Báo cáo khoa học: E2A participates in a fine control of pre-mature B-cell apoptosis mediated by B-cell receptor signaling via transcriptional regulation of survivin, IAP2 and caspase-8 genes

E2A participates in a fine control of pre-mature B-cell apoptosis mediated by B-cell receptor signaling via transcriptional regulation of survivin, IAP2 and caspase-8 genes Kenji Toyonaga1,2,*, Hidehiko Kikuchi1,3,*, Koki Yamashita1, Masami Nakayama1, Kazuo Chijiiwa2 and Tatsuo Nakayama1,3

1 Section of Biochemistry and Molecular Biology, Department of Medical Sciences, Miyazaki Medical College, University of Miyazaki, Japan 2 Section of Surgical Oncology and Regulation of Organ Function, Department of Medical Science, Miyazaki Medical College, University of Miyazaki, Japan 3 Department of Life Science, Frontier Science Research Center, University of Miyazaki, Japan

Keywords apoptosis; B cell; caspase; E2A; survivin

Correspondence T. Nakayama, Department of Life Science, Frontier Science Research Center, University of Miyazaki, 5200 Kihara, Kiyotake, Miyazaki 889-1692, Japan Fax: +81 985 85 6503 Tel: +81 985 85 3127 E-mail: tnakayam@med.miyazaki-u.ac.jp

*These authors contributed equally to this work

(Received 8 May 2008, revised 16 December 2008, accepted 24 December 2008)

doi:10.1111/j.1742-4658.2009.06881.x

Antigen binding to the B-cell receptor (BCR) of pre-mature B lymphocytes induces their apoptotic cell death, but binding to the BCR of mature B lymphocytes triggers activation and proliferation. Binding to pre-mature B lymphocytes is thought not only to function as a mechanism to exclude B-cell clones that possess the ability to react with self-antigen, but also to act as a defense mechanism in auto-immune diseases. Cross-linking of BCR of pre-mature B-cell lines, including the chicken DT40 cell line, with anti-immunoglobulin IgG induces apoptotic cell death. Treatment with phorbol 12-myristate 13-acetate/ionomycin, which mimics BCR stimula- tion, is used to study intracellular signal transduction of B lymphocytes. Here, by analyzing the E2A-deficient DT40 cell line, E2A)/), we show that E2A deficiency prevents certain levels of apoptotic cell death mediated by BCR signaling. In addition, E2A deficiency-linked BCR signaling controls the mimicked pre-mature B-cell apoptosis by PMA/ionomycin through ele- vated survivin plus inhibitor of apoptosis 2 levels, and reduced caspase-3 and caspase-8 activities, resulting in increased amounts of ICAD (inhibitor of caspase-activated DNase), compared with those in the presence of E2A, followed by reduction of DNA fragmentation. These findings will contrib- ute to the resolution of molecular mechanisms of negative selection of B cells and also auto-immune diseases.

Abbreviations AIF, apoptosis-inducing factor; AKT, acutely transforming retrovirus AKT8 in rodent T cell lymphoma; BCR, B-cell receptor; CAD, caspase- activated DNase; EBF, early B-cell factor; FACS, fluorescence-activated cell sorter; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; GATA-3, GATA binding protein-3; HAT, histone acetyltransferase; HDAC, histone deacetylase; IAP, inhibitor of apoptosis; PARP, poly(ADP- ribose)polymerase; PCAF, p300/CBP-associated factor; PMA, phorbol 12-myristate 13-acetate.

FEBS Journal 276 (2009) 1418–1428 ª 2009 The Authors Journal compilation ª 2009 FEBS

1418

It is widely known that B lymphocytes are essential to immune responses in health and disease, and maintain homeostasis by balancing cell viability and cell death [1]. The molecular mechanisms of B-lymphocyte devel- opment have mostly been studied in mammalian bone marrow and/or peripheral lymphoid tissue (e.g. spleen). Their development requires not only controlled lineage- and locus-specific immunoglobulin gene recombination, establishing unique antigen specificity of the B lympho- cytes, but also developmental stage-specific gene expres- sion participating in lymphoid cell proliferation and synthesis of immune mediators [1–3]. In addition, vari- ous factors and/or signals control various aspects of the normal development of B lymphocytes and func- tion of the immune system. Such developmental activi- the ties require numerous transcription factors, i.e.

K. Toyonaga et al.

Fine control of pre-mature B-cell apoptosis by E2A

basic helix-loop-helix transcription factors E2A, early B cell factor (EBF), GATA-binding protein-3 (GATA- 3), Pax5, PU.1, Ikaros and Aiolos, etc [2–4]. Their importance in B-cell development has been established by knockout experiments on mouse hematopoietic stem cells, in which all these functions are associated with cessation of early stages of B-cell differentiation [3,5]. However, the expression of these factors is sustained lymphocytes in normal throughout development of mice after the observed block in their knockout mice [6]. Thus, their physiological functions beyond develop- mental arrest remain poorly understood.

types [5,7]. Recently,

line,

results show that HDAC2 controls the amount of IgM H-chain at two stages: transcription of its gene and alternative processing of its pre-mRNA [21]. Recently, we revealed not only that HDAC2 upregulates gene expression of EBF1, Pax5, Aiolos, Ikaros and HDAC7, and down-regulates those of E2A, p300/CBP-associated factor (PCAF), HDAC4 and HDAC5, but also that E2A upregulates expression of IgM H- and L-chain genes, but downregulates Aiolos, but Aiolos, EBF1, Pax5, and Ikaros downregulate expression of these two genes [22]. These results, together with others, indicate that HDAC2 indirectly controls the expression of IgM H- and L-chain genes, through upregulated transcrip- tional regulation of EBF1, Pax5, Aiolos and Ikaros, and downregulated transcriptional regulation of E2A. Moreover, our results indicated that GCN5 functions as a supervisor in normal cell-cycle progression, with com- prehensive control over the expression of several cell cycle-related genes, as well as apoptosis-related genes, probably through alterations in the chromatin structure, indicated by the changing acetylation status of core histones surrounding these widely distributed genes [23]. Recently, we showed that GCN5 and BCR signaling collaborate to induce apoptotic cell death of the DT40 cell through depletion of ICAD [inhibitor of caspase-activated DNase (CAD)] and inhibitor of apop- tosis 2 (IAP2), and activation of caspase activities [24].

Among these factors, E2A has been directly impli- cated in transcriptional regulation of several B lineage- specific genes, and has been shown to be essential for Ig H- and L-chain recombination [5]. Moreover, E2A is required to initiate the expression of some B lineage- specific genes such as EBF, mb-1 and B29, but not to maintain expression of these genes [5]. In general, E2A has also been shown to promote proliferation and sur- vival of various cell it was revealed that E2A might be a key regulator of apopto- sis versus proliferation in lymphoid cells, in addition to its B lineage-determining function [8–11]. E2A-defi- cient mice develop T cell-derived lymphoma, and enforced expression of an inhibitor of differentiation proteins that indirectly inhibit E2A function [12,13]. In addition, ectopic expression of E47 or E12 induced apoptotic cell death in thymic lymphoma cells derived from E2A-deficient mice [14]. These results indicate that E2A might act as a tumor suppressor.

immune responses [1,15,16]. Recent Based on these results, in this study, as a first step in elucidating the participation of B cell-specific factors in pre-mature B-cell apoptosis mediated by BCR stim- ulation by analyzing the E2A-deficient DT40 mutant E2A)/), we clarified the impact of E2A on apoptotic cell death of the DT40 cell line, and show that E2A is involved in fine control of pre-mature B-cell apoptosis mediated by BCR signaling, via transcriptional regula- tion of survivin, IAP2 and caspase-8 genes.

Results

Insignificant influence of E2A deficiency on gene expression of apoptosis-related factors

It is also accepted that B lymphocytes are suscepti- ble to receptor- and mitochondria-initiated cell death at various stages of peripheral differentiation and dur- genetic ing evidence has contributed to understanding of the BCR-dependent survival mechanism of mature B cells [17,18]. It is also known that E2A plays important roles in the apoptosis of B lymphocytes [9–11]. In addition, Id3 protein, an E protein antagonist, induces growth arrest and apoptosis in B-lymphocyte progeni- tor cells [19]. However, the impact of B cell-specific including E2A, on the BCR-mediated apop- factors, tosis of pre-mature B cells remains unclear, when apoptosis is triggered by antigen stimulation.

histone (HATs),

FEBS Journal 276 (2009) 1418–1428 ª 2009 The Authors Journal compilation ª 2009 FEBS

1419

In order to better understand the roles of histone deacetylases acetyltransferases (HDACs) and B cell-specific transcription factors in B-cell functions, we have systematically generated vari- including HDAC2)/), ous homozygous mutants, GCN5)/), Aiolos)/) and E2A)/), using gene-targeting techniques on the DT40 cell line, which was established [20]. Our from chicken pre-mature B lymphocytes To assess the influence of E2A deficiency on gene expression of apoptosis- and BCR signaling-related fac- tors, and other factors, we performed semi-quantitative RT-PCR on total RNAs prepared from DT40 and three independent E2A)/) clones (Fig. 1). E2A deficiency did not have a significant influence on transcription of most of these genes, except for survivin (to approximately 200%), PKCa (to approximately 60%), PKCg (to approximately 40%), PKCl (to approximately 40%) and PKCf (to approximately 160%). In addition, we performed immunoblot analyses to assess the influence

K. Toyonaga et al.

Fine control of pre-mature B-cell apoptosis by E2A

Fig. 1. Effect of E2A deficiency on gene expression of apoptosis-related factors, caspases, caspase-regulating factors, CAD/ICAD and PKCs. Total RNAs were extracted from DT40 and three independent E2A)/) clones (1–3), and mRNA levels were determined by semi-quantitative RT-PCR using appropriate primers. The chicken GAPDH gene was used as an internal control. The numbers under the panels indicate the number of cycles used for PCR.

of E2A deficiency on the amounts of proteins whose mRNA levels were altered as noted above. Consistent with the results on mRNA levels, the protein levels for survivin and PKCf were increased in E2A)/) and that of PKCg was decreased (Fig. S1); PKCa and PKCl could not be detected using the available antibodies. On the other hand, transcription of various genes encoding membrane-proximal factors, NF-jBs, transcription fac- tors and B cell-related factors, amongst others, was not altered in the E2A-deficient mutants (data not shown). These insignificant effects of E2A deficiency on the expression of numerous genes probably resulted in no changes in the amount of apoptotic cells, as discussed below (see Fig. 2).

Moderate resistance to apoptosis is induced by PMA/ionomycin in E2A-/-

FEBS Journal 276 (2009) 1418–1428 ª 2009 The Authors Journal compilation ª 2009 FEBS

1420

We examined the influences of phorbol 12-myristate on 13-acetate (PMA)/ionomycin etoposide and cell-cycle progression and proliferation of DT40 and E2A)/) cells. Cells cultured in the presence of PMA/ ionomycin for 24 h or etoposide for 6 h were analyzed by fluorescence-activated cell sorter (FACS) after stain- ing with propidium iodide (Fig. 2A). As expected, eto- poside treatment caused apoptotic cell death for the two cell lines at the same level because the drug inhib- its the topoisomerase-2 activity that is essential for DNA replication. Although PMA/ionomycin treatment of DT40 cells resulted in cell-cycle distributions that were quite different from those in the case of the eto- poside treatment, it did induce apoptosis, and these findings agree with those reported previously [24]. On the other hand, although PMA/ionomycin treatment of E2A)/) did not alter the cell-cycle distribution pat- tern up to 24 h (cell growth was slightly delayed there- after), the depletion of E2A slightly prevented apoptotic cell death even in the presence of PMA/ ionomycin. To confirm these findings, we examined the effects of PMA/ionomycin treatment on the viability

K. Toyonaga et al.

Fine control of pre-mature B-cell apoptosis by E2A

A

B

C

Fig. 2. Analyses of apoptosis in E2A)/). (A) Effects of PMA/ionomycin and etoposide treatments on cell-cycle distributions of DT40 and E2A)/). DT40 and E2A)/) cells treated with etoposide (10 lgÆmL)1) for 6 h, or with PMA (10 ngÆmL)1) plus ionomycin (1 lM) for 24 h, were processed for DNA content analysis by propidium iodide staining. Nuclei were analyzed by flow cytometry (FACSCalibur, Becton Dickinson and Company, Franklin Lakes, NJ, USA), and data for DT40 and E2A)/) (clone 1) were plotted on linear histograms as relative cell number (y axis) against red fluorescence intensity (x axis). The percentages of the various cell-cycle phases (sub-G1, G1, S and G2/M) for for DT40 and E2A)/) (clone 1), together with those of two other E2A)/) clones (2 and 3) are indicated in the table. (B) Sensitivity of DT40 (circles) and E2A)/) (squares, triangles and diamonds) to PMA/ionomycin-mediated apoptotic cell death. Cells were resuspended in DMEM containing 10% v/v fetal bovine serum, and treated with (filled symbols) or without (open symbols) 10 ngÆmL)1 PMA plus 1 lM ionomycin at 37 (cid:2)C for up to 48 h. Viable cells were counted by the trypan blue dye exclusion method. Data represent the mean of two separate experiments, and error bars indicate the standard deviation. (C) Morphology of DT40 and E2A)/) (clone 1) cells treated with PMA/ionomycin. Cells were cultured for 24 h without (no treatment) or with PMA/ionomycin (PMA/ionomycin), and their nucleus forms were analyzed by microscopy.

E2A deficiency leads to blockage of the induced apop- totic cell death that is seen in the DT40 cell line when treated with PMA/ionomycin.

Influence of depletion of B cell-specific transcrip- tion factors, HDACs and HATs on resistance to apoptosis induced by PMA/ionomycin treatment

FEBS Journal 276 (2009) 1418–1428 ª 2009 The Authors Journal compilation ª 2009 FEBS

1421

[20,22]. Our previous Using gene-targeting techniques, we systematically gen- erated several homozygous DT40 mutants that lacked genes encoding B cell-specific transcription factors, HDACs or HATs results revealed that GCN5 and PMA/ionomycin treatment of DT40 and E2A)/) (Fig. 2B). As expected, the viabil- ity of the two cell lines did not differ in the absence of in the presence of PMA PMA/ionomycin. However, and ionomycin, the viability of E2A)/) (approximately 50% at 48 h) was slightly higher than that of DT40, which was dramatically reduced (approximately 20% by 48 h). We undertook a comparative analysis of changes in the morphological structure of nucleus as an effect of PMA/ionomycin treatment in DT40 and E2A)/) (Fig. 2C). Nuclear fragmentation, another characteristic of apoptosis, was partially hindered in E2A)/), but it was clearly detected for DT40 in the presence of PMA/ionomycin. These results show that

K. Toyonaga et al.

Fine control of pre-mature B-cell apoptosis by E2A

Table 1. Influences of depletion of B cell-specific transcription fac- tors, HDACs and HATs on resistance to apoptosis induced by PMA/ionomycin treatment.

Mutants

Reference

Resistance for PMA/ionomycin

[22] [22] [22] Unpublished data [22]

› fl fi fi fi

[21] [21] [23] [31] [31]

fi › fi fi fi

B cell-specific factors E2A)/) Aiolos)/) EBF)/) Helios)/) Pax5)/) HDACs HDAC1)/) HDAC2)/) HDAC7)/) SIRT1)/) SIRT2)/) HATs GCN5)/) PCAF)/) HAT1)/) MORF)/) MOZ)/) MOZ)/)/MORF)/)

[23] [23] [32] Unpublished data Unpublished data Unpublished data

› fi fi fi fi fi

mentioned above is influenced by PMA/ionomycin treatment, DT40 and E2A)/) were cultured in the pres- ence of PMA/ionomycin, and RT-PCR was performed (Fig. 3 and Fig. S2). E2A deficiency dramatically decreased the mRNA level of Aiolos (to approximately 30%), but did not have any effect on transcripts of the remaining B cell-specific transcription factors, or HDACs and HATs. On the other hand, PMA/ionomy- cin treatment dramatically altered gene expression of E2A in DT40 (to approximately 320% by 24 h) and that of Aiolos in both DT40 and E2A)/) (to less than 10% by 24 h). However, in the two cell lines, the treat- ment did not have significant effects on mRNA levels of HDAC1, HDAC2, SIRT2, HAT1 and MORF, and slightly distinct but almost similar effects on mRNA levels of EBF, Pax5, HDAC7, SIRT1, GCN5, PCAF and MOZ. These findings, together with those shown in Table 1, indicate not only that HDAC2 and GCN5 are necessary for control of the apoptosis of the DT40 cell line mediated by PMA/ionomycin treatment, but also that their own transcription is not influenced by PMA/ionomycin. Therefore, the apoptotic cell death of DT40 mediated by PMA/ionomycin treatment must be under the control of the elevated or decreased amounts of E2A or Aiolos.

Upregulation of survivin and IAP2 gene expres- sion and no effect on caspase-8 gene expression by PMA/ionomycin treatment in E2A-/-

To further clarify the molecular mechanism linked to the apoptotic induction of the DT40 cell line coopera- tively mediated by E2A and BCR signaling, DT40 and E2A)/) were cultured in the presence of PMA/ionomy- cin, and RT-PCR was performed for various factors (Fig. 3 and Fig. S2). E2A deficiency did not alter the gene expression of the apoptosis-related factors bcl-2, bcl-xL, bak, Apaf-1, cytochrome c, acutely transform- lymphoma ing retrovirus AKT8 in rodent T cell (AIF) and poly (AKT), apoptosis-inducing factor (ADP-ribose)polymerase (also shown in (PARP) Fig. 1). The PMA/ionomycin treatment did not have a significant influence on the mRNA levels of bcl-2, bak, Apaf-1, cytochrome c, AKT and AIF, and had slightly distinct but almost similar effects on the mRNA levels of bcl-xL and PARP in both DT40 and E2A)/). cooperatively induce apoptotic cell death in the DT40 cell line [24]. As a first step in elucidating the participa- tion of E2A in apoptosis of DT40 cells, we examined the effects of PMA/ionomycin treatment for these homozygous DT40 mutants by FACS after staining with propidium iodide and/or determination of cell viability. Detailed information on the generation of mutants lacking Helios, Pax5, MORF, MOZ and MOZ/MORF will be shown elsewhere. As shown in Table 1, depletion of HDAC2, as well as of E2A and GCN5, prevented the apoptosis induced by PMA/ion- omycin treatment, but depletion of Aiolos promoted such apoptosis. No changes were detected in the remaining single mutants lacking EBF, Helios, Pax5, HDAC1, HDAC7, SIRT1, SIRT2, PCAF, HAT1, MORF or MOZ, or in a double knockout mutant lacking MOZ and MORF. These findings suggest that, among the B cell-specific transcription factors, HDACs and HATs examined, E2A, Aiolos, HDAC2 and GCN5 preferentially participate in control of the apop- totic cell death induced by PMA/ionomycin treatment of the DT40 cell line.

Up- and downregulation of expression of the E2A or Aiolos genes by PMA/ionomycin treatment

FEBS Journal 276 (2009) 1418–1428 ª 2009 The Authors Journal compilation ª 2009 FEBS

1422

To determine whether or not expression of the B cell-specific transcription factors, HDACs and HATs Similarly, depletion of E2A showed no effects on gene expression of CAD and ICAD (also shown in Fig. 1). PMA/ionomycin treatment showed similar effects on the expression of CAD and ICAD genes in both DT40 and E2A)/), i.e. the CAD mRNA level decreased by 3 h but thereafter increased to the con- level by 24 h, and the ICAD mRNA level trol

K. Toyonaga et al.

Fine control of pre-mature B-cell apoptosis by E2A

Fig. 3. Effects of PMA/ionomycin treatment on gene expression of B cell-specific factors, HDACs, HATs, apoptosis-related factors, CAD/ ICAD, caspases and caspase-regulating factors. Total RNAs were extracted from PMA/ionomycin-treated DT40 and E2A)/) (clone 1) at indi- cated times up to 24 h, and then the mRNA levels of appropriate genes were determined by RT-PCR. The chicken GAPDH gene was used as a control.

this level remained unchanged at 24 h, but the treat- level of ment showed no change in the transcript caspase-8 in E2A)/). These findings indicate that expression of most caspase genes is not much influ- enced by either E2A or BCR stimulation, except that of caspase-8. decreased by 3 h and thereafter remained unchanged at 24 h in the presence of PMA/ionomycin. These findings indicate that E2A and BCR stimulation have no effects on gene expression of CAD and ICAD, in contrast to the effects of GCN5 and BCR stimula- tion [24].

FEBS Journal 276 (2009) 1418–1428 ª 2009 The Authors Journal compilation ª 2009 FEBS

1423

With regard to caspase-regulating factors, depletion of E2A increased transcription of the survivin gene (to approximately 220%), but did not have a significant effect on expression of the FLIP, IAP1, IAP2 and Smac genes. However, PMA/ionomycin treatment had distinct effects on expression of these caspase inhibi- tors. The IAP1 or Smac mRNA levels increased or decreased slightly by 3 h and thereafter remained unchanged in both DT40 and E2A)/). The FLIP mRNA level was slightly decreased at 3 h (to approxi- mately 60%) and thereafter increased to the control in DT40, but remained unchanged in E2A)/). level With regard to caspases, E2A depletion showed no effects on expression of caspase-3, caspase-6, caspase- 8, caspase-9 and caspase-10 genes (also shown in Fig. 1). On the other hand, in both DT40 and E2A)/), the caspase-6 mRNA level was decreased gradually by PMA/ionomycin treatment by 24 h, and the cas- pase-10 mRNA level was increased by 3 h and there- after decreased dramatically by 24 h. Expression of caspase-3 and caspase-9 remained unchanged in the in DT40, presence of PMA/ionomycin. Interestingly, PMA/ionomycin treatment increased the caspase-8 mRNA level by 3 h (to approximately 160%) and

K. Toyonaga et al.

Fine control of pre-mature B-cell apoptosis by E2A

influenced by the drug treatment

Interestingly, whereas the IAP2 mRNA level in DT40 was significantly decreased by PMA/ionomycin treat- ment at 3 h (to approximately 30%) and thereafter in E2A)/) remained unchanged, the transcript level was unchanged at 3 h, and thereafter decreased by 24 h (to approximately 20%). Furthermore, in E2A)/), the survivin mRNA level was maintained at a high level (approximately 220%) when treated with PMA and ionomycin for 3 h, and thereafter decreased to slightly higher level than that in DT40; the mRNA level was not in DT40. These findings indicate not only that E2A downregulates transcription of the survivin gene and has no effects on that of FLIP, IAP1, IAP2 or Smac, but also that BCR stimulation and E2A cooperatively control expression of FLIP, IAP2 and survivin genes.

Resistance to PMA/ionomycin-mediated apoptosis of E2A-/- is brought about by increased amounts of survivin and IAP2, and reduced activity of caspase-3

slightly decreased by 24 h did not agree with the find- ings that the mRNA levels of IAP2 and ICAD were reduced slowly by 24 h or quickly by 3 h, respectively. To resolve this discrepancy between the results regarding the mRNA and protein levels of ICAD (and also IAP2), we next examined the effects of PMA/ ionomycin treatment on caspase activities (Fig. 4B). in E2A)/), PMA/ionomycin treatment Interestingly, showed slightly decreased activities of caspase-3 (to approximately 50% by 16 h), caspase-8 (to approxi- mately 70% by 16 h) and caspase-9 (to approximately 70% by 16 h), compared with those in DT40, probably due to a balance of the amounts of each of the three caspases and the inhibitors survivin and IAP2 (and also FLIP and IAP1). The slightly decreased caspase-8 activity mediated by PMA/ionomycin treatment in E2A)/) may have resulted from the balanced mRNA (and probably protein) levels of caspase-8, which lead to activation of pro-caspase-9, causing formation of the active form of caspase-3, and of the inhibitors FLIP (for caspase-8), survivin (for caspase-9) and IAP2 (for caspase-3) [25]. In E2A)/), the decreased activity of caspase-3 mediated by PMA/ionomycin treatment must depend on both the reduced amount of activated caspase-3 itself as a result of decreased caspase-8 (and probably caspase-9) activity, and the elevated protein (and also mRNA) levels of the inhibitors survivin and IAP2. As a result, the slow diminution of the protein level of ICAD by PMA/ionomycin treatment in E2A)/) (Fig. 4A) must be due to its slight degradation medi- ated by suppressed caspase-3 activity, although the ICAD mRNA level was decreased when exposed for 3 h but thereafter remained unchanged, as did that in DT40 (Fig. 3 and Fig. S2).

Next we examined the effect of PMA/ionomycin treat- ment on cellular protein levels of survivin, IAP2 and ICAD, which are proximal factors controlling CAD activity for DNA fragmentation, by immunoblotting using their specific antibodies (Fig. 4A). E2A deficiency increased the protein levels of survivin (to approxi- mately 190%), but had no effect on those of IAP2 and ICAD. Consistent with previous results [24], PMA/ion- omycin treatment in DT40 dramatically decreased the protein levels of IAP2 and ICAD (to approximately 40% and less than 10%) by 24 h, but had a moderate influence on that of survivin (approximately 60% at 24 h). Therefore, in DT40 cells treated with PMA/iono- mycin, the time courses of alterations in the protein and mRNA levels of survivin were virtually similar, and the gradual reductions in protein levels of IAP2 plus ICAD compared with acute decreases (unchanged thereafter) of their mRNA levels agreed with previous results [24]. On the other hand,

Because the alterations in the mRNA level of CAD were the same in both DT40 and E2A)/) (Fig. 3 and Fig. S2), and its protein could not be detected by the available antibodies to assess CAD activity, we exam- ined the effects of PMA/ionomycin treatment on DNA fragmentation, a typical result of CAD activity (Fig. 4C). The DNA fragmentation was found to be more moderate for E2A)/) than that observed for DT40, even in the presence of PMA/ionomycin up to 24 h. These results indicate that the CAD activity in E2A)/) is suppressed by the moderately reduced amount of its inhibitor ICAD compared with that in DT40, resulted in decreased DNA fragmentation, a characteristic of apoptosis. in E2A)/),

Discussion

FEBS Journal 276 (2009) 1418–1428 ª 2009 The Authors Journal compilation ª 2009 FEBS

1424

In recent years, numerous studies have been performed to determine the physiological target genes of E2A. in E2A)/), PMA/ionomycin treatment did not influence the protein level of survivin by 8 h and thereafter it decreased gradually. PMA/ion- omycin treatment did not change the protein level of IAP2. Further, the PMA/ionomycin treatment showed a moderate reduction in the protein level of ICAD (to approximately 40%) by 24 h in E2A)/), i.e. the rate of decrease in the amount of ICAD in the mutant was in the slower than that in DT40. Thus, presence of PMA/ionomycin, the time courses of alter- ations in the mRNA and protein levels of survivin were virtually similar up to 24 h, but the findings that the protein levels of IAP2 or ICAD were increased or

K. Toyonaga et al.

Fine control of pre-mature B-cell apoptosis by E2A

A

B

C

Fig. 4. Analyses of mechanisms of the resistance to PMA/ionomycin-mediated apoptosis in E2A)/). (A) Effects of PMA/ionomycin treatment on protein levels of survivin, IAP2 and ICAD. Whole proteins were isolated from PMA/ionomycin-treated DT40 and E2A)/) at indicated times up to 24 h, and subjected to SDS–PAGE followed by immunoblotting. Antibody binding was detected using secondary antibodies conjugated to horseradish peroxidase, and then data analysis was performed using a luminescent image analyzer. Left panel: typical immunoblot pattern (DT40 and E2A)/) clone 1). b-actin was used as a control. The apparent molecular masses of marker proteins are indicated. Right panel: time courses of protein levels for survivin, IAP2 and ICAD after treatment with PMA/ionomycin in DT40 (circles) and three E2A)/) clones (1–3) (squares, triangles and diamonds). Data are expressed as percentages of the control (DT40 at 0 h). (B) Effects of PMA/ionomycin treatment on caspase activities. Cell lysates were prepared from PMA/ionomycin-treated DT40 (circles) and E2A)/) clones (1–3) (squares, triangles and diamonds) at indicated times up to 16 h, and then caspase activity assays were performed using appropriate caspase assay kits. Absorbance at 405 nm was measured to determine activities. Data represent the mean of two separate experiments, and error bars indicate standard deviation. (C) Effects of PMA/ionomycin treatment on DNA fragmentation in DT40 and E2A)/). DNA was isolated from DT40 and E2A)/) cells incubated for 0, 8, 16 and 24 h in the presence of PMA and ionomycin, and analyzed by 1.5% agarose gel electrophoresis. The sizes of k-DNA digested with HindIII are indicated in kb. Left panel: typical electrophoregram of DNA extracted from PMA/ionomycin-treated DT40 and E2A)/) (clone 1). Right panel: electrophoregram of DNA extracted from PMA/ionomycin-treated DT40 and three E2A)/) clones (1–3) at 16 h.

FEBS Journal 276 (2009) 1418–1428 ª 2009 The Authors Journal compilation ª 2009 FEBS

1425

K. Toyonaga et al.

Fine control of pre-mature B-cell apoptosis by E2A

the other hand, in E2A)/), the detected protein level of Aiolos, which was already suppressed to a very low level, is further reduced by the treatment, resulting in suppression of apoptotic cell death compared with that of DT40.

Interestingly, [24].

E2A directly activates the EBF gene [26] and regulates expression of several genes, i.e. k5, Rag-1, Vj1 and jo, that are involved in D-JH rearrangement, cell survival, Igj rearrangement, etc [26,27]. E2A directly controls IgH gene expression, and is involved in repressing the Nfil3 and FGFR2 genes in pre-mature B lymphocytes [28]. Thus, E2A is one of the most essential regulators at multiple stages of B-cell development. In T lympho- cytes, PLCc2, Cdk6, CD25, Tox, Gadd45a, Gadd45b, Gfi1, Gfi1b, Socs1, Socs3, Id2, Eto2, Xbp1 etc have been identified as novel E47 target genes using an E2A- deficient lymphoma cell line [29]. Recently, we reported that apoptosis of the chicken DT40 cell line, a pre- mature B-cell line, is cooperatively controlled by GCN5 and BCR stimulation via complex transcriptional regulation of a number of genes encoding BCR signal- ing-related factors, B cell-specific factors, transcription factors and apoptosis-related factors, indicating that both are necessary for apoptosis of DT40 cells [24]. In DT40 cells, BCR signaling is transduced from BCR and membrane-proximal factors (Syk, BTK, BLNK and PLCc2, etc), via mainly PKCd, PKCe and PKCf, to NF-jBs (probably c-Rel and NFp50). This activated signal mediated by BCR signaling is probably trans- ducted separately into two apoptotic pathways, i.e. direct transduction of the signal into the CAD/ICAD system and transduction of the activated signal into the caspase cascade pathway. However, understanding of the participation of most B cell-specific factors in the apoptotic process has remained elusive.

The alterations in expression of E2A and Aiolos (and probably others) in DT40 when exposed to PMA/ionomycin accompany altered expression of vari- ous genes encoding apoptosis-related factors, caspases, caspase-regulating factors and CAD/ICAD (Fig. 3 and Fig. S2), and almost all of these results agreed with those in a previous report [24]. In E2A)/), the altered in the expression of Aiolos (and probably others) presence of PMA/ionomycin results in (and/or accom- panies) slightly different effects on expression of the genes mentioned above, i.e. the influences of PMA/ion- omycin treatment on expression of the survivin and IAP2 (and probably FLIP) genes were more moderate than in DT40, and no effect was in the mutant observed on expression of caspase-8. However, in DT40, the PMA/ionomycin-induced alterations in gene expression of various B cell-specific factors, HDACs, HATs, apoptosis-related factors, caspases, caspase-reg- ulating factors and CAD/ICAD led to changes in the activities of caspase-3, caspase-8 and caspase-9, and in the protein levels of IAP2 and ICAD (Fig. 4A,B), con- sistent with previous in results E2A)/), the noticeable alterations in gene expression of survivin and IAP2 accompanied by unchanged gene expression of caspase-8 (and also various B cell-specific factors, HDACs, HATs, apoptosis-related factors, caspases, caspase-regulating factors and CAD/ICAD) mediated by PMA/ionomycin results in suppression of activities of caspase-3, caspase-8 and caspase-9, and alterations in the protein levels of survivin, IAP2 and ICAD (and probably FLIP) (Fig. 4A,B).

Finally, in E2A)/), the slightly suppressed degrada- tion of ICAD molecules as an effect of reduced cas- pase-3 activity reduces CAD activity, leading to moderate fragmentation of DNA molecules (Fig. 4C). Thus, progress towards apoptotic cell death in E2A)/) is suppressed by collaboration of both BCR signaling and E2A depletion, mainly via moderate changes in amounts of the inhibitors survivin, IAP2 and ICAD (and probably FLIP). Thus, E2A is involved in fine control of pre-mature B-cell apoptosis mediated by BCR signaling via transcriptional regulation of survi- vin, IAP2, FLIP and caspase-8 genes.

FEBS Journal 276 (2009) 1418–1428 ª 2009 The Authors Journal compilation ª 2009 FEBS

1426

Lack of E2A partially prevents the apoptotic cell death seen in DT40 cells treated with PMA/ionomycin, which mimics BCR stimulation (Fig. 2); such apoptosis is completely prevented by either GCN5 deficiency [24] or HDAC2 deficiency, and is significantly accelerated by Aiolos deficiency (Table 1). By analyzing E2A)/), we revealed that E2A upregulates the expression of PKCa, PKCg and PKCl genes, and downregulates the expres- sion of survivin and PKCf genes, among the numerous factors examined (Fig. 1 and unpublished data). PMA/ ionomycin treatment increased expression of the E2A gene and dramatically suppressed that of the Aiolos gene in DT40, but in E2A)/) had no effects or similar effects on the expression of other disrupted genes (Fig. 3A) and genes encoding B cell-specific factors, HDACs and HATs (our unpublished data). These results suggest not only that, among the B cell-specific factors tested, E2A or Aiolos participates preferentially in suppression or acceleration of apoptosis of the DT40 cell line, but also that, among the HAT and HDAC families tested, gene expression of GCN5 and HDAC2, which are essential for apoptotic cell death, are not influenced directly by PMA/ionomycin treatment. On The observations in this study regarding the par- ticipation in apoptosis of Aiolos and HDAC2, which are now being further studied by us, as well as that of GCN5, amongst others, will be useful in elucidat- ing not only the linkage between BCR signaling and

K. Toyonaga et al.

Fine control of pre-mature B-cell apoptosis by E2A

Cells were treated with 10% trichroloacetic acid, collected by centrifugation 20 000 g for 5 min at 4 (cid:2)C, dissolved in 0.5 m Tris/HCl (pH 6.8) containing 2.5% SDS, 10% glyc- erol and 5% 2-mercaptoethanol, and heated at 100 (cid:2)C for 5 min. Immunoblotting was performed as described previ- ously [24]. b-actin was used as a control.

in pre-mature B-cell Immunoblotting

lines, but apoptosis cascades also the molecular mechanism of negative selection or development of B lymphocytes through cross-talk among B-cell signaling, B cell-specific transcriptional regulation and epigenetic chromatin topology altera- tions. These results, combined with other findings obtained in the future, may contribute to clinical understanding of auto-immune diseases and B-cell lymphomas.

Acknowledgements

Experimental procedures

Materials

We thank Y. Takami and H. Suzuki for technical sup- port and H. K. Barman for editorial reading of the manuscript. This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

References

PMA was purchased from Calbiochem (Darmstadt, Ger- many) and ionomycin was purchased from Sigma (St Louis, MO, USA). The antibodies used were anti-ICAD (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), anti- cIAP-2 (Chemicon, Temecula, CA, USA), horseradish per- oxidase-conjugated goat anti-rabbit immunoglobulin and horseradish peroxidase-conjugated rabbit anti-mouse immu- noglobulin (Dako Inc., Glostrup, Denmark).

1 Cancro MP (2005) B cells and aging: gauging the inter- play of generative, selective, and homeostatic events. Immunol Rev 205, 48–59.

2 Singh H, Medina KL & Pongubala JMR (2005) Contin- gent gene regulatory networks and B cell fate specifica- tion. Proc Natl Acad Sci USA 102, 4949–4953.

3 Kee BL & Murre C (2001) Transcription factor regula- tion of B lineage commitment. Curr Opin Immunol 13, 180–185.

4 Riley RL, Van der Put E, King AM, Fransca D &

Blomberg BB (2005) Deficient B lymphopoiesis in mur- ine senescence: potential roles for dysregulation of E2A, Pax-5, and STAT5. Semin Immunol 17, 330–336.

5 Greenbaum S & Zhuang Y (2002) Regulation of early

DT40 cells and all subclones were cultured essentially as described previously [24]. Apoptosis was induced as follows: cells (2 · 106) in 10 mL of culture medium were incubated with 10 ngÆmL)1 PMA plus 1 lm ionomycin or with 10 lgÆmL)1 etoposide at 37 (cid:2)C. Viable cells were counted by the trypan blue dye exclusion method. Flow cytometric analyses, morphological analyses, the caspase activity assay and the DNA fragmentation assay were performed as described previously [24,30].

lymphocyte development by E2A family proteins. Semin Immunol 14, 405–414.

Cell cultures and apoptosis induction

6 Alinikula J, Lassila O & Nera KP (2006) DT40

mutants: models to study transcriptional regulation of B cell development and function. In Reviews and Protocols in DT40 Research, (Buerstedde JM & Takeda S., eds) pp. 189–205. Springer-Verlag, Berlin, Germany.

7 Riley RL, Blomberg BB & Frasca D (2005) B cells,

E2A, and aging. Immunol Rev 205, 30–47.

8 Ikawa T, Kawamoto H, Wright LYT & Murre C

(2004) Long-term cultured E2A-deficient hematopoietic progenitor cells are pluripotent. Immunity 20, 349–360. 9 Lazorchak AS, Wojciechowski J, Dai M & Zhuang Y (2006) E2A promotes the survival of precursor and mature B lymphocytes. J Immunol 177, 2495–2504. 10 Lietz A, Janz M, Sigvardsson M, Jundt F, Dorken B & Mathas S (2007) Loss of bHLH transcription factor E2A activity in primary effusion lymphoma confers resistance to apoptosis. Br J Haematol 137, 342–348.

Total RNAs were isolated from DT40 and its subclones. Reverse transcription was performed using a first-strand DNA synthesis kit (Toyobo, Osaka, Japan) at 42 (cid:2)C for 20 min, followed by heating at 99 (cid:2)C for 5 min. PCRs were performed as described previously [24] using sense primers and antisense primers synthesized according to the EST data deposited in GenBank for the appropriate genes, and listed in previous reports [22–24], except for SIRT1 (sense primer 5¢-CTGTTTTTACCACCAAATCG-3¢ and antisense primer 5¢-CAACTTGTTGCTTGTTGGAT-3¢) and SIRT2 (sense primer 5¢-ATGTCCCTCATGGGCTTCGG-3¢ and antisense primer 5¢-TCACGGCTCTTTGTCGTCCC-3¢). The chicken glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene was used as an internal control. PCR prod- ucts were subjected to 1.5% agarose gel electrophoresis, and analyzed using an LAS-1000plus luminescent image analyzer (Fujifilm, Tokyo, Japan).

FEBS Journal 276 (2009) 1418–1428 ª 2009 The Authors Journal compilation ª 2009 FEBS

1427

Semi-quantitative RT-PCR

K. Toyonaga et al.

Fine control of pre-mature B-cell apoptosis by E2A

11 Murre C (2005) Helix-loop-helix proteins and lympho-

cyte development. Nat Immunol 6, 1079–1086.

12 Rivera R & Murre C (2001) The regulation and func- tion of the Id proteins in lymphocyte development. Oncogene 20, 8308–8316.

25 Aggarwal BB, Bhardwaj U & Takada Y (2004) Regula- tion of TRAIL-induced apoptosis by ectopic expression of antiapoptotic factors. Vitam Horm 67, 453–483. 26 Kee BL, Quong MW & Murre C (2000) E2A proteins: essential regulators at multiple stages of B-cell develop- ment. Immunol Rev 175, 138–149.

27 Lazorchak A, Jones ME & Zhuang Y (2005) New

13 Mikkers H, Allen J & Berns A (2002) Proviral activa- tion of the tumor suppressor E2a contributes to T cell lymphomagenesis in EmuMyc transgenic mice. Onco- gene 21, 6559–6566.

insights into E-protein function in lymphocyte develop- ment. Trends Immunol 26, 334–338.

28 Greenbaum S, Lazorchak AS & Zhuang Y (2004) Dif-

14 Engel I & Murre C (1999) Ectopic expression of E47 or E12 promotes the death of E2A-deficient lymphomas. Proc Natl Acad Sci USA 96, 996–1001.

15 Carey GB, Donjerkovic D, Liu S, Hinshaw JA, Ton-

ferential functions for the transcription factor E2A in positive and negative gene regulation in pre-B lympho- cyte. J Biol Chem 279, 45028–45035.

netti L, Davidson W & Scott DW (2000) B-cell receptor and Fas-mediated signals for life and death. Immunol Rev 176, 105–115.

16 Sen R (2006) Control of B lymphocyte apoptosis by

29 Schwartz R, Engel I, Fallahi-Sichani M, Petrie HT & Murre C (2006) Gene expression patterns define novel roles for E47 in cell cycle progression, cytokine-medi- ated signaling, and T lineage development. Proc Natl Acad Sci USA 103, 9976–9981.

the transcription factor NF-jB. Immunity 25, 871–883.

17 Moscat J, Diaz-Meco MT & Rennert P (2003) NF-jB

activation by protein kinase C isoforms and B-cell func- tion. EMBO Rep 4, 31–36.

18 Foreman AL, Van de Water J, Gougeon ML &

30 Kikuchi H & Imajoh-Ohmi S (1995) Activation and possible involvement of calpain, a calcium-activated cysteine protease, in down-regulation of apoptosis of human monoblast U937 cells. Cell Death Differ 2, 195–199.

31 Matsushita N, Takami Y, Kimura M, Tachiiri S, Ishiai M, Nakayama T & Takata M (2005) Role of NAD- dependent deacetylases SIRT1 and SIRT2 in radiation and cisplatin-induced cell death in vertebrate cells. Genes Cells 10, 321–332.

Gershwin ME (2007) B cells in autoimmune disease: insights from analyses of immunoglobulin variable (Ig V) gene usage. Autoimmun Rev 6, 387–401. 19 Kee BL, Rivera RR & Murre C (2001) Id3 inhibits B lymphocyte progenitor growth and survival in response to TGF-b. Nat Immunol 2, 242–247.

32 Barman HK, Takami Y, Ono T, Nishijima H,

20 Kikuchi H, Barman HK, Nakayama M, Takami Y & Nakayama T (2006) Participation of histones, histone modifying enzymes and histone chaperones in verte- brate cell functions. In Reviews and Protocols in DT40 Research, (Buerstedde JM & Takeda S., eds) pp. 225–243. Springer-Verlag, Berlin, Germany.

Sanematsu F, Shibahara K & Nakayama T (2006) Histone acetyltransferase 1 is dispensable for replication-coupled chromatin assembly but contributes to recover DNA damage created following replication blockage in vertebrate cells. Biochem Biophys Res Commun 345, 1547–1557.

Supporting information

21 Takami Y, Kikuchi H & Nakayama T (1999) Chicken histone deacetylase-2 controls the amount of the IgM H-chain at the steps of both transcription of its gene and alternative processing of its pre-mRNA in the DT40 cell line. J Biol Chem 274, 23977–23990.

22 Nakayama M, Suzuki H, Yamamoto-Nagamatsu N, Barman HK, Kikuchi H, Takami Y, Toyonaga K, Yamashita K & Nakayama T (2007) HDAC2 controls IgM H- and L-chain gene expressions via EBF1, Pax5, Ikaros, Aiolos and E2A gene expressions. Genes Cells 12, 359–373.

23 Kikuchi H, Takami Y & Nakayama T (2005) GCN5: a supervisor in all-inclusive control of vertebrate cell cycle progression through transcription regulation of various cell cycle-related genes. Gene 347, 83–97.

The following supplementary material is available: Fig. S1. Influences of E2A deficiency on protein levels of survivin, PKCg and PKCf. Fig. S2. Effects of the PMA/ionomycin treatment on gene expression of B cell-specific factors, HDACs, HATs, apoptosis-related factors, CAD/ICAD, caspases and caspase-regulating factors in two E2A)/)clones. This supplementary material can be found in the online version of this article.

24 Kikuchi H & Nakayama T (2008) GCN5 and BCR sig- nalling collaborate to induce pre-mature B cell apopto- sis through depletion of ICAD and IAP2 and activation of caspase activities. Gene 419, 48–55.

FEBS Journal 276 (2009) 1418–1428 ª 2009 The Authors Journal compilation ª 2009 FEBS

1428

Please note: Wiley-Blackwell is not responsible for the content or functionality of any supplementary materials supplied by the authors. Any queries (other than missing material) should be directed to the corresponding author for the article.