From the
CD40 activation is critical for B-cell function, leading to
activation and expression of cell surface markers, proliferation,
immunoglobulin class switching and inhibition of programmed cell death
(PCD). Germinal center B-cells, for example, can be prevented from
undergoing PCD by CD40 activation. The mechanism by which PCD is
inhibited has been an enigma. A potential role for A20, a novel zinc
finger protein, in inhibiting B-cell apoptosis was suggested by our
previous finding that it is induced by the Epstein-Barr virus LMP-1
gene product, a potent cell death inhibitor. We now show that CD40
activation induces A20 and that expression of A20 renders B-cell lines
resistant to PCD. Additionally, we show that CD40 activation of A20
expression is mediated by inducible binding of NF-
Programmed cell death (PCD)
CD40, a member of the tumor necrosis factor (TNF)
receptor family, mediates cognate help of T-cells for the survival,
growth and differentiation of B-cells. The ligand for CD40 (CD40L) is
expressed on activated CD4
We have previously shown that A20, a novel zinc
finger protein, is induced by TNF and confers resistance to TNF killing
(16) and is induced by the LMP1 gene product of EBV
(17) ,
suggesting a potential role in inhibiting B-cell PCD. In this report we
now show that CD40 activation leads to the induction of A20 and that
expression of A20 renders B-cell lines resistant to PCD. Additionally,
evidence is provided for a critical role for Thr
The construction and characterization of the native and
mutant CD40 expression vectors has been described
previously
(22) .
The A20 promoter reporter plasmid A20PstCAT,
which contains nucleotides -233 to +12 of the wild type A20
promoter inserted upstream of the chloramphenicol acetyltransferase
(CAT) gene, has been previously described
(23, 24) . The
A20dmCAT construct is identical except both
293 cell transient transfections were
performed as per published procedures using 5 µg of plasmid/2
We thank Ian Jones for help with the preparation and
Suzanne Suchard for critical reading of the manuscript.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
B complexes to
the A20 promoter and provide evidence for a critical role for
Thr
(in the CD40 cytoplasmic domain) in activating
NF-
B.
(
)
is of
critical importance not only in the maintenance of tissue homeostasis
but also during development and in immune selection. In an attempt to
understand the molecular basis of PCD in lymphoid cells, a number of
factors have been identified that either directly initiate PCD, such as
activation of the Fas receptor
(1, 2) , or inhibit cell
death, such as activation of the CD40 receptor
(3) and
expression of the LMP1 gene product of the Epstein-Barr virus
(EBV)
(4) .
T-cells
(5) and on
binding CD40 expressed on B-cells
(6) there ensues a dramatic
alteration in B-cell physiology that includes activation, induction of
B-cell proliferation, immunoglobulin (Ig) isotype switching and rescue
from PCD
(7, 8, 9) . The importance of CD40 in
B-cell biology has been emphasized by the recent discovery that genetic
mutations in the gene for
CD40L
(10, 11, 12, 13, 14) or
defects in CD40 signaling
(15) result in an immunodeficiency
syndrome characterized by defective Ig class switching and lack of
germinal center formation. This is in keeping with previous studies
showing that CD40 signaling blocks spontaneous apoptosis of germinal
center B-cells and may therefore be instrumental in the selection of
B-cells undergoing somatic hypermutation in germinal
centers
(3) .
in the
CD40 cytoplasmic domain in activating NF-
B and the A20 promoter.
Cell Culture
The EBV-negative cell lines Louckes
and BJAB were maintained in RMPI 1640 and 10% fetal bovine serum
(HyClone, Logan, UT) supplemented with nonessential amino acids and
antibiotics (100 units/ml penicillin, 50 mg/ml streptomycin). The human
embryonic kidney cell line was grown in Dulbecco's modified
Eagle's medium containing 10% fetal bovine serum and nonessential
amino acids plus antibiotics.
CD40 Activation of Louckes and B Cells
Louckes
cells were treated for the indicated periods of time (Fig. 1)
with anti-CD40 ascites (used at a 1:10,000 dilution of the ascites
HG-14, gift from Tom Tedder, Duke University; Ref. 18) or with
isotype-matched contol antibody in the presence or absence of
cycloheximide (CHX, 10 µg/ml).
Figure 1:
CD40 activation induces A20 transcript
(A and B). RNA was isolated from the EBV-negative
Louckes B-cell line or from primary B-cells following exposure to
agonist CD40 monoclonal or isotype-matched control antibody in the
absence or presence of CHX. A20 expression was determined by Northern
blot analysis. RNA isolated from human umbilical vein endothelial cells
treated with TNF, and CHX was run as a positive control for A20
expression. The bottom part of each panel is a photograph of ethidium
bromide stained 18S rRNA to indicate equivalency of
loading.
Mononuclear cells were isolated
from the peripheral blood of healthy volunteers and monocytes removed
by adherence as described in Ref. 19. B-cells were separated from
T-cells by rosetting with 2-aminoethylisothiouronium bromide-treated
sheep red blood cells again as described in Ref. 19. Isolated B-cells
were either treated with monoclonal anti-CD40 ascites (at a dilution of
1:10,000) or with isotype-matched control antibody for 4 h prior to RNA
extraction.
Northern Blot
Total RNA was isolated using a
previously described guanidine hydrochloride procedure
(20) . Ten
µg of RNA was resolved on a 1% agarose-formaldehyde gel, blotted
onto nitrocellulose, and hybridized to a P-labeled A20
cDNA probe using hybridization and washing conditions as described
previously
(20) .
Plasmids
The A20 expression construct was obtained
by cloning the entire coding region of A20 into the pZEMneo vector
(21) which also contained a neoR cassette allowing for selection
in G418.
B sites within this
region of the A20 promoter have been inactivated by site-directed
mutagenesis
(23, 23) .
Transfection and CAT Assay
Louckes and BJAB cells
(5 10
) were transfected by electroporation (200 V,
960 microfarads) and after 2 days in culture, clonal lines were
obtained by selection in medium containing 3.5 mg/ml G418 (Geneticin,
Life Technologies, Inc.).
10
cells
(25) . Transfected cells were
treated for 10 h with a 1:10,000 dilution of the HG-14 anti-CD40
ascites or isotype-matched control antibody 36 h post-transfection.
Cells were harvested, extracts prepared, and CAT enzyme activity
assayed according to standard protocols
(23, 24) . CAT
activity was quantitated using a Betascope 603 Blot analyzer (Betagen,
MA).
Metabolic Labeling and Immunoprecipitation
A20
expression in the stably transfected cell lines was assessed by
metabolic labeling with [S]methionine and
cysteine (100 µCi/ml) followed by immunoprecipitation as described
previously
(17) .
Flow Cytometry Analysis
Vector control and CD40
transfected cells were washed once in phosphate-buffered saline
containing 1% bovine serum albumin, blocked with 10% normal goat serum
for 20 min, and then incubated with a 1:500 dilution of the HG-14
anti-CD40 ascites or isotype-matched control antibody for 30 min at 4
°C. After washing twice in phosphate-buffered saline, cells were
incubated with a 1:50 dilution of fluorescein-conjugated goat
anti-mouse IgG (Cappel) for 20 min. at 4 °C, washed twice and
analyzed by flow cytometry on a FACScan (Becton-Dickinson).
Nuclear Extracts and Electrophoretic Mobility Shift Assay
(EMSA)
Nuclear extracts used in EMSAs were prepared as described
previously
(23, 24) , from 293 cells expressing either
the native or mutant (CD40A) receptor. Cells were treated with a
1:10,000 dilution of the HG-14 anti-CD40 ascites prior to extract
preparation. EMSA was performed as described by us in previous
publications
(23, 24) . Briefly, a 43-base pair
double-stranded oligonucleotide containing residues -74 to
-32 of the A20 promoter (A20B) was fill-in labeled using
Klenow fragment. Binding reactions containing 10 µg of nuclear
protein extract, 1 µg of poly(dI
dC)-(dI
dC), 0.1 ng of
P-end-labeled A20
B probe, and binding
buffer
(23, 24) were incubated 15 min at room
temperature. For competition studies, 10 µg of unlabeled
double-stranded oligonucleotide was added to the binding reaction. The
sequences of these oligonucleotides have been published
previously
(23, 24) . Products were resolved on a 4%
polyacrylamide gel and analyzed by autoradiography.
Apoptosis Assay
Apoptosis was assessed by the use
of the fluorescent DNA-staining dye acridine orange to reveal nuclear
morphology essentially as described in Refs. 4 and 26.
CD40 Activation Induces A20 Expression
The
effect of CD40 activation on A20 expression was examined by Northern
blot analysis. The activation of the CD40 receptor, which abrogates
B-cell apoptosis, resulted in the induction of A20 both in EBV-negative
B-cell lines, Louckes (Fig. 1A) and BJAB (data not
shown) and in primary B-cells (Fig. 1B). The induction
of A20 transcript was rapid, occurring within an hour, and in the
presence of CHX, there was a superinduction of the transcript. This
finding is consistent with A20 being an immediate early or primary
response gene whose induction is independent of intervening protein
synthesis.
A20 Expression Protects B-cells from Apoptosis
To
determine whether or not A20 influenced B-cell survival, the
EBV-negative human B lymphoma cell lines BJAB and Louckes were
transfected with an A20 expression construct or vector alone as a
control. EBV-negative B-cell lines are sensitive to apoptosis following
transfer into medium containing a sub-optimal concentration (0.1%) of
fetal calf serum (FCS) and have been used extensively to characterize
factors that modulate B-cell apoptosis
(4) . An additional
advantage of these cells is that entry into apoptosis is easily
recognized by staining cellular DNA with acridine orange and assessing
nuclear fragmentation by fluorescence microscopy
(Fig. 2A). As shown in Fig. 2(B and
C), four independent A20 transfectants on a BJAB background
and three on a Louckes background expressed A20 protein as confirmed by
metabolic labeling and immunoprecipitation. These A20 transfectants
displayed significant, albeit partial, resistance to PCD when compared
to an equal number of vector control transfectants.
Figure 2:
A20
expression confers resistance to apoptosis. A, acridine orange
staining of vector control transfected Louckes cells maintained in 10%
FCS (leftpanel) or 0.1% FCS (rightpanel) for 5 days. Nuclear condensation characteristic of
apoptosis is clearly evident. B and C, BJAB
(B) and Louckes (C) clonal cell lines transfected
with an A20 expression construct and the corresponding vector control
transfectants (VB and VL) were transferred to 0.1%
FCS containing medium and 6 days later scored for apoptotic
morphological changes following acridine orange staining. A minimum of
100 cells were counted from triplicate cultures and values shown
represent averages ± standard deviations. The bottompanels of B and C show A20expression in transfected and vector control lines as assessed by
metabolic labeling and immunoprecipitation.
Partial
resistance to PCD in B-cells is also provided by Bcl-2
(27) ;
however, contrary to an earlier report, more recent work by the same
authors now indicates that Bcl-2 is not CD40-inducible
(28) .
Nevertheless, it is entirely feasible that the heightened state of
resistance to PCD conferred by CD40 activation is due to a synergistic
interaction between A20 and a Bcl-2-like protein. In fact, we have
recently found that Bcl-x, a related anti-apoptotic gene, is
CD40-inducible.(
)
A20 Induction upon CD40 Activation Is
Mediated by
The exact mechanism
by which CD40 transduces signals is unknown. CD40 does possess a short
cytoplasmic tail (63 amino acid residues) and mutagenesis studies
indicate that ThrB Sites in the A20 Promoter
in the cytoplasmic domain is essential
for signal transduction
(29) . Since CD40 possesses no intrinsic
signaling capacity (e.g. kinase activity), signal transduction
is likely to be mediated by associated molecules
(22) .
Regardless of the exact mechanism, it is now evident that CD40
activation induces the transcription factor NF-
B. NF-
B
consists of a variety of homo- and heterodimeric complexes homologous
to the proto-oncogene c-rel with the ability to
differentially regulate transcription through recognition of variant
B elements. Given that LMP1 induction of A20 is mediated through
the inducible binding of NF-
B to
B sites within the A20
promoter
(23, 24) , we asked whether CD40 activation
similarly induces A20. To address this question and to determine the
exact role of Thr
in CD40 signaling, two stably
transfected cell lines were established that expressed either native
CD40 or mutant CD40 in which the Thr
was converted to Ala
(CD40A). CD40 surface expression in the stably transfected 293 cells
was confirmed by FACS analysis using HG-14, an agonist CD40 monoclonal
antibody
(18) (Fig. 3A). Transient transfections
were performed using these cell lines to determine first, if CD40
induction of A20 was mediated by
B elements within the A20
promoter and second, if Thr
was essential for A20
induction. Transfection of the wild type A20 promoter CAT reporter
construct, A20PstCAT, led to a significant induction of CAT activity on
cross-linking surface expressed CD40 with the agonist monoclonal
antibody (Fig. 3B). In contrast, no significant
difference was seen on transfection with A20dmCAT in which the two
B elements were mutated, confirming the importance of these
B
elements in conferring CD40 responsiveness to the A20 promoter.
Additionally, no significant induction of the native A20PstCAT
construct was observed on cross-linking mutant CD40 (CD40A), indicating
that Thr
was critical for engagement of the
B signal
transduction pathway. Electrophoretic mobility shift assays (EMSA) were
performed to confirm that CD40 activation induced the binding of
NF-
B proteins to
B sites within the A20 promoter. Nuclear
extracts prepared from cells expressing native CD40, in the absence or
presence of agonist CD40 antibody, were incubated with a
double-stranded oligonucleotide probe containing both A20
B
sequences. An electrophoretically retarded complex was clearly induced
on CD40 activation (Fig. 3C) and was effectively
competed by a 50-fold molar excess of double-stranded oligonucleotides
containing the A20 or HIV
B elements, but not by an unrelated IL-2
octamer binding sequence. In contrast, no electrophoretically retarded
complex was evident in nuclear extracts prepared from cells expressing
mutant CD40 (CD40A) in the presence or absence of agonist CD40
antibody. Taken together, these findings indicate that CD40 activation
of A20 expression is mediated by the inducible binding of NF-
B
complexes to the A20 promoter and provide evidence for a critical role
for Thr
in activating NF-
B.
Figure 3:
Thr is required for
CD40-mediated activation of A20 promoter CAT expression. A,
293 cells were stably transfected with either a native or a mutant CD40
(Thr
Ala; designated CD40A) expression construct
and surface expression confirmed by FACS analysis using an agonist CD40
monoclonal (HG-14) or isotype-matched control antibody (C-Ab).
B, representative cell lines expressing either native or
mutant CD40 (CD40A) were transiently transfected with the indicated A20
promoter CAT constructs and CAT activity measured following exposure to
agonist or isotype-matched control antibody. C, activation of
native CD40 induces binding of NF-kB to the A20 promoter. Nuclear
extracts were prepared from cells expressing either the native or
mutant CD40 receptor following stimulation with agonist monoclonal
(+) or isotype-matched control antibody (-), and
electrophoretic mobility assays performed as described previously. For
competition studies, 10 µg of unlabeled double
stranded-oligonucleotide was added to the binding reaction. The
specific DNA-protein complex is indicated by the
arrow.
In summary, these
studies demonstrate that A20 significantly protects B-cell lines from
PCD and suggests that this may be an important mechanism utilized by
physiological inhibitors of PCD like CD40 and viral inhibitors of PCD
such as the LMP-1 gene product of EBV. Finally, these data are the
first to demonstrate that A20 inhibits PCD induced by factors other
than TNF, in this case serum deprivation, and thereby indicates a
broader role for this novel zinc finger protein in inhibiting PCD,
especially in B-cells. A cautionary note, however, is that,
unlike Bcl-2 and related family members, A20 does not appear to be a
general antidote against programmed cell death as certain forms of
death, such as glucocorticoid-induced apoptosis that are potently
inhibited by Bcl-2, are not inhibited by A20.(
)
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.