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From the
Received for publication, February 6, 2002, and in revised form, April 16, 2002
Apoptosis is an integral aspect of B lymphocyte
development and homeostasis and is regulated by the engagement of
antigen costimulatory and cytokine receptors. Although it is well
established that interleukin 4 (IL-4) is a potent anti-apoptotic
cytokine for B lymphocytes, little is known about the IL-4-induced
molecular events regulating cell survival. Stat6 is rapidly activated
after IL-4 stimulation, but its role in B lymphocyte apoptosis has not been explored. In this report we demonstrate that Stat6 is a critical signaling molecule for IL-4 in protecting primary B cells from passive
and Fas-induced cell death. We show that expression of the Bcl-2 family
member, Bcl-xL, is induced maximally by IL-4 and anti-IgM/IL-4 in a
Stat6-dependent manner. Additionally, we demonstrate that
bcl-xL transcription is likely to be directly activated
through a Stat6 binding site in the bcl-xL-flanking region.
Finally, reconstitution of Stat6-deficient splenic B cells with Bcl-xL
was able to protect those cells from Fas-induced cell death. These
results suggest that the anti-apoptotic activity of IL-4 in B cells is
mediated through the activation of Stat6 and subsequent transcription
of Bcl-xL.
Both resting and activated mature B lymphocytes require cytokine
or antigenic survival signals for viability and die passively by
neglect in their absence (for review, see Ref. 1). Primed B lymphocytes
can also be actively programmed to die through engagement of cell
surface Fas by encountering Fas ligand (FasL) on activated T cells.
These highly regulated processes are thought to be important for
removing autoreactive B cells, terminating an immune response, and
maintaining a state of equilibrium.
The activation or suppression of apoptosis is regulated in part by the
members of the Bcl-2 family, proteins found in various cytoplasmic
membranes including mitochondrial membranes (for review, see Refs. 2
and 3). Several members, including Bcl-2 and Bcl-xL, are antagonistic
to apoptotic stimuli and are thought to function by preserving
mitochondrial membrane integrity and preventing cytochrome c
release into the cytoplasm. Other Bcl-2 family members, such as Bax and
Bad, promote apoptosis. These pro-apoptotic proteins are thought to
antagonize the function of Bcl-2 and Bcl-xL through heterodimer
formation. Consequently, the relative levels of intracellular pro-
versus anti-apoptotic Bcl-2 family proteins are critical in
determining the viability of a cell. Clearly it is of interest to
understand how the expression of these proteins is regulated in
response to external stimuli.
The cytokine IL-41 has been
demonstrated to be a potent cofactor for B and T lymphocyte
proliferation and differentiation (for review, see Ref. 4).
Additionally, its role as an anti-apoptotic factor has been studied
extensively. IL-4 has been demonstrated to prevent cell death by
neglect of resting T and B lymphocytes after growth factor withdrawal
in culture (5, 6). It can also prevent apoptosis of B cells induced by
Ig cross-linking and glucocorticoids and render activated B cells
insensitive to Fas ligation (7-9). However, the molecular events
induced by IL-4 that regulate cell survival are poorly understood.
Signal transducer and activator of transcription 6 (Stat6) is a
critical mediator of IL-4 signaling (for review, see Ref. 10). Stat6 is
a latent cytoplasmic transcription factor recruited specifically to the
IL-4 receptor and activated by phosphorylation after IL-4 stimulation.
Activated Stat6 homodimers are capable of translocating to the nucleus
where they can influence the transcription of IL-4-responsive genes.
The importance of Stat6 in IL-4 signal transduction has been
demonstrated in Stat6-deficient lymphocytes, which are unable to
proliferate normally in response to IL-4, are defective in their
ability to activate IL-4 responsive genes, and are unable to undergo
IL-4-dependent Th2 differentiation (11-13). However, Stat6
does not appear to be required for the anti-apoptotic effects of IL-4
in resting or activated T cells or in myeloid cell lines (5, 14-16).
This raises the question of whether Stat6 is a required intermediary
for the anti-apoptotic effect of IL-4 on B cells.
In this study we demonstrate that in contrast to T lymphocytes, Stat6
signaling is involved in the anti-apoptotic activities of IL-4 in
primary B cells that were committed to die by either growth factor
withdrawal or by Fas ligation. We find that Stat6-deficient B cells are
defective in their ability to maximally induce expression of the
anti-apoptotic Bcl-2 family member, Bcl-xL, in response to IL-4
stimulation. Additionally, we have identified a Stat6-responsive element upstream of the bcl-xL, gene suggesting that Stat6
is directly responsible for activating the IL-4-induced transcription of this anti-apoptotic factor. Finally, reconstitution of Bcl-xL expression in Stat6-deficient primary B cells renders the cells resistant to Fas-induced cell death.
Mice--
Stat6-deficient and insulin receptor substrate 2 (IRS-2)-deficient mice were generated and maintained as described
previously (13, 17).
Lymphocyte Culture--
Naïve Th cells were purified
from lymph nodes by cell sorting (Mo Flo) using anti-CD4 and anti-CD62L
(BD PharMingen) to 98% purity. Splenic B lymphocytes were purified
to 95% purity using Macs magnetic beads specific for B220 (Miltinyi
Biotec) per the manufacturer's instructions. Both T and B lymphocytes
were cultured at 1-2 × 106 cells/ml in RPMI 1640 supplemented as described previously (13). Recombinant IL-4 (Peprotech)
was added to the indicated cultures at a concentration of 10 ng/ml.
Anti-IgM (Jackson Laboratories) was added to the indicated cultures at
a concentration of 5 µg/ml. Anti-CD40 (BD PharMingen) was added to
the indicated cultures at a concentration of 5 µg/ml.
Immunoblot Analysis--
Whole cell extracts were prepared by
lysing cells in 50 mM Tris, 0.5%Nonidet P-40, 5 mM EDTA, 50 mM NaCl and clearing the lysates by
microcentrifugation. 4 µg of protein were separated on a 10%
polyacrylamide gel and transferred to an Optitran membrane (Schleicher
& Schuell). The immunoblots were blocked for 1 h at room
temperature in 5% dry milk in TBST (50 mM Tris, pH 7.5, 100 mM NaCl, 0.03% Tween 20) and incubated with either
Bcl-xL- or Bcl-2-specific antibodies (Santa Cruz Biotechnology) diluted
1:1000 in blocking buffer overnight at 4 °C. The blots were washed
with TBST and incubated with either horseradish peroxide-conjugated anti-mouse or anti-rabbit antibody (Santa Cruz Biotechnology) in
blocking buffer for 1 h at room temperature. After washing the
blots with TBST, detection was carried out using enhanced chemiluminescence (ECL, Amersham Biosciences) according to
manufacturer's instructions.
Northern Analysis--
Total RNA was isolated using TRIzol RNA
isolation reagent (Invitrogen). The RNA was separated on a 1.5%
agarose, 6% formaldehyde gel and transferred to GeneScreen
(PerkinElmer Life Sciences) membrane. The membrane was hybridized with
radiolabeled cDNA probes for bcl-xL, bcl-2,
and Propidium Iodide Analysis--
Lymphocytes were cultured as
described in the figure legends, pelleted by centrifugation, and fixed
in 40% EtOH. The cells were treated with RNase A (50 µg/ml) for 45 min at 37 °C and subsequently stained with 700 mM
propidium iodide in 3.8 × 10 Transient Transfections and Luciferase Assays--
5 × 106 B lymphoma cells were combined with 10 µg of both the
reporter and a Stat6 expression plasmid (18) in 0.4 ml supplemented RPMI medium. The cells were transfected using a Bio-Rad electroporator (280 V, 975 microfarads) and placed on ice for 10 min. The individual transfectants were then split into two cultures and cultured overnight in 2.5 ml of supplemented RPMI medium. Recombinant IL-4 (10 ng/ml) was
added to the indicated cultures 24 h after transfection, and the
cells were harvested after an additional 24 h. Luciferase assays
were performed using the luciferase assay system per the manufacturer's instructions (Promega). The 4×Stat6 gene constructs were generated by synthesizing double-stranded oligos that span the
bcl-xL Stat6 site and cloning them into the BglII
site of pTkluc. The sequence of the bcl-xL Stat6 wild type
oligo is 5'-GATCCCCCGGTCTTCTTCAGGGGAAACTGAGGCCGGCTTCA-3' and the
sequence of the bcl-xL Stat6mut oligo is
5'-GATCCCCCGGTCTTCTAGAGGGCTAACTGAGGCCGGCTTCA-3'. The bcl-xL
promoter region was cloned using primers 5'-CTAAACCCATACCTCCGGGA-3' and
5'-GCGCAAGCTTGGGCTCAACCAGTCCATTGTC-3', digested with
HindIII, and cloned into pGL2 (Promega). Single copies of
the 40-bp wild type and mutant Stat6 sites were cloned upstream of
the promoter into the Bglll site.
Retroviral Transduction of B Cells--
The GFP-RV
bicistronic vector was obtained from K. Murphy (19), and the
Phoenix-Eco packaging cell line was obtained from G. Nolan (20). The
bcl-xL cDNA was ligated with XhoI linkers and
cloned into the XhoI cloning site of GFP-RV. Transfection of
the packaging cell line was performed using Effectene (Qiagen), and
viral supernatants were harvested 48-72 h later.
Lipopolysaccharide-activated (25 µg/ml, 24 h) purified splenic B
cells were retrovirally transduced by incubation of 1 × 107 B cells at 1 × 106/ml with an equal
volume of viral supernatant, 8 µg/ml Polybrene, and 25 µg/ml
lipopolysaccharide. The cultures were centrifuged at 500 × g for 40 min at room temperature. Infections were repeated 24 h later with a resulting transduction efficiency as assessed by
GFP expression of 10-35%. GFP+ cells were sorted by
fluorescence-activated cell sorter (BD PharMingen) 48 h later and
cultured in 5 µg/ml anti-CD40 and lipopolysaccharide for 48 h.
Anti-Fas (BD PharMingen) was added at 0.06 µg/ml to the cultures, and
cells were analyzed for DNA content by propidium iodide analysis.
Electrophoretic Mobility Shift Assay--
Nuclear extracts were
prepared from BJAB B cells cultured in the presence or absence of IL-4
for 1 h as described previously (21). 1 µg of nuclear extracts
were used in electrophoretic mobility shift assay as described
previously (22). Double-stranded oligos spanning the 40-bp of
bcl-xL genomic sequence described under "Transient
Transfections and Luciferase Assays" were used in the assay. The
Stat6 and Stat5 antibodies were obtained from Santa Cruz Biotechnology.
Stat6 Signaling Contributes to IL-4-mediated Rescue of Primary B
Cells from Apoptosis--
To examine the contribution of Stat6 in
regulating cell death in lymphocytes, we utilized mice that were
genetically deficient for this protein (13). We initially studied the
requirement for Stat6 in the rescue of B and T cells from death after
cytokine withdrawal. Purified splenic B or T cells from Stat6-deficient and wild type mice were cultured in the presence or absence of IL-4
overnight and subsequently analyzed for the presence of apoptotic cells
by propidium iodide staining. As previously reported, a significant
proportion of both wild type and Stat6-deficient T and B lymphocytes
were found to be subdiploid after culture without cytokine (Fig.
1) (5, 6). In agreement with previously
published reports, IL-4 served as an effective anti-apoptotic factor
for resting T cells, rescuing 62% of those cells from apoptosis,
regardless of Stat6 expression (5, 14) (Fig. 1A). The
addition of IL-4 to the wild type B cell culture also rescued ~62%
of those cells from apoptosis (Fig. 1B). In contrast to T
cells, however, the addition of IL-4 to the Stat6-deficient B cell
culture resulted in only a partial rescue from apoptosis (Fig.
1B).
IRS-2 is a second, well characterized signaling mediator that directly
binds to the IL-4 receptor (23). In vitro transfection studies of IL-4 receptor mutants in myeloid cells suggests that IRS-2
is involved in regulating mitogenic and anti-apoptotic signals from
IL-4 (24, 25). The role IRS-2 plays in regulating IL-4 responses in
primary B cells has not been reported. To determine whether IRS-2 plays
a role in protecting primary B lymphocytes from apoptosis after IL-4
stimulation, we repeated these studies in B lymphocytes from
IRS-2-deficient mice (17). In contrast to what we find for Stat6, B
lymphocytes purified from IRS-2-deficient mice were still efficiently
rescued from apoptosis by IL-4 (Fig. 1C). These results
suggest that Stat6, but not IRS-2, plays an important role in the
rescue of primary B cells from apoptosis induced by growth factor withdrawal.
It has been previously demonstrated that splenic B cells stimulated
through CD40 up-regulate Fas expression and become susceptible to
Fas-induced apoptosis in vitro, whereas treatment of
CD40-stimulated B cells with either IL-4 or anti-IgM results in
resistance to Fas-mediated cell death (8, 26). To determine whether
Stat6 is required for IL-4-induced Fas resistance, we purified splenic B cells from both wild type and Stat6-deficient mice and cultured the
cells for 48 h in the presence of stimulatory antibodies to CD40.
Fluorescence-activated cell sorter analysis of these cells indicated
that cell surface Fas expression was induced similarly on both cell
populations (data not shown). As expected, the subsequent culture of
these cells with antibodies to Fas resulted in signaling for cell
death, and combined treatment of the wild type cells with IL-4 in
conjunction with anti-CD40 resulted in protection from Fas-induced
apoptosis (40% rescue from apoptosis) (Fig.
2). In contrast, IL-4 had no effect on
the viability of CD40-stimulated Stat6-deficient B cells after
treatment with anti-Fas (Fig. 2). Both wild type and Stat6-deficient
cells were equally protected from apoptosis by the addition of anti-IgM
to the cell cultures (55% rescue from apoptosis), indicating that the
defect observed in the Stat6-deficient B cells is specific to IL-4
signaling (Fig. 2). Additionally, wild type B cells treated with both
IL-4 and anti-IgM were afforded even greater protection from
Fas-induced apoptosis than were B cells treated with either alone,
whereas Stat6-deficient B cells did not respond with the same effect
(Fig. 2). These results were duplicated in a related experimental
system using CD40L to up-regulate Fas expression and Fas ligand
(FasL)-dependent Th1 cell-mediated cytotoxicity to kill the
cells as previously described (26) (data not shown). The above results
indicate that Stat6 signaling is involved not only in IL-4-induced
rescue from passive cell death by growth factor withdrawal but is also absolutely required for IL-4-induced protection from Fas-mediated apoptosis in B cells.
Stat6 Is Required for Maximal IL-4-induced Bcl-xL
Expression--
Based on the above observations, we presumed that
IL-4-dependent Stat6 activation induces the transcription
of a factor that protects splenic B cells from apoptosis. It has been
shown in a number of systems that the expression of anti-apoptotic
Bcl-2 family members is modulated by the addition of mitogens and
growth factors. For example, Bcl-2 expression in T cells has been
demonstrated to be induced by IL-2 and T cell receptor engagement,
whereas A1, another anti-apoptotic Bcl-2 family member, has been shown to be induced by Rel proteins after mitogen stimulation in lymphocytes (27-29). Bcl-xL expression in particular has been shown to be
up-regulated by a number of different cytokines in a variety of cell
types (27, 30). Furthermore, granulocyte-macrophage colony-stimulating factor, IL-3, and Epo were all shown to induce Bcl-xL transcription through the cytokine receptor-associated Stat5 protein (31, 32). These
observations made Bcl-xL an attractive candidate for a potential
Stat6-dependent, IL-4-inducible gene in primary B cells.
To determine whether IL-4 induces Bcl-xL expression in a
Stat6-dependent manner, we analyzed Bcl-xL protein and
mRNA expression from wild type and Stat6-deficient B cells treated
in culture with IL-4. We found that the inclusion of IL-4 in the wild
type B cell cultures resulted in a small but reproducible increase in
Bcl-xL protein expression (Fig.
3A). IL-4 could significantly induce Bcl-xL expression in both wild type and Stat6-deficient B cells,
but maximal induction only occurred in the presence of Stat6,
suggesting that both Stat6-dependent and -independent
pathways are involved in Bcl-xL expression. Anti-IgM stimulation led to a comparable increase in Bcl-xL protein and mRNA expression in both
wild type cells and in Stat6-deficient cells (Fig. 3, A and B). Notably, the addition of both IL-4 and anti-IgM resulted
in a synergistic induction of Bcl-xL expression in normal B cells. In
contrast, the maximal induction of Bcl-xL by IL-4 and anti-IgM was
especially compromised in Stat6-deficient B cells (Fig. 3, A
and B). In agreement with previously published results,
Bcl-2 protein and message levels were minimally affected by these
treatments (Fig. 3A) (33). We also found that the induction
of bcl-xL mRNA occurred as early as 2 h after IL-4
stimulation in anti-IgM-treated wild type B cells and was not inhibited
by cycloheximide (Fig. 3C). These results suggest
that IL-4 is capable of rapidly inducing bcl-xL mRNA and
protein expression and that maximal induction is dependent on the
presence of Stat6.
Stat6 Directly Activates a bcl-xL Regulatory Element--
The
above results imply that a consequence of IL-4 treatment of B cells is
the induction of Bcl-xL expression and that this induction is dependent
on the activation of Stat6. Furthermore, the activation of Bcl-xL
expression in response to IL-4 is quite rapid and does not require the
de novo synthesis of additional proteins. These observations
suggest that Bcl-xL may be a direct transcriptional target of Stat6. To
formally test this possibility we analyzed the murine Bcl-xL genomic
sequence for the presence of a Stat6-responsive element.
Most Stat proteins recognize a promoter element defined as the
We also tested the ability of the Stat6 site to provide IL-4
inducibility to the native bcl-xL promoter. A bcl-xL
promoter fragment, extending 576 bp upstream of the starting methionine and including the two Stat5 sites described previously, was cloned upstream of the luciferase reporter gene along with the
bcl-xL Stat6 site (32). A single Stat6 site was able to
reproducibly provide IL-4-induced reporter gene activity to the
bcl-xL promoter (Fig. 4C). The IL-4-induced
reporter activity was also dependent on an intact Stat6 site and was
not able to directly activate transcription through the Stat5 sites
(Fig. 4C and data not shown). Additionally, using
electrophoretic mobility shift assay analysis, we were able to detect
an IL-4-induced protein complex from B cell nuclear extracts bound to
the bcl-xL Stat6 site (Fig. 4D). The formation of
this complex was reduced when the nuclear extracts were preincubated
with antibodies to Stat6, but not to Stat5, suggesting that Stat6
directly binds to an element in the bcl-xL-flanking region.
These results combined with the findings above strongly suggest that
Stat6 is a direct activator of a bcl-xL regulatory element
in B cells.
Ectopic Bcl-xL Expression Rescues Stat6-deficient B Cells from
Fas-induced Apoptosis--
The studies above suggest that Bcl-xL is a
potential downstream target of Stat6 and may be in part responsible for
the protection of IL-4-treated B cells from apoptosis. It has
previously been demonstrated that overexpression of a Bcl-xL transgene
in developing B cells increased their resistance to Fas-induced
apoptosis (39). To determine whether reconstitution of Bcl-xL
expression could protect Stat6-deficient B cells from undergoing
apoptosis, we used a retroviral gene expression system to overexpress
Bcl-xL. We cloned Bcl-xL cDNA into a bicistronic retroviral
expression construct, allowing for the coexpression of Bcl-xL and GFP
within the same cell. Purified wild type and Stat6-deficient B cells were activated in vitro by lipopolysaccharide for 24 h
and then transduced with retroviral supernatants containing the empty
GFP vector or the vector including GFP and Bcl-xL (GFP BclxL). The transduced cells were activated with anti-CD40 for 48 h to
up-regulate Fas expression and subsequently treated with antibodies to
Fas to trigger cell death. The extent of apoptosis was examined by comparing propidium iodide staining of GFP only to
GFP/Bcl-xL-expressing transduced cells by fluorescence-activated cell
sorter analysis. Similar to the data in Fig. 1B, a large
percentage of wild type and Stat6-deficient cells transduced with the
control GFP vector were sensitive to Fas-mediated apoptosis, and the
addition of IL-4 to the wild type culture was able to rescue 46% of
those cells from death (Fig. 5). IL-4 was
unable to rescue any of the anti-Fas treated Stat6-deficient B cells
from apoptosis and, if anything, enhanced cell death (Fig. 5). However,
ectopic expression of Bcl-xL in either wild type or Stat6-deficient B
cells led to a marked increase in the resistance of both wild type and
Stat6-deificient cells to Fas-induced apoptosis, comparable with that
observed for IL-4 and wild type B cells. Thus, Bcl-xL expression is
sufficient to confer Fas-resistance in Stat6-deficient B cells.
In this report we have demonstrated that IL-4 fails to efficiently
rescue Stat6-deficient B cells from apoptosis produced by growth factor
withdrawal or Fas engagement. We find that the anti-apoptotic Bcl-2
family member, Bcl-xL, is one likely candidate target gene directly
activated by Stat6 that can modulate the response of B cells to
apoptotic stimuli.
In contrast to what we observed in B cells, Stat6 is not a critical
regulator of the anti-apoptotic activity of IL-4 in T cells (Fig.
1A and Refs. 5, 13, and 14). This difference is reminiscent
of the recent observation that IL-6 is a potent anti-apoptotic factor
for resting T cells but not for activated T cells (40). In this
situation, T cell activation appears to alter the cellular environment
so that IL-6-induced Stat signaling is less potent. Although robust
Stat6 activation is observed in both T cells and B cells after IL-4
exposure, the cellular context of this signal is clearly important in
determining the anti-apoptotic outcome of IL-4 signaling.
A number of cytokines are capable of suppressing apoptosis, and Bcl-xL
expression has been demonstrated to be cytokine-inducible in a variety
of systems. For example, IL-3 is required as a survival factor for a
number of cytokine-dependent cell lines and is capable of
inducing Bcl-xL expression in both myeloid and pro-B cell lines in a
Jak kinase-dependent manner (30). Additionally, IL-3 and granulocyte-macrophage colony-stimulating factor are both capable of
inducing Bcl-xL expression in wild type mouse bone marrow cells but not
in Stat5a/b-deficient bone marrow cells, and the deficient cells are
characterized by increased apoptosis (31). Stat5 and Stat3 have also
been recently demonstrated to activate the bcl-xL expression
directly although through a different Stat response element than the
one we have described here for Stat6 (32, 41). The Stat5-responsive
element is composed of two tandem GAS sites in the first intron of
bcl-xL (Fig. 4A). Not surprisingly we found that
Stat6 was incapable of activating transcription through the Stat5 sites
in our reporter assays since these are GAS elements with only a
three-base pair spacer within the palindromes (Fig. 4C and
data not shown). These findings suggest that bcl-xL
transcription can be regulated by a number of different cytokines
through different Stat transcription factors, but this regulation is
not necessarily mediated by the same Stat response elements in the
bcl-xL gene. Interestingly, a recent report suggests that
IL-15 is able to activate Stat6 specifically in mast cells and that in
this cell type Stat6 is able to direct IL-15-induced bcl-xL
transcription through the Stat5 sites (42). This result implies that
the context of the cytokine-Stat signal can also strongly influence
downstream changes in gene regulation.
We have demonstrated here that both IL-4 and IgM stimulation induce
Fas-resistance in activated B cells in a manner that correlates with
increased bcl-xL mRNA levels. It has been previously
shown that IL-4- and anti-IgM-induced Fas resistance are mediated by signaling pathways that differ both in their time course and dependence on protein kinase C activation (8). Both IL-4 and anti-IgM stimulation
ultimately result in increased bcl-xL transcription, although the mechanism of promoter activation is likely to be different
for the two stimuli. Here we show that Stat6 is an IL-4-induced transcription factor that directly activates a bcl-xL
regulatory element. Additionally we observed that the synergistic
induction of Bcl-xL expression after IL-4 and antigen receptor
stimulation is especially compromised in the absence of Stat6 (Fig. 3).
Interestingly, anti-IgM has been shown to induce Stat6
phosphorylation, although this apparently plays no role in Ig-mediated
Fas resistance or Bcl-xL expression (Figs. 2 and 3) (46). Anti-IgM
stimulation has also been shown to induce the activation of NF Even in the absence of Stat6, however, we observed that IL-4 was
capable of partially rescuing B cells from passive cell death (Fig.
1B) and inducing low levels of Bcl-xL expression (Fig. 3). Clearly other signal transduction pathways can be activated in response
to IL-4 in addition to Stat6, and these may also contribute to the
anti-apoptotic effects of IL-4. IRS-2 is an additional signaling
protein that associates with the IL-4 receptor and is rapidly
phosphorylated in lymphocytes after IL-4 treatment (23). IRS-2
activation results in the recruitment and activation of phosphatidylinositol 3-kinase and ultimately Akt activation. This cascade has been suggested to lead to protection from apoptosis, and
furthermore, B cells lacking the regulatory subunit of
phosphatidylinositol 3-kinase have been shown to be deficient in their
response to the anti-apoptotic effects of IL-4 (25, 47). However, we do not find that IRS-2 activation or Akt activation is absolutely required
for IL-4-mediated rescue from growth factor withdrawal in that B cells
from IRS-2-deficient mice are competent in their anti-apoptotic
response to IL-4. (Fig. 1C). An additional pathway from the
IL-4 receptor has also been implicated in rescue from apoptosis in
myeloid cell lines (15). This pathway emanates from a different
phosphorylated tyrosine residue on the IL-4 receptor that is not
recognized by Stat6 or IRS-2. The nature of this pathway has not yet
been defined, and its role in opposing apoptosis in primary B cells has
not been reported.
Although our results suggest that Bcl-xL is an important regulator of B
cell apoptosis, we do not observe a direct correlation between Bcl-xL
expression and protection from apoptosis. Both IL-4 and anti-IgM
stimulation induce similar amounts of Bcl-xL protein in Stat6-deficient
B cells, but only anti-IgM stimulation is protective of Fas-mediated
apoptosis (Fig. 3A). Additionally, overexpression of Bcl-xL
in our retroviral gene expression experiments as well as in other
reports resulted in only partial protection from Fas-induced apoptosis.
These results suggest that other important factors are also induced by
IL-4 or antigen receptor stimulation that modulate apoptosis. One
likely candidate is a recently described anti-apoptotic factor, FAIM,
which is induced specifically by anti-IgM stimulation in primary B
cells and is capable of blocking Fas-induced cell death when
overexpressed in B cell lines (48). Also, antigen receptor stimulation
has been demonstrated to block the association of Fas with its direct
downstream effector, FAS-associated death domain protein (FADD), in a
manner that is independent of new protein synthesis (49). Additionally,
we have found that BAG-1, a Bcl-2-associating protein with
anti-apoptotic activities, is induced in primary B cells by IL-4 in a
Stat6-dependent manner (50) (data not shown). Clearly, the
coordinated expression of a number of pro- and anti-apoptotic
factors that are regulated by multiple signals is critical for
determining the appropriate cellular response to a death signal.
Because Bcl-2 family members are important regulators of cell survival,
there has been a great deal of interest in understanding the role of
these proteins in the generation of an oncogenic state. Bcl-xL in
particular was shown to be specifically activated by retroviral
insertion in a number of murine myeloid and T cell leukemias (30).
Because Stat proteins play important roles in cellular proliferation
and apoptosis, they have also been implicated in playing a causative
role in oncogenesis. Indeed, a mutant form of Stat3 was recently
demonstrated to directly mediate cellular transformation (51).
Interestingly, a link between constitutive Stat3 activation and
disregulated Bcl-xL expression was reported in squamous cell carcinomas
(52). Constitutive Stat6 activation has also been associated with
specific tumor cells (10). In the future, it will be interesting to
determine whether disregulated Stat6 can affect cellular transformation
through the induction of Bcl-xL expression.
We thank Dr. Stanley Korsmeyer,
Dr. Uli Schindler, Dr. Gary Nolan, Dr. Ken Murphy, and Dr. Mirav
Socolovsky for plasmids and reagents. We also thank Jyothi
Rengarajan and Devangi Mehta for thoughtful review of the manuscript.
*
This work was supported by National Institutes of Health
Grants AI40171 (to M. J. G.) and AI40181 (to T. L. R.) and by the Mathers Foundation (to M. J. G.).The costs of publication of this article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
**
A scholar of the Leukemia and Lymphoma Society. To whom
correspondence should be addressed. Tel.: 617-432-1240; Fax:
617-432-0084; E-mail: mgrusby@hsph.harvard.edu.
Published, JBC Papers in Press, May 22, 2002, DOI 10.1074/jbc.M201207200
The abbreviations used are:
IL-4, interleukin 4;
Stat6, signal transducer and activator of transcription 6;
IRS-2, insulin receptor substrate 2;
GFP, green fluorescent protein;
GAS,
Interleukin-4-mediated Protection of Primary B Cells from
Apoptosis through Stat6-dependent Up-regulation of
Bcl-xL*
,,
**
Department of Immunology and Infectious
Diseases, Harvard School of Public Health and § Howard
Hughes Medical Institute, Joslin Diabetes Center, Harvard Medical
School, Boston, Massachusetts 02115, ¶ Departments of Medicine and
Microbiology and the Evans Memorial Department of Clinical Research,
Boston University Medical Center, Boston, Massachusetts 02118, and
Department of Medicine, Harvard Medical School,
Boston, Massachusetts 02115
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-actin. The relative expression of bcl-xL compared
with bcl-2 was determined by densitometry using a
ChemiImager 4000 (AlphaInotech).
2 M sodium
citrate. Analysis was performed on a FACScan flow cytometer (BD PharMingen).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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[in a new window]
Fig. 1.
Stat6 is required for IL-4 to block passive
cell death of B cells in vitro. Purified
naïve CD4+ T cells (A) or splenic B cells
(B and C) were cultured for 18-24 h in the
presence or absence of 10 ng/ml IL-4. The resulting populations were
analyzed for apoptosis by propidium iodide staining. The data represent
the average of two experiments and are representative of four
independent experiments.
View larger version (19K):
[in a new window]
Fig. 2.
Stat6 is required for IL-4 to block
Fas-induced cell death of B cells. Purified splenic B cells were
stimulated with anti-CD40 for 48 h. IL-4 and anti-IgM were added
for the final 12 h of the culture where indicated. The cells were
then induced to undergo Fas-mediated apoptosis by the inclusion of
anti-Fas antibody (0.06 µg/ml) overnight. The cells were subsequently
analyzed for apoptosis by propidium iodide staining. The results shown
are in duplicate and representative of four independent
experiments.
View larger version (32K):
[in a new window]
Fig. 3.
IL-4-induced Bcl-xL expression is
Stat6-dependent. A, purified splenic B
cells were cultured in the presence or absence of IL-4 and/or anti-IgM.
Total protein extracts were prepared from the cultures at 48 h and
analyzed for Bcl-xL and Bcl-2 protein expression by immunoblot. The
results shown are representative of three independent experiments.
B, total RNA was prepared from 7 h cultures and
analyzed for the presence of bcl-xL, bcl-2, and
-actin transcripts by Northern analysis. bcl-xL mRNA
expression was induced 3.3- and 1.3-fold by IL-4 in wild type and
Stat6-deficient cells, respectively. The results shown are
representative of three independent experiments. C, wild
type B cells were stimulated with anti-IgM or anti-IgM/IL-4 for 2 h. Cyclohexamide 10 µg/ml (CHX) was included 30 min
before stimulation in the indicated cultures.
-activating sequence (GAS), which is composed of a palindromic sequence separated by three nucleotides (TTCNNNGAA) (10, 34). Stat6 is
unusual in that it only recognizes the GAS at low affinity and
demonstrates instead a marked preference for a variant sequence with a
four-base pair spacer (TTCNNNNGAA) (18). In fact, most naturally
occurring IL-4-responsive promoters analyzed to date contain
Stat6-responsive elements with the four-base pair spacer (35, 36).
Previous studies of the bcl-xL genomic sequence revealed
several functional GAS elements in the first intron of the Bcl-xL gene
that are regulated by Epo- and IL-3-activated Stat5 (Fig.
4A) (31, 32, 37, 38). Our
analysis of the genomic sequence of the bcl-xL-flanking
region revealed in addition a perfect Stat6-responsive element 928 bp
upstream of the first major transcriptional start site and 1600 bp
upstream of the starting methionine (Fig. 4A) (37). The 40 base pairs surrounding this Stat6 site was multimerized, and the
compound element was cloned upstream of a minimal thymidine kinase
promoter driving the expression of a luciferase reporter gene. The
activity of this element was tested by transiently transfecting M12 B
cells with the reporter construct in the presence and absence of IL-4.
We found that in the absence of IL-4 the reporter construct was
essentially inactive, but in the presence of IL-4 reporter gene
activity was induced ~20-fold (Fig. 4B). This robust
IL-4-induced reporter activity was dependent on an intact Stat6
response element since specific mutations introduced into our reporter
construct that destroy the palindrome resulted in a complete loss of
IL-4 induction (Fig. 4, A and B).
View larger version (20K):
[in a new window]
Fig. 4.
Stat6 directly activates a Bcl-xL promoter
element. A, schematic of the bcl-xL
promoter. The previously identified Stat5 binding sites are shown in
the first intron. The asterisks represent the
transcriptional start sites reported previously (37). The Stat6 site
described in this study is shown 928 base pairs upstream of the first
exon. The sequence of the wild type Stat6 site identified in the
bcl-xL promoter and the mutated site used in this study are
also indicated. B, the genomic sequence surrounding and
including the wild type Stat6 binding site from the Bcl-xL promoter and
the genomic sequence including a mutant Stat6 binding site were each
multimerized four times and cloned upstream of the minimal thymidine
kinase promoter driving the expression of a luciferase reporter gene.
The reporter constructs were transfected along with a Stat6 expression
construct into the B cell lymphoma M12 in the presence and absence of
recombinant IL-4. The experiments were carried out in duplicate, and
the data shown are representative of three independent experiments.
C, a single copy of the bcl-xL genomic sequence
including wild type and mutant Stat6 binding sites described in
B were cloned upstream of the 576-bp bcl-xL
promoter region driving the expression of a luciferase reporter gene.
The reporter constructs were transfected into the B cell lymphoma BJAB
as described in B. D, nuclear extracts from
untreated ( 
) and IL-4-treated (+) BJAB cells were incubated with a
radiolabeled, double-stranded oligo including the wild type
bcl-xL Stat6 site and analyzed by electrophoretic mobility
shift assay. Extracts were preincubated with antibodies to Stat5
(
S5) and Stat6 (S6) where indicated.
View larger version (18K):
[in a new window]
Fig. 5.
Ectopic expression of Bcl-xL rescues
Stat6-deficient B cells from Fas-induced cell death. Purified
splenic B cells were stimulated as described under "Experimental
Procedures" and transduced with packaging cell supernatants
containing virus expressing GFP or GFP/Bcl-xL. The resulting transduced
cells were sorted for GFP expression, stimulated with anti-CD40 for
48 h, and induced to die by exposure to anti-Fas for 6 h.
Apoptosis was assessed by propidium iodide staining. Where indicated,
IL-4 was included for the last 24 h of culture. The data represent
the percentage of GFP-only expressing cells protected from apoptosis
when either exposed to IL-4 or compared with cells transduced with
GFP/Bcl-xL. The results are representative of four independent
experiments.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B and
NFAT transcription factors, and a number of binding sites for these factors are present in the bcl-xL promoter region (37,
43-45). However, we have yet been able to identify an anti-IgM
responsive element in the bcl-xL promoter region (data not
shown), suggesting that regulatory elements outside of the proximal
promoter element may contribute to anti-IgM-induced bcl-xL
expression. It will be interesting to further define the regulatory
elements that are involved in anti-IgM-induced bcl-xL
transcription and how these elements potentially cooperate with Stat6
to activate bcl-xL transcription synergistically.
ACKNOWLEDGEMENTS
FOOTNOTES
ABBREVIATIONS
-activating sequence.
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E. V. Acosta-Rodriguez, C. L. Montes, C. C. Motran, E. I. Zuniga, F.-T. Liu, G. A. Rabinovich, and A. Gruppi Galectin-3 Mediates IL-4-Induced Survival and Differentiation of B Cells: Functional Cross-Talk and Implications during Trypanosoma cruzi Infection J. Immunol., January 1, 2004; 172(1): 493 - 502. [Abstract] [Full Text] [PDF]
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G. Stassi, M. Todaro, M. Zerilli, L. Ricci-Vitiani, D. Di Liberto, M. Patti, A. Florena, F. Di Gaudio, G. Di Gesu, and R. De Maria Thyroid Cancer Resistance to Chemotherapeutic Drugs via Autocrine Production of Interleukin-4 and Interleukin-10 Cancer Res., October 15, 2003; 63(20): 6784 - 6790. [Abstract] [Full Text] [PDF]
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M. W. Wynes and D. W. H. Riches Induction of Macrophage Insulin-Like Growth Factor-I Expression by the Th2 Cytokines IL-4 and IL-13 J. Immunol., October 1, 2003; 171(7): 3550 - 3559. [Abstract] [Full Text] [PDF]
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 A. E. Kelly-Welch, E. M. Hanson, M. R. Boothby, and A. D. Keegan Interleukin-4 and Interleukin-13 Signaling Connections Maps Science, June 6, 2003; 300(5625): 1527 - 1528. [Abstract] [Full Text] [PDF]
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 C. Vasu, S. R. Gorla, B. S. Prabhakar, and M. J. Holterman Targeted engagement of CTLA-4 prevents autoimmune thyroiditis Int. Immunol., May 1, 2003; 15(5): 641 - 654. [Abstract] [Full Text] [PDF]
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H. A. Bruns, U. Schindler, and M. H. Kaplan Expression of a Constitutively Active Stat6 In Vivo Alters Lymphocyte Homeostasis with Distinct Effects in T and B Cells J. Immunol., April 1, 2003; 170(7): 3478 - 3487. [Abstract] [Full Text] [PDF]
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E. V. Acosta Rodriguez, E. Zuniga, C. L. Montes, and A. Gruppi Interleukin-4 biases differentiation of B cells from Trypanosoma cruzi-infected mice and restrains their fratricide: role of Fas ligand down-regulation and MHC class II-transactivator up-regulation J. Leukoc. Biol., January 1, 2003; 73(1): 127 - 136. [Abstract] [Full Text] [PDF]
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