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
Trang 1apoptosis 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
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 varivari-ous aspects of the normal development of B lymphocytes and func-tion of the immune system Such developmental activi-ties require numerous transcription factors, i.e the
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-caspase-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.
Trang 2basic 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
throughout development of lymphocytes in normal
mice after the observed block in their knockout mice
[6] Thus, their physiological functions beyond
develop-mental arrest remain poorly understood
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 types [5,7] Recently, 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
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-ing immune responses [1,15,16] Recent genetic
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
factors, including E2A, on the BCR-mediated
apop-tosis of pre-mature B cells remains unclear, when
apoptosis is triggered by antigen stimulation
In order to better understand the roles of histone
acetyltransferases (HATs), histone deacetylases
(HDACs) and B cell-specific transcription factors in
B-cell functions, we have systematically generated
vari-ous homozygous mutants, including HDAC2)/),
GCN5)/), Aiolos)/) and E2A)/), using gene-targeting
techniques on the DT40 cell line, which was established
from chicken pre-mature B lymphocytes [20] Our
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 line, through depletion of ICAD [inhibitor of caspase-activated DNase (CAD)] and inhibitor of apop-tosis 2 (IAP2), and activation of caspase activities [24] 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
To assess the influence of E2A deficiency on gene expression of apoptosis- and BCR signaling-related fac-tors, and other facfac-tors, 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
Trang 3of 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 facfac-tors, 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
-/-We examined the influences of phorbol 12-myristate
13-acetate (PMA)/ionomycin and etoposide on
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
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.
Trang 4of DT40 and E2A)/)(Fig 2B) As expected, the
viabil-ity of the two cell lines did not differ in the absence of
PMA/ionomycin However, in the presence of PMA
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
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 Using gene-targeting techniques, we systematically gen-erated several homozygous DT40 mutants that lacked genes encoding B cell-specific transcription factors, HDACs or HATs [20,22] Our previous results revealed that GCN5 and PMA/ionomycin treatment
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 l M ) 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)1PMA plus 1 l M ionomycin at 37 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.
Trang 5cooperatively 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
To determine whether or not expression of the
B cell-specific transcription factors, HDACs and HATs
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 expresexpres-sion
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-ing retrovirus AKT8 in rodent T cell lymphoma (AKT), apoptosis-inducing factor (AIF) and poly (ADP-ribose)polymerase (PARP) (also shown in 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)/) 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-trol level by 24 h, and the ICAD mRNA level
Table 1 Influences of depletion of B cell-specific transcription
fac-tors, HDACs and HATs on resistance to apoptosis induced by
PMA/ionomycin treatment.
Resistance for PMA/ionomycin
B cell-specific factors
HDACs
HATs
Trang 6decreased 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]
With regard to caspases, E2A depletion showed no
effects on expression of 3, 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
presence of PMA/ionomycin Interestingly, in DT40,
PMA/ionomycin treatment increased the caspase-8
mRNA level by 3 h (to approximately 160%) and
this level remained unchanged at 24 h, but the treat-ment showed no change in the transcript level of 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
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 level in DT40, but remained unchanged in 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.
Trang 7Interestingly, whereas the IAP2 mRNA level in DT40
was significantly decreased by PMA/ionomycin
treat-ment at 3 h (to approximately 30%) and thereafter
remained unchanged, the transcript level in E2A)/)
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 influenced by the drug treatment 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
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, 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
slower than that in DT40 Thus, in E2A)/), in the
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
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) Interestingly, in E2A)/), PMA/ionomycin treatment 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 approxiapproxi-mately 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)
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
Discussion
In recent years, numerous studies have been performed
to determine the physiological target genes of E2A
Trang 8B
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.
Trang 9E2A 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
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 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
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 expression of Aiolos (and probably others) in the 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
in the mutant than in DT40, and no effect was 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 results [24] Interestingly, in 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
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 signalelucidat-ing and
Trang 10apoptosis cascades in pre-mature B-cell lines, but
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
Experimental procedures
Materials
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)
Cell cultures and apoptosis induction
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 37C 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]
Semi-quantitative RT-PCR
Total RNAs were isolated from DT40 and its subclones
Reverse transcription was performed using a first-strand
DNA synthesis kit (Toyobo, Osaka, Japan) at 42C for
20 min, followed by heating at 99C 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)
Immunoblotting Cells were treated with 10% trichroloacetic acid, collected
by centrifugation 20 000 g for 5 min at 4C, dissolved in 0.5 m Tris/HCl (pH 6.8) containing 2.5% SDS, 10% glyc-erol and 5% 2-mercaptoethanol, and heated at 100C for
5 min Immunoblotting was performed as described previ-ously [24] b-actin was used as a control
Acknowledgements
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
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 lymphocyte development by E2A family proteins Semin Immunol 14, 405–414
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