial membrane potential; CDK, cyclin-dependent kinase; dn, dominant-negative; DiOC6, 3,3'dihexyloxacarbocyanine iodide; mCICCP, carbonyl cyanide m-chlorophenylhydrazone; PI, propidium iodide; pNA, p-nitroanilide; Z-VAD-fmk, benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone; wt, wild type. Back

Received for publication November 5, 1999. Accepted for publication November 9, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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