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-Mediated NF-
B Activation and Apoptosis in Pre-B Cells1
* Department of Environmental Health, Boston University School of Public Health, Boston, MA 02118; and
Lady Davis Institute for Medical Research, McGill University, Montreal, Quebec, Canada
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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(PPAR) in adipocyte physiology has been exploited for the treatment
of diabetes. The expression of PPAR in lymphoid organs and its
modulation of macrophage inflammatory responses, T cell proliferation
and cytokine production, and B cell proliferation also implicate it in
immune regulation. Despite significant human exposure to PPAR
agonists, little is known about the consequences of PPAR activation
in the developing immune system. Here, well-characterized models of B
lymphopoiesis were used to investigate the effects of PPAR ligands
on nontransformed pro/pre-B (BU-11) and transformed immature B
(WEHI-231) cell development. Treatment of BU-11, WEHI-231, or primary
bone marrow B cells with PPAR agonists (ciglitazone and GW347845X)
resulted in rapid apoptosis. A role for PPAR and its dimerization
partner, retinoid X receptor (RXR)
, in death signaling was supported
by 1) the expression of RXR mRNA and cytosolic
PPAR protein, 2) agonist-induced binding of PPAR to a PPRE, and
3) synergistic increases in apoptosis following cotreatment with
PPAR agonists and 9-cis-retinoic acid, an RXR
agonist. PPAR agonists activated NF-
B (p50, Rel A, c-Rel) binding
to the upstream B regulatory element site of c-myc.
Only doses of agonists that induced apoptosis stimulated NF-B-DNA
binding. Cotreatment with 9-cis-retinoic acid and
PPAR agonists decreased the dose required to activate NF-B. These
data suggest that activation of PPAR-RXR initiates a potent
apoptotic signaling cascade in B cells, potentially through NF-B
activation. These results have implications for the nominal role of the
PPAR in B cell development and for the use of PPAR agonists as
immunomodulatory therapeutics. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, 
, and . PPAR is expressed highly in
metabolically active tissues such as liver, heart, and kidney and
regulates the uptake, activation, and 
-oxidation of fatty acids
(1). Recently, PPAR has been found to be expressed in
myeloid and lymphoid cells (2). PPAR is ubiquitously
expressed (3) and plays a role in embryonic development
and adipocyte physiology (4). Alternative splicing and
promoter use result in the formation of the PPAR isoforms (5, 6). While the distribution of PPAR2 is largely restricted to
adipocytes, PPAR1 is more widely expressed and is the isoform found
in tissues of the immune system (3, 7). PPAR plays an
important role not only in regulating adipocyte differentiation
(8, 9) and insulin responsiveness (10), but
also in immune function, particularly in macrophage inflammatory
responses (11, 12, 13, 14, 15); T cell activation, proliferation, and
production of inflammatory cytokines (16, 17, 18); endothelial
cell production of chemokines (19); dendritic cell
maturation (20); and B cell proliferative responses
(21).
PPAR is a ligand-inducible transcription factor. Natural ligands for
this receptor include
15-deoxy-
12,14-PGJ2
(15d-PGJ2) (22) and 9- and 13-hydroxyoctadienoic acid
(23). Synthetic ligands for PPAR include
thiazolidinediones and tyrosine analogs, developed for the
treatment of type II diabetes and investigated as chemotherapeutics
(23), as well as a metabolite of
di[2-ethylhexyl]phthalate, a major environmental pollutant
(24). Following ligand binding, PPAR forms a
heterodimeric complex with retinoid X receptor (RXR), allowing
binding to PPAR response elements
(5'-AACTAGGNCAAAGGTCA-3', consensus
sequenceunderlined) (25) within promoters of
PPAR target genes. Ligand binding initiates a conformational change
that results in the dissociation of corepressors and the
association of coactivators, allowing ligand-induced
trans-activation (26, 27). Providing ligands
for both PPAR and RXR results in additive or synergistic
activation of transcription (28, 29, 30, 31).
PPAR mRNA and/or protein are expressed widely throughout the immune
system, including in mature B cell lines and primary populations
(2, 7, 32), splenic white pulp, Peyer’s patches germinal
centers, and human lymphoid follicles (7). PPAR’s
dimerization partner RXR has been shown to be expressed in mature B
cells (33). A physiological role for PPAR in B cell
function is suggested by an increased proliferative response and
enhanced Ag-specific immune response of mature B cells from PPAR
haploinsufficient mice (21). However, little is known of
the expression or function of PPAR or its dimerization
partner, RXR, in primary pro- or pre-B cells. The effects of PPAR
agonists on these developing lymphocytes are of particular concern
given the increased sensitivity of the developing immune system to
other environmental chemicals (34, 35, 36, 37) and therapeutics
(38), and the recent consideration of PPAR agonists as
tumor-specific therapeutics (39, 40, 41, 42, 43, 44, 45).
Also of note is the ability of PPAR to modify the activity of other
transcription factors involved in lymphocyte function and survival,
particularly NF-B. Our laboratory and those of other investigators
have demonstrated that modulation of NF-B in pro/pre-B and immature
B cells through a variety of stimuli results in apoptosis (46, 47). Most studies with PPAR agonists suggest that PPAR
activation down-regulates NF-B-DNA binding and
trans-activation stimulated by activators such as LPS,
TNF-, PMA, and IL-1 in a variety of cell types (48),
including macrophages and T cells (13, 14, 15). However, in
some circumstances, PPAR can bind DNA cooperatively with NF-B
(49, 50) and enhance NF-B-DNA binding
(51). Because of these results, the expression of PPAR
in mature B cell populations, and the suggestion that PPAR plays a
role in mature B cell responses, we sought to determine whether PPAR
agonists adversely affect the developing immune system by inducing bone
marrow-derived B cell apoptosis, potentially through NF-B
dysregulation. These issues were addressed using two PPAR agonists
of significantly different molecular structures: ciglitazone (a
thiazolidinedione), and GW7845 (a tyrosine analog), a previously
described bone marrow stromal cell-dependent pro/pre-B cell line
(BU-11), and a well-characterized transformed immature B cell line
(WEHI-231). The data show that the developing B cell compartment is
exquisitely sensitive to PPAR agonists and are consistent with a
role for PPAR-RXR-mediated modulation of NF-B activity in the
induction of an extremely rapid and potent apoptosis signal.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Ciglitazone, 9-cis-retinoic acid, and
poly(ADP-ribose) polymerase (PARP)-specific Ab were obtained
from Biomol (Plymouth Meeting, PA). GW347845X was provided by Dr. T.
Willson (GlaxoSmithKline, Research Triangle Park, NC).
PPAR-specific Ab was obtained from Calbiochem (San Diego, CA).
NF-B-specific Abs were purchased from Santa Cruz Biotechnology
(Santa Cruz, CA). Propidium iodide and Protease Inhibitor Cocktail for
Mammalian Cells were obtained from Sigma (St. Louis, MO). Annexin V-PE,
annexin V-FITC, and the PE-labeled B220-specific Ab were purchased from
BD PharMingen (San Diego, CA). Murine IL-7 was obtained from Research
Diagnostics (Flanders, NJ). Cell Proliferation Kit I was purchased from
Roche (Indianapolis, IN). Plasmocin was obtained from Invitrogen (San
Diego, CA). All other reagents were purchased from Fisher Scientific
(Suanee, GA) unless otherwise noted.
B cell isolation
B cells were isolated from the bone marrow of 8- to 10-wk-old C57BL/6 male mice by the method of Tze et al. (52). Cultures were determined to be >80% B220+ following preparation by this method.
Cell lines
The stromal cell-dependent, C57BL/6-derived BU-11 cell line has been characterized previously (35, 37). BU-11 cells represent B cells at the transition between the pro- and early pre-B cell stages as they are CD43+/B220+/IgM- with rearranged Ig heavy chain genes (53). BMS2 is a culture dish adherent cloned bone marrow stromal cell line that supports BU-11 cell growth (54). Cultures of BU-11 cells maintained on BMS2 cell monolayers were grown in an equal mixture of DMEM and RPMI 1640 medium with 5% FBS, Plasmocin, L-glutamine, and 2-ME. For experiments, BU-11 cells were plated without BMS2 in the presence of IL-7. WEHI-231 (immature B) cells were grown in DMEM with 5% FBS, Plasmocin, L-glutamine, and 2-ME. All cultures were maintained at 37°C in a humidified 7.5% CO2 atmosphere, and all experiments were conducted under these conditions. Cell cultures were determined to be mycoplasma negative by PCR (Mycoplasma Detection kit; American Type Culture Collection, Manassas, VA).
Determination of apoptosis
Primary bone marrow B cells were treated in 24-well plates with ethanol (vehicle; final concentration, <0.1%), ciglitazone, or GW7845 (20–100 µM) for 4 h. For B220/annexin V double staining, the cells were collected and washed in cold PBS containing 5% FBS and 10 mM azide. PE-labeled B220-specific Ab was added, and the cells were incubated in the dark at 4°C for 30 min. Cells were resuspended in 150 µl annexin V binding buffer containing 10 mM HEPES (pH 7.4), 140 mM NaCl, and 2.5 mM CaCl2. Annexin V-FITC (2.5 µl) was added. The cells were incubated in the dark at room temperature for 15 min and were analyzed in a FACScan flow cytometer (BD Biosciences, Mountain View, CA).
BU-11 and WEHI-231 cells were treated in 24-well plates with ethanol
(vehicle; final concentration, <0.1%), ciglitazone (80 µM), or
GW7845 (60 µM). For propidium iodide staining, cells were treated for
4–6 h, harvested by gentle pipetting, and washed once with cold PBS
containing 5% FBS and 10 mM azide. Cells were resuspended in 0.25 ml
hypotonic buffer containing 50 µg/ml propidium iodide, 1% sodium
citrate, and 0.1% Triton X-100 and were analyzed by flow cytometry.
Cells undergoing apoptosis have a weaker propidium iodide fluorescence
than those in the G0/G1
phase of the cell cycle (35, 37). For studies of PPAR
and RXR agonist synergy, BU-11 were treated with ethanol (vehicle;
final concentration, <0.1%) or ciglitazone (10–20 µM) and
9-cis-retinoic acid (10–20 µM). Eighteen hours later
cells were harvested, stained with propidium iodide, and analyzed by
flow cytometry as described above.
For annexin V staining, cells were treated for 4–6 h, harvested by gentle pipetting, and washed once with cold PBS containing 5% FBS and 10 mM azide. Cells were resuspended in 150 µl annexin V binding buffer, and 2.5 µl of annexin V-PE was added. The cells were incubated in the dark at room temperature for 15 min and were analyzed by flow cytometry within 1 h.
For determination of DNA fragmentation, cells were treated for 2–6 h, harvested by gentle pipetting, and washed once with cold PBS. Cells were lysed in 0.4 ml of lysis buffer containing 10 mM Tris (pH 8), 1 mM EDTA, ad 0.2% Triton X-100. Samples were centrifuged, and DNA was extracted from the supernatant using phenol and chloroform and precipitated with ethanol. DNA was recovered by centrifugation, dried, and quantified. Before loading on a 1.5% agarose gel, the DNA was treated with 10 µg/ml RNase A at 37°C for 10 min. DNA was visualized using ethidium bromide staining.
For detection of PARP cleavage, cells were treated for 2–6 h, harvested by gentle pipetting, and washed once with cold PBS. PARP cleavage was detected by Western blotting using specific pAb (SA-253). Samples were prepared, electrophoresed, and immunoblotted according to the manufacturer’s instructions.
A 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay (Cell Proliferation Kit I) was used to rapidly quantitate cell death in multiple samples simultaneously. BU-11 and WEHI-231 cells were plated in 96-well plates and were treated with ethanol (vehicle; final concentration of <0.1%), ciglitazone (1–100 µM), or GW7845 (1–100 µM) for 2 h. MTT reagent was added, and the incubation was continued for 4 h. Solubilization buffer was added, and the cells were allowed to solubilize overnight at 37°C. Absorbance was determined at 562 nm using a spectrophotometric plate reader.
Protein isolation
Cytoplasmic and nuclear proteins were isolated as follows. Cells were resuspended in P10EG lysis buffer containing 10 mM sodium phosphate (pH 7.5), 0.75 mM EDTA, 10% glycerol, 0.75% Triton X-100, 0.5% protease inhibitor cocktail for mammalian cells, and 1 mM PMSF. Plasma membranes were broken by pipetting, and the quality of nuclear isolation was determined by visual inspection. Nuclei were pelleted by centrifugation for 2 min at 13,000 x g at 4°C. The resulting supernatant containing cytoplasmic proteins was collected and stored at -80°C. Nuclear pellets were washed in P10EG and resuspended in cold nuclear lysis buffer containing 20 mM HEPES (pH 7.9), 420 mM sodium chloride, 1.5 mM magnesium chloride, 1 mM EDTA, 25% glycerol, 0.1% Triton X-100, 1 mM DTT, 1 µg/ml aprotinin, 1 mM sodium orthovanadate, 10 µg/ml leupeptin, and 1 mM PMSF. Nuclei were incubated for 30 min on ice and centrifuged for 15 min at 13,000 x g at 4°C. The supernatant containing nuclear proteins was collected and stored at -80°C. Protein content was determined by the Bradford method.
PPAR and RXR expression
Cytoplasmic proteins (70 µg) were resolved on 10% SDS-PAGE
gels, electrophoretically transferred to 0.2-µm pore size
nitrocellulose membranes, and incubated with pAb specific for
PPAR (no. 516555). The secondary Ab was HRP-linked goat
anti-rabbit IgG (Bio-Rad, Hercules, CA). Immunoreactive bands were
visualized using enhanced chemiluminescence.
RXR expression was determined by Northern blot. Total RNA was
isolated using RNeasy (Qiagen, Valencia, CA). Ten micrograms of total
cellular RNA were electrophoresed on a 1% formaldehyde agarose gel and
blotted onto a 
probe transfer membrane (Bio-Rad, Mississauga,
Canada). The membranes were hybridized to DNA probes labeled by
Ready-To-Go DNA labeling beads (Amersham Pharmacia Biotech, Piscataway,
NJ) and purified with ProbeQuant G-50 Micro Columns (Amersham Pharmacia
Biotech). Hybridizations and autoradiography were performed as
previously described (55). The radiolabeled probe was
isolated from the plasmid containing a 1.8-kb EcoRI fragment
of RXR cDNA (56) .
EMSA
BU-11 cells were plated in T75 flasks and treated with ethanol (vehicle; final concentration, <0.1%), ciglitazone (10–70 µM), GW7845 (10–60 µM) and 9-cis-retinoic acid (40 µM) for 15 min to 8 h. Cells were collected and washed in PBS. Nuclear proteins were extracted as described above.
For determination of NF-B activation, a double-stranded
oligonucleotide containing the NF-B binding site from the upstream
B regulatory element (URE) of c-myc
(5'-GATCCAAGTCCGGGTTTTCCCCAACC-3') (57)
was used. The consensus sequence is underlined. The DNA probe was
end-labeled using T4 polynucleotide kinase (Promega, Madison, WI) and
[-32P]ATP, and was purified using a
Centrispin-20 column (Princeton Separations, Adelphia, NJ). EMSA was
performed as follows. The 32P-labeled DNA (
0.5
ng, 50,000 cpm) and 2 µg of nuclear protein were combined with buffer
(final concentrations: 10 mM Tris-HCl (pH 7.5), 1 mM EDTA, 100 mM
sodium chloride, 0.5 mM magnesium chloride, 1 mM DTT, 10% glycerol,
and 0.5% Triton X-100) and poly(dI-dC) (1 µg) in a final volume of
20 µl. The mixture was incubated at room temperature for 30 min.
Polyacrylamide gels (16 cm, 5%) were prerun at 200 V for 30 min.
Mixtures were electrophoresed at 200 V for 1.5 h in 0.5x TBE
(final concentrations, 44 mM Tris-base (pH 8), 44 mM boric acid, and
0.8 mM EDTA). The gels were dried and exposed to film. For
quantification, the gels were analyzed by phosphorimaging on a
PhosphorImager (Molecular Dynamics, Sunnyvale, CA). The specificity of
the shifted bands was determined by including 100x cold
oligonucleotides containing the NF-B site for the URE of
c-myc or an unrelated site
(5'-GAGCCGCAAGTGACTCAGCGCGGATCAATTA-3'). The identities of the
NF-B subunits were determined by including Abs specific for p50
(sc-114), p52 (sc-848), Rel A (sc-372), or c-Rel (sc-71) and looking
for the presence of supershifted bands. For determination of Oct-1-DNA
binding, the above procedure was used with the consensus Oct-1 binding
sequence (5'-TGTCGAATGCAAATCACTAGAA-3'; Promega).
For determination of PPAR-DNA binding, a double-stranded
oligonucleotide containing the PPRE from the acyl-coenzyme A (acyl-coA)
oxidase gene
(5'-GTCGACAGGGGACCAGGACAAAGGTCACGTTCGGGAGTCGAC-3')
(22) was used. The consensus sequence is underlined.
PPAR binds to this site with significantly greater affinity than
PPAR (58). The DNA probe was end labeled and
purified as described above. EMSA was performed as follows.
32P-labeled DNA (0.5 ng, 60,000 cpm) and 4
µg of nuclear protein were combined with buffer (final
concentrations, 20 mM HEPES (pH 8), 0.2 mM EDTA, 60 mM sodium chloride,
0.1% Nonidet P-40, 1 mM DTT, and 10% glycerol) and poly(dI-dC) (1
µg) in a final volume of 20 µl. The mixture was incubated at room
temperature for 30 min. The gel was run as described above, dried, and
exposed to film. The specificity of the shifted bands was determined by
including Abs specific for PPAR, 100x cold oligonucleotides
containing the PPRE from the acyl-coA oxidase gene, the PPAR
half-site of the PPRE from the acyl-coA oxidase gene
(5'-AGCTGGACCAGGACAAA-3'), or an unrelated site
(5'-GATCTGGCTCTTCTCACACAACTCCGGATC-3').
Statistics
Statistical analyses were performed with SPSS 8.0 for Windows (SPSS, Chicago, IL). One-factor ANOVAs were used to analyze the data. Dunnett’s multiple comparisons test was used to determine significant differences.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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agonists induce apoptosis in pro/pre-B, immature B, and
primary bone marrow B cells
Previous studies suggested that B cells in the later stages of
development are susceptible to PPAR-mediated apoptosis
(45). Information on PPAR and its role in the
developing immune system is very limited. The effects of PPAR
agonists on developing lymphocytes are of particular concern given the
increased sensitivity of the developing immune system to other
environmental chemicals (34, 35, 36, 37) and therapeutics
(38). Therefore, we designed these studies to investigate
the effects of PPAR agonists on B cells at early stages of
development.
Ciglitazone is a thiazolidinedione, a class of chemicals currently in
use as a treatment for type II diabetes (23). BU-11 cells
exhibited a relatively low level of background apoptosis (<19%), as
measured by propidium iodide staining and flow cytometric analysis
(Fig. 1
A). Treatment with 80
µM ciglitazone rapidly (i.e., within 4 h) and consistently
induced apoptosis in a large fraction (>85%) of these pro/pre-B
cells. Apoptosis induction was not development stage-specific, since
>65% of the WEHI-231 immature B cells similarly underwent apoptosis
within 6 h (Fig. 1A). Analyses of an early marker of
apoptosis, annexin V staining, indicated that >90% of either the
BU-11 or WEHI-231 population was induced to undergo apoptosis within
4–6 h (Fig. 1B). Detection of apoptotic WEHI-231 cells with
the annexin stain at a somewhat later time point (6 h) could reflect a
slightly delayed kinetics of apoptosis in these immature B cells. As
expected, DNA fragmentation, a hallmark of apoptosis, was readily
demonstrable in both cell populations following a 6- to 8-h exposure to
ciglitazone (Fig. 1C). Finally, the activation of effector caspase 3
(59) was substantiated by the demonstration of PARP
cleavage following ciglitazone exposure (Fig. 1D).
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agonist, it is possible that its
ability to induce pro/pre-B cell apoptosis in these cell lines is
related to other pharmacologic activities. To address this possibility
and to extend studies to a more potent PPAR agonist, BU-11 and
WEHI-231 cells were exposed, as described above, to GW7845, a PPAR
agonist and tyrosine analog whose chemical structure is considerably
different from that of ciglitazone (23). In this series of
experiments, GW7845 induced greater than 80 and 65% of BU-11 and
WEHI-231 cells, respectively, to undergo apoptosis within 4 h
(Fig. 2A). Greater than 90%
of BU-11 or WEHI-231 cells had entered the early phase of apoptosis by
4 h as measured by annexin V staining (Fig. 2B). In
keeping with the greater potency of GW7845 (23), DNA
fragmentation (Fig. 2C) and PARP cleavage (Fig. 2D) were seen at an earlier time point, i.e., 2 h, than
following ciglitazone treatment. PARP cleavage at both the 2- and 4-h
time points was more profound in the pro/pre-B BU-11 cells than in the
immature WEHI-231.
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). In the
presence of serum, the EC50 in BU-11 cells
following GW7845 exposure was significantly lower than that for
ciglitazone (24 vs 51 µM; p < 0.05). Similarly, in
WEHI-231 cells, the EC50 for GW7845 was 38 µM,
and that for ciglitazone was 52 µM (p <
0.05). Interestingly, the EC50 for
ciglitazone-treated BU-11 cells was reached at 10-fold lower
concentrations (5 µM) in the absence of serum (data not shown). This
may be an important consideration when comparing results from different
studies.
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agonists, ciglitazone, or
GW 7845, rapidly (i.e., within 4 h) induced a significant increase
in annexin V staining (Fig. 4).
Consequently, it was concluded that bone marrow B cells, the majority
of which are surface Ig-, pre-B cells, are
exquisitely sensitive to PPAR agonists.
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expression and relationship to apoptosis
PPAR along with its dimerization partner RXR are expressed
in mature B cell lines (45). To determine whether PPAR
is constitutively expressed at the pro/pre-B cell stage, protein was
extracted from BU-11 cells and, as a positive control, from WEHI-231
cells and was analyzed by Western blot. Both cell types express
significant levels of PPAR (Fig. 5A). Similarly, RXR mRNA
was present in both BU-11 and WEHI-231 (Fig. 5B).
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and RXR in the BU-11 pro/pre-B
cell line, it was predicted that both ciglitazone and GW7845 would
induce binding of the PPAR complex to its DNA recognition element,
with GW7845 probably being a more potent inducer of PPAR-PPRE
binding. Indeed, treatment of BU-11 cells with ciglitazone and GW7845
resulted in a time-dependent increase in binding to the PPRE sequence
from the acyl-coA oxidase gene (Fig. 6A). As with other assays,
GW7845 was the more potent agonist, inducing PPAR complex-PPRE
binding within 15 min. The specificity of DNA binding was confirmed by
competition from 100x cold sequence containing the PPRE from the acyl
co-A oxidase gene and the lack of competition by the PPAR half-site
of the PPRE from the acyl-coA oxidase gene or an unrelated sequence
(Fig. 6B). In addition, the PPAR complex-PPRE band could
be supershifted with an Ab specific for PPAR. These results
demonstrate that PPAR was activated in these cells following agonist
exposure.
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signaling for a number of biologic
outcomes (31, 60), the role of the RXR in delivering
the apoptosis signal in B lymphocytes has not been addressed. RXR
signaling has been found to be involved in T lymphocyte
(61) and leukemic myeloid precursor cell apoptosis
(62). To evaluate the contribution of the RXR to
pro/pre-B cell apoptosis, BU-11 cells were treated with limiting doses
of ciglitazone (10 or 20 µM) with or without 10 or 20 µM
9-cis-retinoic acid. Addition of 10–20 µM
9-cis-retinoic acid or 10 µM ciglitazone to BU-11 cell
cultures had no significant affect on the viability of BU-11 cells
(Fig. 7). In contrast, cotreatment with
10 µM 9-cis-retinoic acid and 20 µM ciglitazone
synergistically increased the incidence of BU-11 apoptosis from a
relatively low background level of 3–6 to 21% (Fig. 7). Similarly,
addition of 20 µM 9-cis-retinoic acid along with 20 µM
ciglitazone increased apoptosis from 7% in cultures treated with 20
µM ciglitazone alone to 56% (p < 0.01).
These data are consistent with a role for the RXR in
ciglitazone-mediated pro/pre-B cell apoptosis.
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and NF-B activation
Previous studies suggested that activation of PPAR can either
down-regulate NF-B (13, 14, 15) or cooperatively enhance
NF-B-mediated transcription (49). Since we have shown
that NF-B modulation in WEHI-231 and BU-11 cells activates the cell
death signaling pathway (46, 47), it was postulated that
PPAR activation would affect NF-B activity either before or
concomitant with apoptosis induction. To test this hypothesis, BU-11
cells were treated with ciglitazone for 30 min to 8 h, and nuclear
proteins were extracted and assayed by EMSA for binding to the URE of
c-myc. As predicted, analysis of NF-B-DNA band densities
indicated that treatment with ciglitazone resulted in a time-dependent
increase in NF-B-DNA binding (Fig. 8A). NF-B-DNA binding
increased as early as 1 h post-treatment and climbed to a maximum
of 3.6-fold at 6–7 h post-treatment (Fig. 8B). The failure
to detect a change in binding of the constitutively active Oct-1
transcription factor with its cognate DNA binding element demonstrated
the specificity of the NF-B response (data not shown). Only doses of
ciglitazone that induced apoptosis (i.e., >40 µM) induced an
increase in NF-B-DNA binding (Fig. 8C). Competition
studies confirmed the specificity of the bands, and supershift assays
determined that binding of p50, p65, and c-rel contributed to the
increase (Fig. 8D).
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B-DNA binding, and the
effect was more rapid than that observed with ciglitazone (Fig. 9). That is, NF-B-DNA binding
increased as early as 15 min post-treatment and peaked at 1 h,
with a maximum 2.5-fold increase in binding. NF-B-DNA binding
dropped rapidly after 1 h, coincident with a decrease in Oct-1-DNA
binding that presumably reflected the rapid demise of GW7845-treated
cells. Only doses of GW7845 that caused significant apoptosis (>30
µM) stimulated NF-B-DNA binding.
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B is involved in apoptosis induction, it would be predicted
that addition of 9-cis-retinoic acid would increase
NF-B-DNA binding synergistically, as it increased the induction of
apoptosis. A dose of 9-cis-retinoic acid (40 µM) that had
no effect on apoptosis (data not shown) similarly had no effect on
NF-B-DNA binding (Fig. 10A). Consistent with
results presented in Figs. 3
and 8, 30 µM ciglitazone had no
significant effect on either apoptosis or NF-B-DNA binding (Fig. 10A). However, doses of ciglitazone as low as 30 µM, in
combination with 40 µM 9-cis-retinoic acid, synergized to
generate an increased level of NF-B-DNA binding. Similarly, a
combination of 20 µM GW7845 and 40 µM 9-cis-retinoic
acid synergistically increased NF-B binding (Fig. 10B).
The combination of 30 µM GW7845 and 40 µM 9-cis-retinoic
acid resulted in a significant reduction in Oct-1 binding (data not
shown), suggesting that there was an increase in PPAR-mediated cell
death at this dose as well.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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has the potential to influence multiple functions of the
immune system. It has been shown to regulate macrophage inflammatory
responses (11, 12, 13, 14, 15); T cell activation, proliferation, and
production of inflammatory cytokines (16, 17, 18); endothelial
cell production of chemokines (19); and dendritic cell
maturation (20). A growing body of evidence suggests that
PPAR may play a role in B cell function as well (21, 45). Here we show for the first time that cells in the
developing immune system are sensitive to the effects of PPAR
agonists. We show that PPAR agonists rapidly induce apoptosis in
primary B cells from the bone marrow and in a B cell line representing
the pro/pre-B cell stage. The role of PPAR in stimulating apoptosis
is substantiated by the facts that pro/pre B cells express PPAR and
RXR, that PPAR-DNA binding increases in response to agonist
exposure, and that cotreatment with PPAR and RXR agonists
synergistically increases the incidence of apoptosis. Furthermore,
apoptosis is preceded by a profound increase in NF-B-DNA binding,
suggesting a role for this transcription factor in the death of early B
cells.
PPAR agonists, including 15d-PGJ2 and glitazone drugs, induce
apoptosis in cells of the immune system, including monocytes
(14) and T cells (63). Critical initial
studies by Padilla et al. (45) suggested that the
mechanism of B cell death was PPAR-dependent, since
PPAR-selective glitazone drugs induced apoptosis. In addition,
15d-PGJ2, but not a control prostanoid PGF2,
induced apoptosis; 15d-PGJ2 had greater potency in inducing apoptosis
than any other compound tested, potentially reflecting the greater
affinity of 15d-PGJ2 for PPAR binding and/or its ability to directly
interact with NF-B family members (64). The potential
for 15d-PGJ2-mediated NF-B down-regulation to induce apoptosis in
pro/pre-B cells is currently under investigation.
The data presented here demonstrate the high level of sensitivity of
the developing immune system to PPAR agonists. It was shown that
pro/pre-B cells are exquisitely sensitive to PPAR agonists. Primary
bone marrow B cells, as well as an established, stromal cell responsive
pro/pre-B cell line, undergo apoptosis as early as 2 h after
exposure to PPAR agonists. Our results provide substantial evidence
that the mechanism of death is PPAR-dependent. First, apoptosis was
induced by two structurally diverse PPAR ligands: ciglitazone, a
thiazolidinedione, and GW7845, a tyrosine analog (23). The
concentrations required to induce death in B cells were significantly
greater (10-fold) than those reported previously (45).
However, in the previous studies serum was not present during the
initial exposure to the agonists, a factor we have shown here to
significantly reduce the concentration required to induce apoptosis. A
similar phenomenon has been noted for ligands of the aryl hydrocarbon
receptor (45). GW7845 induced B cell death both at a lower
dose and in a more rapid manner than ciglitazone, potentially
reflecting a higher affinity for the receptor (23).
Evaluation of apoptosis at early time points (i.e., 2–4 h) revealed
the extremely rapid manner in which PPAR agonists induce apoptosis,
a previously unrecognized feature of PPAR-mediated apoptosis.
Second, not only are PPAR and its dimerization partner RXR
expressed in B cells in early stages of development, they are capable
of activation and DNA binding. Studies have shown that PPAR
(3, 7, 45) and RXR (33, 65) are expressed
in B lineage cell lines and mature B cells in vivo, but have not
addressed their expression in the developing immune system or their
competence for activation in B cells. We show here that B cell lines
representing the pro/pre- and immature B cell stages express PPAR
and RXR. In addition, we demonstrate that the PPAR:RXR
heterodimer is capable of DNA binding in B cells. The increase in
PPAR-DNA binding following agonist exposure indicates that PPAR
has been activated in these B cells and potentially could participate
in initiating a death program.
Finally, the participation of the PPAR:RXR heterodimer in
initiation of apoptosis is indicated by the synergistic increase in the
incidence of apoptosis in the presence of agonists for both receptors.
The PPAR:RXR heterodimer is known as a permissive heterodimer in
which RXR ligands alone can activate transcription by the heterodimer
(66). Cotreatment with ligands for both PPAR and RXR
results in a synergistic increase in PPAR-dependent
trans-activation of reporter genes, adipocyte
differentiation in cultured cells (67), and
differentiation of liposarcomas in vivo (60). Synergy
results from increased recruitment of coactivators and an RXR-dependent
conformational change in PPAR (31). In addition, RXR
signaling has been found to be involved in T lymphocyte
(61) and leukemic myeloid precursor cell apoptosis
(62).
A physiological role for PPAR in B cells is suggested by studies
with PPAR haploinsufficient mice (21). There is an
increased proliferative response of B cells from PPAR
haploinsufficient mice following stimulation by LPS or cross-linking of
Ag-specific receptors. In addition, haploinsufficient mice show
increased levels of specific Abs following immunization with
either OVA or mBSA. These effects may be linked to a dysregulation of
the NF-B pathway. There was an increase in spontaneous NF-B
activation as well as increased IB phosphorylation and
NF-B-DNA binding following LPS exposure in B cells from
PPAR+/- mice.
A growing number of studies have highlighted the interaction of PPAR
and NF-B. The majority of research has suggested that both natural
and synthetic PPAR agonists antagonize NF-B-DNA binding and
trans-activation stimulated by activators such as LPS,
TNF-, PMA, and IL-1 (13, 14, 15, 16, 48, 68, 69, 70, 71, 72, 73). Most
studies show a decrease in NF-B-DNA binding or
trans-activation that may result from IB kinase
inhibition (73) or from the physical interaction of
PPAR and NF-B (15). However, the results presented
here suggest that PPAR agonists alone can strongly stimulate
NF-B-DNA binding. The PPAR dependence of the activation of
NF-B is suggested by the fact that cotreatment with an agonist for
RXR decreases the dose required to stimulate NF-B activation.
There is other evidence for a positive interaction between PPAR and
NF-B. IL-1-induced type II secreted phospholipase A2 expression
requires the binding of both NF-B and PPAR, and they are
suggested to cooperate at the enhanceosome-coactivator level
(49). NF-B and PPAR are coordinately activated in
human neuroblastoma cells following exposure to an Alzheimer’s peptide
(50). Finally, rosiglitazone stimulates TNF--induced
cyclooxygenase-2 expression in human colorectal carcinoma cells by
up-regulating the TNF- pathway potentially via NF-B activation
(51).
The strong activation of NF-B following exposure to PPAR agonists
may reflect an attempt to override the potent death signal induced by
those agonists. Generally NF-B is thought of as a survival factor,
typified by its protection against TNF--, anti-B cell receptor-,
or environmental pollutant-induced cell death (46, 47, 74). However, the activation of NF-B also could initiate the
death signal. Indeed, NF-B was initially described as a
pro-apoptotic factor (75, 76, 77) required for bacteria- and
virus-induced cell death (73, 78), Fas ligand-induced
apoptosis in T lymphocytes (79), and p53-mediated cell
death initiated by such stimuli as UV radiation (80).
Interestingly, activation of NF-B via the mitogen-activated protein
kinase pathway is required for p53-mediated apoptosis
(80). PPAR ligands, both 15d-PGJ2 and glitazone drugs,
have been shown to up-regulate the same pathway (81). It
remains to be determined whether the PPAR agonists used in these
studies up-regulate mitogen-activated protein kinase in BU-11 and
whether this may be related to the increase in NF-B activation.
Attempts to confirm a causal relationship between PPAR
agonist-induced NF-B up-regulation and cell death have been hampered
by the fact that concentrations of NF-B inhibitors required to block
such a strong and rapid activation of NF-B tend to rapidly reduce
the baseline levels of NF-B, a signal that also induces BU-11 cell
apoptosis (46).
While the results presented here suggest that ciglitazone and GW7845
initiate apoptosis via a PPAR-dependent mechanism, they do not rule
out the possibility that PPAR also may participate in initiating
apoptosis in B cells. PPAR and PPAR are both expressed in
lymphocytes and myeloid cells. While PPAR is expressed more highly
than PPAR in myeloid lineage cells, PPAR is the dominant form in
lymphocytes (2). The potential for interaction between
PPAR and PPAR is suggested by overlapping agonist specificities
(82).
In general, the results presented here and previously (21, 45) suggest that PPAR may be involved in B cell development,
proliferation, and immune responsiveness. The demonstration here that
the developing B cell compartment is particularly sensitive to
PPAR agonists emphasizes a mechanism through which
environmental PPAR agonists could suppress the immune system and
suggests possible debilitating side effects of PPAR agonist
therapeutics. Additional research is required to define the nominal
role of PPAR in early B cells, particularly with regard to its
influence on NF-B, a critical element in normal B cell development
and function.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Jennifer J. Schlezinger, Department of Environmental Health, Boston University School of Public Health, 15 Stoughton Street, Housman R405, Boston, MA 02118. E-mail address: jschlezi{at}bu.edu
3 Abbreviations used in this paper: PPAR, peroxisome proliferator-activated receptor; acetyl-coA, acyl-coenzyme A; MTT, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide; 15d-PGJ2, 15-deoxy-12,14-PGJ2; RXR, retinoid X receptor; URE, upstream B regulatory element; PARP, poly(ADP-ribose) polymerase.
Received for publication July 23, 2002. Accepted for publication October 9, 2002.
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