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Departments of
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Microbiology and
Immunology, University of Washington, Seattle, WA 98195
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Quiescent B cells express a distinct set of genes from either anergic or activated B cells (5), suggesting that the decision to remain quiescent is an actively regulated process. A number of proteins with known or predicted inhibitory functions have been implicated in this process (5, 6). For example, lung Kruppel-like factor and special AT-rich sequence-binding protein 1 (SATB1)4 function as transcriptional repressors that maintain T cells in a quiescent state (7, 8). It remains unclear whether lung Kruppel-like factor or SATB1 plays a similar role in B cell homeostasis, which is regulated separately from T cell homeostasis (4).
Another mechanism known to contribute to the homeostatic regulation of B cell populations is programmed cell death or apoptosis. The main effectors of cell death are a family of cysteine proteases called caspases (reviewed in Refs. 9 and10). The best-characterized pathway for caspase activation is via the CD95/Fas receptor. Activated CD95/Fas trimers recruit the pro form of caspase-8 via the adapter protein Fas-associated death domain protein (FADD) (9, 10). The resulting oligomerization of pro-caspase-8 results in autoprocessing and activation. Caspase-8 then cleaves and activates downstream caspases such as caspase-3, -6, and -7. Once activated, caspases target cleavage of a highly specific and restricted group of cellular proteins that regulate cell morphology, signal transduction, and survival (9). Among the known targets of caspases are a number of proteins that function to regulate the cell cycle. These include the retinoblastoma protein (Rb), a critical regulator of cell cycle progression (11), and the cyclin-dependent kinase inhibitors, p21Cip1/Waf1 and p27Kip1 (12). Cleavage of these proteins contributes to cell cycle arrest and serves a permissive role in the induction of apoptosis.
Several reports suggest that caspases can function to regulate processes besides apoptosis, including proliferation and differentiation (13). One of the first hints that caspase-dependent pathways may regulate proliferation came from analyses of FADD-deficient mice and mice expressing dominant-negative FADD in the T cell compartment. Using both these approaches, it was shown that FADD is required for T cell activation and IL-2-dependent proliferation (14, 15, 16). Subsequent studies suggested a positive role for caspases during proliferation of CD3-stimulated normal human T cells (17, 18). These studies found that caspase-8 activity is associated with T cell proliferation, and that either a broad specificity caspase inhibitor or an inhibitor selective for caspase-8 could block proliferation. More recently, Chun et al. (19) identified a caspase-8 mutation in patients with autoimmune lymphoproliferative syndrome-related symptoms. T, B, and NK cells from these patients exhibited defects in activation consistent with a role for caspase-8 in lymphocyte activation. However, which caspases function downstream of caspase-8 and how caspases might act as positive regulators of the cell cycle has not been determined.
In this study we investigated the role of caspases in the activation of normal quiescent human B cells. We found that both caspase-8 and -6 activities increased following B cell activation by a variety of proliferative stimuli, and that inhibitors selective for these caspases blocked B cell proliferation. Inhibition of caspase-6 antagonized both mRNA and protein expression of cyclin-dependent kinase 4 (cdk4) and cyclin D, suggesting that resting B cells may require caspase-6 activity to enter into the G1 phase of the cell cycle. Neither CD40-mediated IL-6 production nor cellular inhibitor of apoptosis 2 (cIAP2) expression was affected by caspase inhibition, suggesting that caspase activity is required for cell cycle entry, but not cytokine or survival programs. We propose a model that caspases may regulate cell cycle entry by targeting proteins that actively maintain the quiescent state, thereby activating transcription of early cell cycle genes.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Dense tonsillar B cells were prepared as described previously (20, 21); briefly, E rosette-negative (Er-) cells were layered over a Percoll step gradient, and, after centrifugation, cells were isolated that were >60% Percoll density (>98% CD20+, >98% in G0 based on acridine orange staining). Mouse dense B cells were purified and stimulated as previously described (22). The G28-5 mAb to human CD40 (23) and the G28-8 mAb to Bgp95, recently designated CD180/RP105 (20, 24), were used alone or together to activate resting B cells. The 1C10 mAb to mouse CD40 was used to stimulate mouse B cells. In some experiments F(ab')2 of goat anti-mouse µ chains or goat anti-human µ sera (Jackson ImmunoResearch Laboratories, West Grove, PA) were used to stimulate B cells.
Reagents
The peptide caspase inhibitors, benzyloxycarbonyl (Cbz)-Val-Ala-Asp(Ome)-fluoromethylketone (ZVAD-fmk), N-acetyl-Val-Ala-Asp-al (ZVAD-CHO), benzyloxycarbonyl (Cbz)-Ile-Glu-Thr-Asp(Ome)-fluoromethylketone (IETD-fmk), benzyloxycarbonyl (Cbz)-Asp-Glu-Val-Asp(Ome)-fluoromethylketone (DEVD-fmk), N-acetyl-Asp-Glu-Val-Asp-al (DEVD-CHO), and benzyloxycarbonyl (Cbz)-Val-Glu-Ile-Asp(Ome)-fluoromethylketone (VEID-fmk), were purchased from Kamiya Biomedical (Seattle, WA). The caspase peptide substrates, acetyl Asp-Glu-Val-Asp 7-amido-4-methylcoumarin (Ac-DEVD-AMC), scetyl-Val-Glu-Ile-Asp-7-amino-4-methyl coumarin (Ac-VEID-AMC), and acetyl-Ile-Glu-Thr-Asp-7-amino-4-methyl coumarin (Ac-IETD-AMC), were purchased from Enzyme System Products (Livermore, CA). Human rIL-4 was obtained from R&D Systems (Minneapolis, MN).
Proliferation analysis
Dense tonsillar B cells were cultured at 1–2 x 106 cells/ml in triplicate at 37°C and 5% CO2 in complete RPMI medium (10% FCS). Cells were then stimulated with anti-CD180 (5 µg/ml), anti-CD40 (1 µg/ml), anti-µ (5 µg/ml), IL-4 (10 ng/ml), or combinations of stimuli. After 2 days cells were pulsed with 0.5 µCi of [3H]thymidine for 18–24 h (for human cells) or 8 h (for mouse cells). Identical conditions were employed for [3H]uridine incorporation. Cells were then harvested on to glass-fiber filters with a cell harvester, and radioactivity was measured in a liquid scintillation counter. In some experiments cells were pretreated for 3 h with caspase inhibitors, z-VAD-fmk, z-IETD-fmk, z-DEVD-fmk, and z-VEID-fmk, at doses ranging from 5–80 µM. As a control for vehicle, cells were treated with DMSO at the same concentration as the highest dose of inhibitor (80 µM).
Cell death analysis
Human tonsillar B cells (1 x 106 cells) were rinsed with cold PBS and then resuspended in 1 ml of PBS containing 0.1% Triton X-100, 0.1 mM EDTA, and 10 U RNase A. After 25 min at room temperature, 100 µl of propidium iodide (PI) solution (0.5 µg/ml) was added, and cells were incubated for another 5 min. Cells were then analyzed on a FACScan analyzer (BD Biosciences, Franklin Lakes, MJ) using the CellQuest program, and DNA content was measured. Cells with sub-G0/G1 DNA content were considered apoptotic.
Western blot analysis
Human tonsillar B cells were pretreated where indicated with caspase inhibitors for 3 h before stimulation. At the indicated time, 1.5 x 107 cells were rinsed with cold PBS and lysed in a buffer containing 1% SDS and 50 mM Tris, pH 7.6. Samples were passed through a 25-gauge needle 10 times, and then insoluble matter was removed by centrifugation. A small sample of the resulting supernatant was reserved for protein determination; the rest was diluted 1/1 in sample buffer containing 5% 
-ME and boiled for 5 min. The protein concentration of each sample was determined using a bicinchoninic acid assay (Pierce, Rockford, IL) with BSA as a standard. Equal amounts of total protein were loaded onto polyacrylamide gels, electrophoresed, and transferred to nitrocellulose. The blot was then immunostained using Abs specific for cyclin D3 (Upstate Biotechnology, Lake Placid, NY), caspase-3, caspase-8 (BD PharMingen, San Diego, CA), caspase-6 (MLB International, Watertown, MA), cyclin A, SATB1 (Santa Cruz Biotechnology, Santa Cruz, CA), phospho-Rb, phospho-extracellular signal-regulated kinase (phospho-ERK), Akt/protein kinase B (Cell Signaling Technology, Beverly, MA), or p27Kip1 (BD Biosciences). Briefly, the blot was first incubated in 5% nonfat dried milk in TBS-T (10 mM Tris (pH 7.6), 150 mM NaCl, and 0.1% Tween 20) for 1 h. The blot was then incubated with primary Ab diluted in TBS-T buffer for 1 h. After rinsing, the blot was exposed to a HRP-conjugated secondary Abs diluted in TBS-T. After rinsing, blots were incubated in ECL (Amersham Pharmacia Biotech) reagent for 1 min, blotted dry, and then exposed to film until a signal was detected.
Caspase activity assays
Following stimulation, cells were washed once in PBS and resuspended at 2 x 108/ml in hypotonic lysis buffer (50 mM NaCl, 40 mM -glycerophosphate, 10 mM HEPES (pH 7.0), 5 mM EGTA, and 2 mM MgCl2). The lysate was then subjected to four freeze-thaw cycles before centrifugation at 10,000 x g for 10 min (100 mM HEPES (pH 7.5), 10% sucrose, 0.1% 3-[(3-cholamidopropyl)dimethylammonio]propanesulfonate, 10 mM DTT, and 0.1 mg/ml OVA). Protein concentrations were determined, and 25 µg of cell extract was incubated for 4 h at 37°C with 50 µM Ac-DEVD-AMC, Ac-VEID-AMC, or Ac-IETD-AMC. Assays were performed in the presence or the absence of 1 µM ZVAD-CHO or DEVD-CHO inhibitor as a control for broad specificity caspase activity and caspase-3 activity, respectively. Protease activity was determined by monitoring the release of 7-amino-4-trifluoromethyl coumarin at an excitation wavelength of 400 nm and an emission wavelength of 510 nm with the use of a CytoFluor II 96-well plate spectrofluorometer (PerSeptive Biosystems, Framingham, MA). Relative caspase activity was determined by dividing the activity observed at each time point by the values detected at time zero.
Cytokine assays
B cells were stimulated as described above. Then supernatants were removed at 24 h, and the concentrations of IL-6, IL-10, and IL-1 were determined by immunoassays. Matched pairs of Abs were as follows: IL-10, JES3-19F1 (capture) and JES3-6B11 (detection); IL-6, MQ2-13A5 (capture) and MQ2-39C3 (detection; BD PharMingen); and IL-1, 508A7G8 and 508A4A2 (capture) and 508A3H12 (detection; BioSource International, Camarillo, CA). IL-6, IL-10, and IL-1 were detected with ExtrAvidin-HRP (1/1000 dilution), followed by 3,3',5,5'-tetramethylbenzidine substrate (Sigma-Aldrich, St. Louis, MO). Results were expressed as mean concentrations extrapolated from a standard curve prepared with recombinant cytokines (BD Biosciences), for each stimulation condition performed in triplicate. The limit of sensitivity of each assay was 15 pg/ml.
RNase protection assay (RPA)
RPA were performed as previously described (25) using the RiboQuant Multiprobe RNase Protection Assay System (BD PharMingen). Total RNAs were extracted from Er--dense tonsillar B cells that were first pretreated with caspase inhibitors for 3 h, then stimulated with CD40 plus CD180 mAb for 24 h using TRIzol reagent (Life Technologies, Grand Island, NY). RPAs were then performed according to the manufacturer’s instructions, using a RiboQuant RNase protection kit and hCY-1 and hCC-1 template sets (BD PharMingen). Briefly, probes from the template sets were labeled with [
-32P]UTP using T7 RNA polymerase and conditions supplied by the manufacturer. Five micrograms of the sample RNAs were resuspended in 8 µl hybridization buffer, followed by the addition of 2 µl of 32P-labeled probe (2–4 x 105 cpm/µl). Following the addition of the reagents, the RNA samples were then quickly denatured at 90°C and then allowed to anneal at 56°C for 12–16 h. The samples were then treated with RNase A and RNase T1 mixture following the protocol described in the kit. The samples were resolved by a 5% acrylamide sequencing gel, which was prepared in 1x TBE (89 mM Tris, 89 mM boric acid, and 2 mM EDTA, pH 8.3). The gels were dried and analyzed by autoradiography and densitometry using National Institutes of Health Image. Values were normalized relative to GAPDH.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In initial experiments we compared various signals for their ability to stimulate human dense G0 tonsillar B cells to enter the cell cycle and proliferate (Fig. 1A). The B cells used, as previously reported (20, 21), were >98% CD20+ and >98–99% in G0. These G0 B cells, when left unstimulated for 3 days in culture, exhibited little or no proliferation. After 3 days in culture the cells were 12–15% annexin V positive, reflecting a basal level of apoptosis in the unstimulated normal cells (Fig. 1B). Stimulation with Abs to surface IgM, CD40, or CD180 (RP105/Bgp95) or with rIL-4 increased B cell proliferation at 72 h 4- to 5-fold as determined by thymidine incorporation (Fig. 1A). Combining anti-CD40 with other signals (anti-CD180, anti-IgM, or IL-4) led to a significant increase in proliferation relative to that induced by single stimuli (Fig. 1A).
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To determine whether caspase activity was also increased by proliferative stimuli, we performed caspase activity assays on lysates prepared from B cells that were either unstimulated or stimulated via CD40/CD180 for 12–48 h. For these experiments we used the peptide reporter substrates DEVD-AMC, VEID-AMC, and IETD-AMC, which are preferred substrates for caspase-3, -6, and -8, respectively. These substrates have been widely used to assess caspase activity. All peptide cleavage activity was completely inhibited by the broad specificity caspase inhibitor ZVAD-CHO and therefore attributable to caspases. In unstimulated B cells, DEVDase (caspase-3-like) activity was detectable at all times measured and increased over the time course of the assay (Fig. 2A). This activity was completely blocked by 1 µM DEVD-CHO, supporting the idea that caspase-3 was probably responsible (data not shown). To exclude contaminating caspase-3 activity, assays for caspase-6 and -8 were performed in the presence of 1 µM DEVD-CHO. In contrast to DEVDase activity, little or no VEIDase (caspase-6-like) activity or IETDase (caspase-8-like) activity was detectable in unstimulated cells at any time point (Fig. 2, B and C). Stimulation via CD40/CD180 led to decreased DEVDase activity by 24 and 48 h, but increased VEIDase and IETDase activity (Fig. 2). Cleavage of VEID-AMC was detected as early as 12 h after stimulation and peaked at 24 h, while IETD-AMC cleavage was detected first at 24 h and peaked 48 h after stimulation (Fig. 2, B and C). These data are consistent with an increase in caspase-8 and -6, but a decrease in caspase-3, activity following the proliferative stimulation of normal human B cells.
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Caspase activity is required for G0 B cells to proliferate
We next determined whether the caspase activity induced after stimulating resting B cells is required for B cell proliferation. Dense human B cells were pretreated for 3 h with the broad spectrum caspase inhibitor ZVAD-fmk and then stimulated with anti-CD40 in combination with anti-CD180, anti-IgM, or IL-4. Pretreatment with ZVAD-fmk inhibited B cell proliferation in response to all stimuli used (data not shown). Thus, caspase activity is required for B cells to be induced to proliferate via these stimuli. We compared the dose-response of caspase-selective inhibitors for their ability to inhibit human B cell proliferation. Dense human B cells were pretreated for 3 h with the indicated concentration of either ZVAD-fmk (a broad specificity inhibitor), IETD-fmk (selective for caspase-8), VEID-fmk (selective for caspase-6), or DEVD-fmk (selective for caspase-3). The cells were then stimulated with anti-CD40 in combination with anti-CD180, and thymidine incorporation was determined after 48 h of stimulation (Fig. 4A). VEID-fmk inhibited CD40/CD180-mediated B cell proliferation over a dose range of 20–80 µM, with complete inhibition seen at 80 µM (Fig. 4A). ZVAD-fmk was effective over a similar concentration range as VEID-fmk, with 50% inhibition occurring between 40 and 50 µM. In contrast, DEVD-fmk had no effect on CD40/CD180-mediated proliferation at any concentration examined. The caspase-8 inhibitor IETD-fmk achieved 
50% inhibition at 80 µM and was consistently less effective than either ZVAD-fmk or VEID-fmk.
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Caspase activity is required for entry into the cell cycle
Since the B cell population used in this study is highly quiescent, we examined the effects of caspase inhibitors on various parameters of cell cycle entry. Two early changes that accompany cell cycle entry include induction of RNA synthesis and an increase in cell size. To test whether caspase inhibitors blocked the induction of RNA synthesis we labeled cells with [3H]uridine following 72 h of stimulation in the presence or the absence of caspase inhibitors (Fig. 4D). While DEVD-fmk had little effect, pretreatment with VEID-fmk almost completely abolished the incorporation of [3H]uridine induced by anti-CD40 and anti-CD180 (Fig. 4D, right panel). Similar results were obtained at 48 h (data not shown). In addition, analysis of forward vs side scatter FACS profiles indicated that the early increase in cell size was also blocked by caspase inhibitors (data not shown).
To more precisely determine the mechanism by which caspase inhibitors blocked cell cycle entry of stimulated B cells, we measured the induction of critical cell cycle regulators. Since the levels of cell cycle regulators can be influenced by both transcriptional and post-translational mechanisms, we measured both mRNA and protein levels. Stimulation of quiescent B cells with anti-CD40/anti-CD180 for 24 h significantly increased the levels of mRNAs for cyclins D1 and D2 and the cyclin-dependent kinase cdk4 (Fig. 5, A and B). Pretreatment with VEID-fmk blocked induction of D-type cyclin and cdk4 mRNA by anti-CD40/CD180 almost to basal levels (Fig. 5, A and B). However, DEVD-fmk did not inhibit the induction of any of these mRNAs. In contrast to its effect on D-type cyclins, VEID-fmk did not influence message levels for the cyclin-dependent kinase inhibitors p21 or p27 (Fig. 5, A and B). These, findings implicate caspase-6, but not caspase-3, in induction of D-type cyclin and cdk4 mRNA by anti-CD40/CD180.
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Caspase activity is not required for all CD40-dependent activation events
We next tested whether the caspase inhibitors selectively blocked other signaling events associated with the stimulation of quiescent B cells. CD40 ligation has been previously shown to up-regulate the expression of cIAP2 (28), a protein that inhibits apoptosis by selectively binding and inhibiting caspase-3 and caspase-7, but not caspases-6 and -8 (29). Western blot analysis of lysates from CD40/CD180-stimulated B cells showed that CD40/CD180 stimulation increased cIAP2 expression. Pretreatment with ZVAD-fmk had no effect on cIAP2 protein levels (Fig. 6A). We also examined the rapid phosphorylation of the ERK/MAPK and protein kinase B/Akt kinases following treatment with the caspase inhibitor ZVAD-fmk. An increase in ERK phosphorylation was observed as early as 15 min after combined stimulation via CD40/CD180 (Fig. 6B), and ERK remained highly phosphorylated for at least 6 h following stimulation. Treatment with ZVAD-fmk did not affect ERK phosphorylation. ZVAD-fmk also had no effect on the phosphorylation of Akt induced by CD40/CD180 stimulation (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Our results demonstrate that treatment of dense human B cells with proliferative stimuli, such as ligation of CD40 and CD180, leads to the selective activation of caspase-8 and caspase-6, while caspase-3 activity and apoptosis are reduced. These conclusions are based upon collective data including the use of peptide substrates that are selective for caspases-3, -6, and -8. Proliferative stimuli increased VEIDase and IETDase activities, while DEVDase activity was reduced. Although these substrates are modeled after preferred substrate sequences for the respective caspases, their specificity is not absolute. However, in addition to this evidence, Western blotting using Abs specific for these caspases as well as for preferred substrates was also performed. Cleavage of caspase-8, caspase-6, and SATB1, but not caspase-3 or PARP, was observed after B cells were activated. Together these data provide strong evidence to suggest that a pattern of caspase activation distinct from that involved in apoptosis, involving increased caspase-8 and caspase-6 activity, is induced by proliferative stimuli.
How might B cells activate caspase-8 and caspase-6 while preventing caspase-3 activation and apoptosis? The decrease in apoptosis may result from both the up-regulation of prosurvival factors and the selective inactivation of effector caspases. In this respect, CD40 ligation of human B cells not only increases the expression of antiapoptotic members of the Bcl-2 family, such as Bcl-2, Bcl-X, and A1 (32, 33, 34), but also up-regulates the expression of IAPs such as cIAP1 and cIAP2 (28). IAPs selectively bind and inhibit caspase-3 and -7, but not caspase-6 and -8 (29). In addition, cIAP2 has been shown to ubiquitinate caspase-3 and stimulate its degradation by the proteasome (35). Thus, IAPs may selectively inactivate caspase-3 without affecting the caspases required for B cells to enter the cell cycle. Interestingly, antisera specific for caspase-3 consistently recognized several species that migrated below the pro form in proliferating cells (Fig. 3A). The fact that these bands are also observed upon treatment of B cells with proteasome inhibitors (J. D. Graves, unpublished observations) supports the idea that caspase-3 might be inactivated by ubiquitination in proliferating cells. The precise mechanism by which the specificity of caspase activation is maintained in response to proliferative stimuli is currently under investigation.
Caspase activity is required for B cell proliferation
Our data based upon the use of cell-permeable caspase inhibitors demonstrate that caspase activity is required for dense B cells to proliferate. The broad specificity caspase inhibitor ZVAD-fmk, which is an efficient inhibitor of apoptosis in many systems, blocked B cell proliferation in response to ligation of CD40 and CD180. VEID-fmk, an inhibitor selective for caspase-6, was an especially effective inhibitor of proliferation, while DEVD-fmk, which is selective for caspase-3, was ineffective (Fig. 3B). While it is important to recognize that these peptide caspase inhibitors are not absolutely specific, several lines of evidence suggest that they exhibit a high degree of selectivity in this system. VEID-fmk completely blocked cleavage of the caspase-6 substrate SATB1, but only partially blocked cleavage of the caspase-3 substrate PARP, while DEVD-fmk had no effect on SATB1 cleavage, but abolished that of PARP (Fig. 3B). These data also argue against differences in permeability making a significant contribution to the differential effectiveness of VEID-fmk and DEVD-fmk. The fact that caspase-6 and not caspase-3 activity is elevated in these cells provides further evidence suggesting that VEID-fmk is likely to be exerting its effects via inhibition of caspase-6.
Based upon its apical position in the apoptotic caspase cascade induced by CD95/Fas, it is tempting to conclude that caspase-8 functions upstream of caspase-6 in regulating B cell proliferation. In this respect, Kennedy et al. (18) showed that caspase-8 is activated in CD3-stimulated normal T cells and that a caspase-8 inhibitor efficiently blocked CD3-mediated proliferation. Further evidence for an important role for caspase-8 can be derived from a recent study that identified a functional deficit in caspase-8 in patients with a lymphoproliferative disorder (19). However, while VEIDase activity was maximal 12 h after CD40/CD180 stimulation of quiescent B cells, caspase-8 activity did not peak until 24 h (Fig. 2, B and C). Furthermore, IETD-fmk was a weak and inconsistent inhibitor of B cell proliferation (Fig. 4B and data not shown). An alternative possibility is suggested by a recent study that indicates caspase-6 functions upstream of caspase-8 in the apoptotic pathway initiated by cytochrome c (36). Thus, it is possible that caspase-6 functions upstream of caspase-8 in response to proliferative stimuli in B cells. In support of this hypothesis, IETD-fmk did not block processing and activation of caspase-6 in response to anti-CD40/CD180 (data not shown).
Although ZVAD-fmk is a potent antagonist of B cell proliferation, caspase activity is not required for all activation events. For example, caspase inhibitors had no effect on ERK/MAPK phosphorylation induced via CD40/CD180 (Fig. 6B). Since the ERK pathway is required for B cell proliferation (37), this argues against the possibility that caspase inhibitors might exert a nonspecific or toxic effect blocking the activation of signaling pathways. Also, no significant decrease in cell viability was observed following incubation with VEID-fmk for periods up to 72 h (data not shown). Further evidence for the specificity of caspase involvement comes from data showing that VEID-fmk antagonized anti-CD40/CD180-induced IL-10 secretion, but not secretion of IL-6 (Fig. 6C). These data support the idea that caspases function downstream of, or parallel to, early signaling events that regulate the proliferation, survival, and function of quiescent B cells.
While the identity of the caspase(s) required for B cell proliferation cannot be definitively deduced from our present data, our results are consistent with the hypothesis that caspase-6 and/or another caspase, sensitive to VEID-fmk yet relatively insensitive to DEVD-fmk, is required for human B cell proliferation. Interestingly, a recent study involving the generation of a caspase-6-deficient chicken cell line has provided evidence that caspase-6 may be important for B cell proliferation and/or survival (38). Experiments in caspase-6-deficient mice assessing the role of caspase-6 as a critical effector of B cell proliferation and maturation are underway.
Caspase-6 activity is required for entry into the cell cycle
Pretreatment of quiescent B cells with VEID-fmk blocked induction of RNA synthesis, increases in cell size, and induction of cyclin D2, cyclin D3, and cdk4 proteins while increasing levels of the cdk inhibitor p27Kip1 in response to anti-CD40/CD180 (Figs. 4D and 5). This suggests that caspase-6 activity is required at the G0/G1 restriction point of cell cycle entry. Although p27Kip1 is cleaved during apoptosis, several reports also suggest that caspase cleavage of p27 may serve a regulatory function during B cell proliferation (39). Thus, one mechanism by which caspase-6 might regulate primary B cell proliferation is via cleavage of p27Kip1. Another potential mechanism is suggested by the fact that VEID-fmk blocks the induction of mRNA encoding cdk4 and D-type cyclins (Fig. 5, A and B). Further evidence to suggest that caspase inhibitors block early events can be inferred from the fact that VEID-fmk or ZVAD-fmk must be added within the first 4 h following stimulation to block proliferation (data not shown). Accordingly, caspase-6 might target transcriptional activators or repressors that regulate early cell cycle gene expression. Of the several defined substrates for caspase-6, SATB1 was of particular interest. SATB1 is a matrix attachment region DNA-binding protein (40), is expressed in B cells (5) (Fig. 3B), is known to down-regulate gene transcription (8, 41), and after caspase cleavage loses its ability to bind to chromatin (19). Activation of human B cells leads to increased cleavage of SATB1 that is antagonized by VEID-fmk. This raises the possibility that caspase-6 functions to down-regulate transcriptional repressors such as SATB1 during proliferation.
The homeostatic regulation of B cell populations is tightly regulated by a balance among cell death, survival, and proliferation. Our data indicate that caspases, already established as critical effectors of cell death, also play an important role in B cell proliferation. While further analysis is required to understand the mechanism by which caspases contribute to these contrasting cell fates, these findings raise the possibility that caspase-6 might constitute an attractive therapeutic target for lymphoproliferative or autoimmune diseases.
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Acknowledgments |
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Footnotes |
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2 N.E.O., J.D.G., and G.L.S. contributed equally to this work.
3 Address correspondence and reprint requests to Dr. Edward A. Clark, Department of Microbiology, Box 357242, University of Washington, Seattle WA 98195. E-mail address: eclark{at}bart.rprc.washington.edu
4 Abbreviations used in this paper: SATB1, special AT-rich sequence-binding protein 1; Ac-DEVD-AMC, acetyl Asp-Glu-Val-Asp 7-amido-4-methylcoumarin; Ac-IETD-AMC, acetyl-Ile-Glu-Thr-Asp-7-amino-4-methyl coumarin; Ac-VEID-AMC, scetyl-Val-Glu-Ile-Asp-7-amino-4-methyl coumarin; cdk, cyclin-dependent kinase 4; DEVD-CHO, N-acetyl-Asp-Glu-Val-Asp-al; DEVD-fmk, benzyloxycarbonyl (Cbz)-Asp-Glu-Val-Asp(Ome)-fluoromethylketone; ERK, extracellular signal-regulated kinase; FADD, Fas-associated death domain protein; IAP, inhibitor of apoptosis; IETD-fmk, benzyloxycarbonyl (Cbz)-Ile-Glu-Thr-Asp(Ome)-fluoromethylketone; MAPK, mitogen-activated protein kinase; PARP, poly-ADP ribose polymerase; Rb, retinoblastoma protein; RPA, RNase protection assay; VEID-fmk, benzyloxycarbonyl (Cbz)-Val-Glu-Ile-Asp(Ome)-fluoromethylketone; ZVAD-CHO, N-acetyl-Val-Ala-Asp-al; ZVAD-fmk, benzyloxycarbonyl (Cbz)-Val-Ala-Asp(Ome)-fluoromethylketone; cIAP, cellular IAP.
Received for publication December 2, 2002. Accepted for publication April 10, 2003.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, a mammalian homolog of CED-3, is a CrmA-inhibitable protease that cleaves the death substrate poly(ADP-ribose) polymerase. Cell 81:801.[Medline]
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J. W. Darnowski, F. A. Goulette, Y.-j. Guan, D. Chatterjee, Z.-F. Yang, L. P. Cousens, and Y. E. Chin Stat3 Cleavage by Caspases: IMPACT ON FULL-LENGTH Stat3 EXPRESSION, FRAGMENT FORMATION, AND TRANSCRIPTIONAL ACTIVITY J. Biol. Chem., June 30, 2006; 281(26): 17707 - 17717. [Abstract] [Full Text] [PDF]
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M. Lamkanfi, K. D'hondt, L. Vande Walle, M. van Gurp, G. Denecker, J. Demeulemeester, M. Kalai, W. Declercq, X. Saelens, and P. Vandenabeele A Novel Caspase-2 Complex Containing TRAF2 and RIP1 J. Biol. Chem., February 25, 2005; 280(8): 6923 - 6932. [Abstract] [Full Text] [PDF]
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M. Lamkanfi, M. Kalai, X. Saelens, W. Declercq, and P. Vandenabeele Caspase-1 Activates Nuclear Factor of the {kappa}-Enhancer in B Cells Independently of Its Enzymatic Activity J. Biol. Chem., June 4, 2004; 279(23): 24785 - 24793. [Abstract] [Full Text] [PDF]
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, 
, and 
) or stimulated with anti-CD40/CD180 (•, 
, and 
) for the indicated time. Cell extracts were prepared and caspase activity measured using DEVD-AMC (A), VEID-AMC (B), or IETD-AMC (C) as substrate. DEVD-AMC activity was completely antagonized by the inclusion of 1 µM DEVD-CHO (data not shown), while data presented for VEIDase and IETDase activity were measured in the presence 1 µM DEVD-CHO. DEVDase (caspase-3-like) activity was decreased by anti-CD40/CD180 stimulation, while VEIDase (caspase-6-like) and IETDase (caspase-8-like) activities were increased. The results presented are representative of three independent experiments.
), or VEID-fmk (•) before stimulation with anti-CD40 in combination with anti-CD180. B, Human tonsilar B cells or E+ rosette-forming T cells were either untreated (
) or VEID-fmk (
), DEVD-fmk (
), or VEID-fmk (
