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B/Rel Is Apoptogenic in Cytokine Withdrawal-induced Programmed Cell Death1
Departments of Microbiology and Immunology [U. S. S., C-L. C., D. J. H., L. D. K.], and Cell Biology [F. E. Y., L. D. K.], and the Vanderbilt Cancer Center [L. D. K.], Vanderbilt University School of Medicine, Nashville, Tennessee 37232
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ABSTRACT |
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 Top ABSTRACT IntroductionMaterials and MethodsResults and DiscussionREFERENCES |
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B (NF-B) promotes apoptosis
during cytokine withdrawal-induced PCD. In the progenitor B lymphocyte
model FL5.12, whereas NF-B has an antiapoptotic function in response
to tumor necrosis factor-
, cytokine withdrawal causes nuclear
translocation of NF-B/cRel, where it is apoptogenic. Inhibition of
NF-B activation delays cytokine withdrawal-induced PCD in both
FL5.12 and transgenic early B cells. Additionally, reconstituting a
bone marrow microenvironment ex vivo abrogates the
differential apoptotic pattern between control and transgenic early B
cells. |
Introduction |
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TopABSTRACTIntroductionMaterials and MethodsResults and DiscussionREFERENCES |
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B3
proteins, which include RelA (p65), c-Rel, v-Rel, RelB, NF-B1
(p105/p50), and NF-B2 (p100/p52), are transcription factors that
regulate genes by binding to recognition sequences (canonical site,
GGGACTTTCC) in promoter/enhancer regions. NF-B molecules form homo-
or heterodimers with each other and remain bound in the cytoplasm in an
inactive complex with inhibitory proteins called IB. The removal of
IB exposes the nuclear localization sequence of the NF-B complex,
thereby mobilizing it for nuclear translocation. The NF-B family is
able to effect a wide variety of responses in the development and
function of the immune system because of the unique characteristics of
each member and their combinatorial possibilities (1
and
references therein).
NF-B factors have been implicated both as activators and repressors
of PCD, depending on the stimulus and cell type examined. For example,
NF-B p50/RelA is protective in a TNF- model of PCD
(2, 3, 4)
. On the other hand, there is evidence that NF-B
may be involved in promoting PCD. v-rel is cytopathic in murine
fibroblasts (5)
, and if expressed in avian cells, it
causes a transforming phenotype. In addition, cRel expression in the
avian embryo is correlated with cells undergoing PCD (6)
.
Finally, the anti-inflammatory drug aspirin (sodium salicylate)
protects neuronal cells by down-regulation of NF-B, thereby
implicating this family of factors in the promotion of cell death
during inflammation (7)
. Taken together, these
observations indicate that NF-B members can have dramatically
different effects during PCD in different cell systems.
PCD after growth factor or cytokine withdrawal is a physiological process that occurs during morphological development, neurogenesis, and lymphopoiesis (Ref. 8 and references therein). In these microenvironments, lack of cytokines and other growth factors causes apoptosis of developing cells. It is estimated that 75% of progenitor and precursor B cells die in the bone marrow because of lack of survival signals (Ref. 9 and references therein). Very little is known about the regulation of PCD during these processes. The evolution of B-lineage cells represents an ideal candidate cell type in which to examine the regulation of physiologically relevant developmental PCD.
To identify and characterize early pathways in factor
withdrawal-induced PCD, we selected the nontransformed progenitor
B-lymphocytic cell line, FL5.12 (10
, 11)
. In these cells,
the NF-B member RelA is constitutively present in the nucleus.
Between 2 and 8 h after cytokine withdrawal, the major NF-B
inhibitor, IB-, is degraded and a different NF-B member, cRel,
is translocated to the nucleus, and cells die by apoptosis. We show
that in FL5.12 cells, the stable expression of a transdominant
inhibitor of NF-B activity, termed IB-
N
(12)
, significantly delays death after cytokine
withdrawal. In addition, in FL5.12 cells transient overexpression of
IB-N delays PCD after cytokine withdrawal, whereas RelA has no
effect and cRel precipitates PCD after cytokine withdrawal. Finally,
bone marrow-derived B cells from transgenic mice expressing
IB-N die more slowly than nontransgenic controls when cultured
in the absence of survival factors. This role of NF-B in PCD is
specific to cytokine withdrawal because when cytokines are provided
exogenously, no difference in rates is observed.
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Materials and Methods |
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TopABSTRACTIntroductionMaterials and MethodsResults and DiscussionREFERENCES |
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B-N, stable clones were isolated under G418 selection,
and individual IB-N-expressing clones were identified by
Western blot analysis. Two independent expressing clones were
characterized.
For luciferase assays, parental FL5.12 cells and FL5.12 clones
expressing IB-N were transiently transfected with either 10
µg of 2XBtk/Luc (IL-2 receptor-B enhancer) or 10 µg of
Mut-2XBtk/Luc (a DNA-binding mutant of the IL-2R-B motif) and
2 µg of SV40-ßgal as an internal transfection control, via the
DEAE-trypsin protocol. Forty-eight h post transfection, multiple
independent transfectants were pooled, the cytokine was withdrawn by
washing the cells three times with PBS, and the cells were resuspended
in complete medium without IL-3. The time t = 0 was taken as the mid-point between the time when the medium
containing cytokine was withdrawn and the cytokine without medium was
added. Cells (1 x 106) were
removed, processed into cell lysates, and analyzed for luciferase
activity according to the manufacturer’s instructions (Analytical
Bioluminescence).
For dose-dependence analysis, 5 x 106 cells were transfected with 3 µg of CMV
EGFP (Clontech) and pSFFV-IB-N, RSV-RelA, or RSV-cRel
in varying amounts as indicated. The total DNA transfected was
normalized with empty vector. Thirty-six to 48 h after
transfection, cells were resuspended in medium without IL-3. The ratio
of GFP+ cells to the total number of cells
counted was determined at t = 0 and
t = 1 day after cytokine withdrawal. The
ratio at t = 0 was normalized to 100%, and
the corresponding value was determined at t = 1 day for each individual experiment. A minimum of 200 cells were
counted in at least three independent experiments.
Bone marrow B cells were purified from femurs by an established
macrophage depletion and complement lysis protocol (13)
.
Characterization of the IB-N transgenic mice will be described
elsewhere.4
Protein Collection and Western Analysis.
At indicated times, cells were washed once with ice-cold PBS (all
centrifugations were 800 x g for 5
min), and cell pellets stored at -80°C until further use. Protein
isolation was carried out at 4°C. Pellets were thawed on ice in 1.0
ml of 1x Wu buffer [Ref. 14
; 2x Wu buffer stock: 20
mM HEPES (pH 7.40), 3 mM
MgCl2, 0.2 mM EGTA, 10%
glycerol, 100 mM ß-glycerophosphate, 1
mM DTT, 2 µM
pepstatin A, 1.0 mM phenylmethylsulfonyl
fluoride], pipetted vigorously, allowed to stand for 10 min,
and pipetted again. The samples were centrifuged at 11,000 x g for 30 min. Supernatants were collected as
cytoplasmic fractions. Nuclei were washed in 1.0 ml of 1x Wu buffer,
centrifuged for 15 min, resuspended by shearing through an 18-gauge
needle in 1x Wu buffer supplemented with 450 mM
KCl, and centrifuged for 30 min. These supernatants and the cytoplasmic
fractions were dialyzed overnight with buffer D [20
mM HEPES (pH 7.90), 20% glycerol, 0.1
M KCl, 0.2 mM EDTA, 0.5
mM phenylmethylsulfonyl fluoride, 0.5
mM DTT, 50 mM
ß-glycerophosphate, 1.0 µM pepstatin A].
Dialysates were centrifuged for 30 min, and Bradford analysis (Bio-Rad)
was conducted to determine the protein concentration. Equal
concentrations of cytoplasmic (100–150 µg) or nuclear extracts
(50–75 µg) were fractionated by SDS-PAGE prior to electroblot
transfer to polyvinylidene fluoride Immobilon membrane (Millipore).
Membranes were immunoblotted with the following antisera: IB-
(sc-203), cRel (sc-71), RelA (sc-109). Cytoplasmic and nuclear lysates,
as indicated, were fractionated by SDS-PAGE prior to electroblot
transfer to polyvinylidene fluoride membrane. Individual membranes were
processed by immunoblot with the indicated antisera, and
immunoglobulin/protein complexes were visualized by chemiluminescent
detection (Renaissance; NEN Life Science Products).
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Results and Discussion |
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TopABSTRACTIntroductionMaterials and MethodsResults and DiscussionREFERENCES |
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; Refs. 10
, 11
). To examine whether nuclear
B-enhancer activity is regulated during the process of cytokine
withdrawal-induced apoptosis, FL5.12 cells were transfected with
reporter constructs bearing two NF-B-inducible enhancer elements
from the IL-2 receptor- chain gene that drives expression of the
Photinus luciferase cDNA. A transient rise in nuclear
NF-B enhancer activity resulted in a 7-fold increase in
B-activity, which peaked 6–8 h post cytokine withdrawal and
gradually diminished to baseline over 48 h (Fig. 1B)
. A
reporter construct bearing the HIV-1 3' long terminal repeat, another
B-responsive promoter that drives the luciferase cDNA, gave
qualitatively similar results (data not shown). No transcriptional
activity was detected using the mutant Btk-enhancer/reporter, a
construct with two DNA-binding mutants of the B-enhancer elements.
Transfected control reporter plasmids driven by the activator protein-1
enhancer construct (2XTRE/Luc) or the SP-1 enhancer construct
(SP-1/Luc) were also unaffected (data not shown).
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B factors are necessary for cytokine
withdrawal-induced apoptosis, a transdominant inhibitor of the NF-B
complex, IB-N (9)
, was stably transfected into
the FL5.12 cell line, and two separate expressing clones were analyzed
for their response to cytokine deprivation. Fig. 1B
compares
the expression of the 2xBtk/Luc reporter in wild-type
versus stably expressing IB-N cells and confirms
that IB-N inhibits the transactivating capabilities of
NF-B. A comparison of cell survival rates after cytokine withdrawal
between wild-type and stably transfected IB-N FL5.12 cells
showed that inhibition of NF-B-induced activity significantly
decreased the onset of apoptosis (Fig. 1C)
. These results
demonstrate that cytokine withdrawal induces NF-B activity, which
may regulate downstream target genes critical in modulating a
proapoptotic pathway. Recent reports have implicated NF-B as an
antiapoptotic factor in the TNF--induced PCD pathway
(2, 3, 4)
. Consistent with previous observations, inhibition
of NF-B activity in FL5.12 post TNF- treatment resulted in cells
that died twice as fast as parental cells (Refs. 2, 3, 4
;
Fig. 1D
). Taken together, these data suggest that there are
multiple pathways leading to death within the same cell type and that
transcription factor NF-B can either promote or attenuate PCD,
depending on the pathway activated.
To identify the NF-B family member responsible for apoptosis
after cytokine withdrawal, immunoblot analyses were performed on
protein extracts prepared from FL5.12 cells at various times after
cytokine withdrawal. Consistent with current models for activation of
NF-B, the cytoplasmic protein levels of the major inhibitor
molecule, IB-, decreased beginning 
2 h after the removal of
cytokine (Fig. 2)
. Reduced levels of IB- are observed for 4 h. No significant
alteration was observed in the cytoplasmic protein levels of the other
major NF-B inhibitor molecule, IB-ß (data not shown).
Coordinately with the disappearance of IB-, the nuclear
translocation of the c-rel proto-oncogene product, cRel, was
detectable 3 h after cytokine withdrawal. Detection of nuclear cRel
peaked at 6 h and diminished concomitant with the resynthesis of
IB- (by 8 h). No significant alteration in the nuclear level
of RelA protein was observed until 23 h post cytokine
withdrawal. No alteration in cytoplasmic IB-, nuclear cRel,
or RelA proteins was detected in extracts from FL5.12 cells stably
expressing IB-N, after cytokine withdrawal (data not shown).
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B-N
clones, increasing levels of IB-N expression vector resulted
in a dose-dependent increase in GFP+ cells
counted 1 day post cytokine withdrawal (Fig. 3A)
. Because the presence of IB-N blocks NF-B
activation, these results confirm that NF-B acts in a proapoptotic
manner in FL5.12 cells. Whereas RelA has been hypothesized to a
generate antiapoptotic signals (2, 3, 4)
, in this model of
progenitor B-cell apoptosis, increasing concentrations of RelA
expression vector were shown to have no significant effect on cytokine
withdrawal-induced apoptosis (Fig. 3B)
. This finding
correlates well with the observation that RelA levels did not change
throughout the early stages of cytokine withdrawal-induced apoptosis
and that this high endogenous level of nuclear RelA is unable to
activate an exogenous B-enhancer/reporter (Fig. 1B)
.
These results would argue for a minimal function of RelA in progenitor
B-cell cytokine withdrawal-induced apoptosis, consistent with
observations from early B-cell lineages isolated from RelA -/- mice
that were at the same levels as wild-type controls (16)
.
Consistent with our hypothesis that cRel promotes apoptotic cell death
in progenitor B cells, there was a dose-dependent decrease in
GFP+, i.e., viable, cells 1 day post
cytokine withdrawal with increasing levels of c-rel
expression vector (Fig. 3C)
. In this regard, it is
interesting to note that we were unable to generate stable clones that
expressed cRel because these cells underwent PCD even in the presence
of
cytokine.5
In summary, (a) cRel translocates to the nucleus in
the early phase after cytokine withdrawal (Fig. 2)
; (b)
sequestering of NF-B in the cytoplasm delays apoptosis (Fig. 1C
and Fig. 3A
); and (c) increasing
levels of cRel decrease viability of cells after cytokine withdrawal in
a dose-dependent manner (Fig. 3C)
. Taken together, these
data demonstrate that cRel promotes death in early progenitor B cells
after cytokine withdrawal.
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B during cytokine
withdrawal in early B cells in a physiological context, we generated
four transgenic mouse lines that expressed the transdominant negative
NF-B inhibitor IB-N in B cells.4
To mimic
cytokine withdrawal, we purified bone marrow B cells from nontransgenic
and transgenic mice, plated them in a rich B-cell medium, and monitored
their viability over time. As would be predicted from the data obtained
in FL5.12 cells, IB-N transgenic B cells from all four lines
had a statistically significant delayed death profile
(P < 0.0001), compared with nontransgenic
bone marrow B cells (Fig. 4A)
. Survival of precursor B cells in the bone marrow is
supported by cytokines and factors provided by the bone marrow stroma
(17)
. We tested the effects of such factors on the delayed
death rate of IB-N transgenic and control precursor B cells.
Bone marrow B cells were cultured on an S10 stromal layer supplied with
IL-7, which previously has been shown to mimic the bone marrow
microenvironment (18)
. When transgenic and wild-type early
B cells were cultured under these conditions, no difference was
detected between these two groups (Fig. 4B)
. Thus, only in
the absence of given cytokines did NF-B appear to promote apoptosis.
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B activity correlated with
a resistance to cytokine withdrawal-induced PCD. In addition, the data
suggest that c-Rel may be the NF-B family member responsible for
promotion of apoptosis. Our studies suggest that the critical
physiological function of B-cell development is regulated, in part, by
activities controlled through NF-B.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by grants from NIH (R01GM51249), the
American Cancer Society (JFRA 516), and the Elsa Pardee Foundation (to
L. D. K.) and a Center Grant from the National Cancer
Institute (CA 68485). U. S. S. was supported by the
Vanderbilt Medical Scientist Training Program and Vanderbilt University
Dissertation Enhancement Award. L. D. K. is a recipient of an
ACS Junior Faculty Research Award and a Cancer Research Institute
Investigator Award. 
2 To whom requests for reprints should be
addressed, at Vanderbilt University School of Medicine, MCN A-4314,
1161 21st Avenue South, Nashville, TN 37232-2363. Phone:
(615) 343-2568; Fax: (615) 343-2569; E-mail: d.kerr{at}mcmail.vanderbilt.edu
3 The abbreviations used are: NF-B, nuclear
factor-B; PCD, programmed cell death; TNF-, tumor necrosis
factor-; IL, interleukin; GFP, green fluorescent protein.
4 F. E. Yull, C-L. Chen, U. S. Sohur,
D. J. Hicks, H. Li, J. O. Price, and L. D. Kerr.
Inhibition of Rel/NF-B activity results in aberrant development and
function of the B cell compartment in vivo, manuscript
in preparation.
5 U. S. Sohur and L. D. Kerr,
unpublished material.
Received 8/ 3/99. Accepted 1/17/00.
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REFERENCES |
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TopABSTRACTIntroductionMaterials and MethodsResults and DiscussionREFERENCES |
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B and rel proteins: evolutionary conserved mediators of immune responses. Annu. Rev. Immunol., 16: 225-260, 1998.[Medline]
B in preventing TNF--induced cell death. Science (Washington DC), 274: 782-784, 1996. apoptosis by NF-B. Science (Washington DC), 274: 787-789, 1996. and cancer therapy-induced apoptosis: potentiation by inhibition of NF-B. Science (Washington DC), 274: 784-786, 1996.B activation. Science (Washington DC), 274: 1383-1385, 1996. B ß with rel/NF-B transcription factors: role of ankyrin repeats in the inhibition of DNA binding activity. Proc. Natl. Acad. Sci. USA, 89: 4333-4337, 1992.B RelA-deficient lymphocytes: normal development of T cells and B cells, impaired production of IgA and IgG1 and reduced proliferative responses. J. Exp. Med., 185: 953-961, 1997.This article has been cited by other articles:
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, viability; •, apoptotic
index. B, cytokine withdrawal-induced
and 
) were transfected transiently with a 
