The molecular mechanisms regulating monocyte differentiation to
macrophages remain unknown. Although the transcription factor NF-
B
participates in multiple cell functions, its role in cell differentiation is ill defined. Since differentiated macrophages, in
contrast to cycling monocytes, contain significant levels of NF-B in
the nuclei, we questioned whether this transcription factor is involved
in macrophage differentiation. Phorbol 12-myristate 13-acetate
(PMA)-induced differentiation of the promonocytic cell line U937 leads
to persistent NF-B nuclear translocation. We demonstrate here that
an increased and persistent IKK activity correlates with monocyte
differentiation leading to persistent NF-B activation secondary to
increased IB
degradation via the IB signal response domain
(SRD). Promonocytic cells stably overexpressing an IB transgene
containing SRD mutations fail to activate NF-B and subsequently fail
to survive the PMA-induced macrophage differentiation program. The
differentiation-induced apoptosis was found to be dependent on tumor
necrosis factor alpha. The protective effect of NF-B is mediated
through p21WAF1/Cip1, since this protein was found to be
regulated in an NF-B-dependent manner and to confer survival
features during macrophage differentiation. Therefore, NF-B plays a
key role in cell differentiation by conferring cell survival that in
the case of macrophages is mediated through p21WAF1/Cip1.
 |
INTRODUCTION |
The molecular mechanisms regulating
cell differentiation involve a fine and complex balance of proteins and
signal transduction pathways that modulate the progression through the
cell cycle and control of cell survival (11, 48). Human
macrophages are differentiated noncycling cells arrested at the
G1 checkpoint that derive from peripheral blood cycling
human monocytes. These cells are present in multiple body tissues and
play a key role in various immune functions and thus are relevant to a
number of human diseases (26, 45). Unlike the
well-characterized process of B-lymphocyte differentiation, little is
known regarding the molecular mechanisms governing the monocyte
differentiation to macrophage, Promonocytic human cell lines, like
human primary monocytes, can be triggered to differentiate to human
macrophages by defined stimuli (45). Agents such as
phorbol esters (phorbol 12-myristate 13-acetate [PMA]) induce their
exit from the cell cycle at G1, leading to their
differentiation, which is characterized by the appearance of
macrophage-like features such as cell surface integrins, adherence to
plastic, and the production of reactive oxygen intermediates (4,
8, 21).
A number of transcription factors such as PU.1 have been implicated in
monocyte differentiation, as have other proteins, including p21WAF1/Cip1 (21, 32, 43, 44, 48, 60).
p21WAF1/Cip1 is a nuclear protein of the family of
cyclin-dependent kinase (CDK) inhibitors (17) which
functions to inhibit CDK1, -2, -4, and -6 (9) and, hence,
its induction by a number of stimuli causes cell cycle arrest at the
G1/S boundary (8, 20, 37, 51, 57), allowing
the cell to exit the cell cycle and differentiate. Interestingly in
differentiated human macrophages, p21WAF1/Cip1 is
predominantly located in the cytosol where it exerts an antiapoptotic function by means of inhibiting the proapoptotic kinase Ask1
(1). Whether the critical role that
p21WAF1/Cip1 plays in macrophage differentiation is
dependent on its ability to inhibit the cell cycle or protect the cell
from death while it is undergoing differentiation is unclear.
Members of the NF-B/Rel family of transcription factors are key
regulators of a variety of genes involved in cell growth and survival
(3, 4, 6). NF-B is generally found as an inactive dimer
sequestered in the cytoplasm by inhibitor proteins termed IB.
However, in a few exceptions, such as terminally differentiated B
lymphocytes and plasma cells, as well as human macrophages, NF-B is
constitutively present at very high levels in the nuclei of these cells
(7, 35, 40). At least two potential mechanisms involving
IB regulation can result in the nuclear translocation of NF-B.
Acute stimuli such as interleukin-1 (IL-1) or tumor necrosis factor
alpha (TNF-) lead to the rapid proteolysis of IB molecules via a
common, terminal transduction pathway leading to the rapid
phosphorylation of IB by two highly related serine kinases, IKK1 and
IKK2. These kinases phosphorylate IB on critical serine residues,
such as Ser32 and Ser36, within the N-terminal signal response domain
(SRD) of IB (31, 61). In addition, IB molecules
have a C-terminal PEST domain, which is a highly negative charged
region conferring protein instability and hence favoring a slow but
continuous level of IB degradation (55, 56). Cycling
primary human monocytes have no NF-B in their nuclei. However, their
differentiation to macrophages triggered by plastic adherence or
phorbol esters leads to a progressive and significant degree of NF-B
nuclear translocation, a hallmark of differentiated macrophages. The
molecular mechanisms regulating NF-B translocation to the nucleus
during macrophage differentiation and the role NF-B plays in the
relevant cellular processes is unknown (24, 25, 34, 53).
The distinct changes in the cellular localization observed for
p21WAF1/Cip1 and NF-B during macrophage differentiation
and their potential influence in cell survival and cell cycle control
raise the possibility that these key proteins may regulate each other,
leading to a coordinated process of differentiation. Likewise, previous
observations in a number of systems suggest that NF-B may regulate
p21WAF1Cip1 expression (5, 29, 49). The recent
availability of genetic tools to inhibit the expression or function of
such proteins has enabled us to address their role in macrophage differentiation.
| |
MATERIALS AND METHODS |
Cells, cell culture, and induction of differentiation.
The
human promonocytic cell line U937 was purchased from the American Type
Culture Collection and grown in RPMI 1640 (BioWhitaker) plus 5%
(vol/vol) heat-inactivated fetal bovine serum (FBS; Intergen), penicillin and streptomycin (100 U/ml; Gibco-BRL), and glutamine (0.3 mg/ml; Gibco-BRL). U937 clones expressing FLAG-IB transgenes were
previously described (2). Results shown herein were
similar for each of three separate clones. U937 cells stably expressing either empty vector (pREP4) or p21(AS) constructs (F4 and B8) were
previously described (60) and were maintained in RPMI 1640 supplemented with 10% FBS, penicillin-streptomycin, glutamine, minimal
essential medium (MEM) nonessential amino acids (0.1 mM; Gibco-BRL),
MEM sodium pyruvate (1 mM; Gibco-BRL), and 200 µg of hygromycin B
(Sigma) per ml. All cells were maintained at 37°C and 5%
CO2. Cells were seeded at 2 × 105
cells/ml and induced to differentiate with PMA (2 ng/ml; Sigma) or
treated with vehicle control (dimethyl sulfoxide [DMSO]). TNF (10 ng/ml; Genzyme) was used at the indicated times as a positive control
for IB degradation and subsequent NF-B activation.
For the inhibition of differentiation-induced apoptosis, cells were
seeded as described above and induced to differentiate with PMA. At
time of culture initiation, TNF receptor (TNFR)-Fc (5.5 ng/ml; Immunex
Corp., Seattle, Wash.) was added. An equivalent quantity of IL-4
receptor (IL4R)-Fc was added to parallel cultures as a control.
Evaluation of differentiation and survival.
Cells were
seeded and induced to differentiate with PMA as described above. At the
indicated time points, cells were evaluated for differentiation
markers. Cells were examined microscopically for differentiation
morphology such as increased cell volume, granularity, and the
appearance of irregularly shaped cellular processes. Adherence was
determined by gently rocking the plates and by collecting nonadherent
cells in the culture supernatant. Phosphate-buffered saline (PBS) was
added back to the culture flasks, and adherent cells were lifted by
gentle scraping. Viable cells were scored by trypan blue exclusion.
Surface expression of CD11c was determined by staining with fluorescein
isothiocyanate-conjugated CD11c antibody (BioSource) detected by
fluorescence-activated cell sorter (FACS) and analyzed with CellQuest
Software (Becton Dickinson). The cell cycle profile was determined by
propidium iodide (PI) staining. In brief, cells were washed twice in
ice-cold PBS and resuspended in approximately 100 µl of PBS. Cells
were fixed and permeabilized with ice-cold 70% ethanol at least
overnight. Fixed cells were pelleted by cold centrifugation and
resuspended in sample buffer containing 50 µg of PI and 100 Kunitz U
of RNase A per ml. Cells were analyzed by FACS and CellQuest Software. The sub-G0 population was scored as apoptotic.
Nuclear and cytoplasmic extract preparation,
immunoprecipitations, and in vitro kinase assays.
Nuclear and
cytosolic extracts were prepared by a modification of the method of
Dignam et al. (14). Briefly, cells were washed twice in
buffer A (10 mM HEPES [pH 7.9], 1.5 mM MgCl2, 10 mM KCl).
Cells were lysed two times for 5 min on ice in buffer A containing
0.1% NP-40; 0.5 mM dithiothreitol (DTT); 0.5 mM phenylmethlysulfonyl fluoride (PMSF); 2 µg each of aprotonin, leupeptin, and pepstatin per
ml; and 1 mM sodium orthovanadate. After centrifugation, the cells were
washed twice in buffer A, and the nuclei were collected by
centrifugation. Pelleted nuclei were lysed in 10 to 15 µl of buffer C
(20 mM HEPES [pH 7.9], 25% glycerol, 0.42 M NaCl, 1.5 mM
MgCl2, 0.2 mM EDTA [pH 8.0], DTT, PMSF, aprotinin,
leupeptin, pepstatin, and sodium orthovanadate) by rotation at 4°C
for 30 min. After centrifugation, the supernatants were diluted in 2× volumes of buffer D (20 mM HEPES [pH 7.9], 20% glycerol, 50 mM KCl,
0.2 M EDTA, DTT, PMSF, aprotinin, leupeptin, pepstatin, and sodium orthovanadate).
For immunoprecipitations, cells were washed twice in 50 mM Tris-HC (pH
7.5) and 150 mM NaCl and then lysed for 5 min on ice in wash buffer
plus 0.1% Trition X-100; 2 µg each of aprotinin, leupeptin, and
pepstatin per ml; and 0.5 mM PMSF. Following clarification by
centrifugation, cytoplasmic extracts (50 µg) were rotated at 4°C
for 1 to 2 h in the presence of antibodies to the N and C termini
of RelA (Santa Cruz) and 10 µl of protein A-conjugated agarose beads
(Gibco-BRL). Precipitates were washed three times with lysis buffer and
eluted with 2× Laemmli sample buffer at 95°C. Subsequent
immunoprecipitations on supernatants demonstrated complete
precipitation of all RelA. Eluted proteins were resolved by sodium
dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on a 10%
polyacrylamide gel and transferred to Immobilon-P membranes (Millipore).
For in vitro kinase assays, whole-cell extracts were prepared by
washing the cells twice in ice-cold PBS, followed by lysis for 5 min on
ice with 40 mM Tris-HCl (pH 8.0), 0.3 M NaCl, 0.1% NP-40, 6 mM EDTA, 6 mM EGTA, 10 mM NaF, 10 mM p-nitrophenyl phosphate (P-NPP), 10 mM 
-glycerophosphate, 0.3 mM sodium
orthovanadate, DTT, PMSF, aprotinin, leupeptin, and pepstatin. Lysates
were clarified by centrifugation. Then, 100 µg of total cell extract
was used in subsequent kinase assays. Lysates were immunoprecipitated
with antibody to the IKK complex (IKK or MKP1; Santa Cruz) for
1 h, after which protein A-agarose was added for 1 h. Beads
were washed three times with lysis buffer, followed by one wash with
buffer A. Beads were incubated in 15 µl of kinase buffer (20 mM HEPES [pH 7.4]; 2 mM MgCl2; 2 mM MnCl; 10 mM ATP; 10 mM NaF; 10 mM P-NPP; 10 mM -glycerophosphate; 0.3 mM sodium
orthovanadate; PMSF; 2 µg each of aprotinin, leupeptin, and pepstatin
per ml; 1 mM DTT) with 2 µg of glutathione S-transferase
(GST)-IB1-53 and 0.1 µCi of
[
-32P]ATP. The kinase reaction was carried out for 30 min at 30°C, and samples were resolved by SDS-PAGE.
EMSA, Northern, and Western analyses.
For electrophoretic
mobility shift assays (EMSAs), 4 µg of nuclear extract was incubated
with a [-32P]ATP-labeled double-stranded probe as
previously described (2). Components of the probe binding
complex were identified by preincubation of the extract with antibodies
to specific components of NF-B (Santa Cruz) prior to probe exposure.
DNA-protein complexes were resolved on a 6% polyacrylamide gel, dried,
and visualized by autoradiography. Western blotting was performed as
recommended by the ECL Kit (Amersham) package insert. Antibodies to
p21WAF1/Cip1 (Transduction Laboratories), IB (Santa
Cruz), FLAG (Sigma), -actin (Sigma), and IKK and IKK (Santa
Cruz) were used. Where indicated, the intensities of the bands were
quantified by densitometry and analyzed by using the Ambis software
package. For Northern analysis, cells were treated with either DMSO,
PMA, or TNF for 6 h. Total RNA was isolated by RNAzol B (TelTest)
according to the manufacturer's instructions. RNA was resolved on an
agarose-formamide gel and transferred to a HyBond-N+
membrane (Amersham) p21WAF1/Cip1 cDNA, and
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) probes were prepared
by using the Random Primed DNA Labeling Kit (Roche) as described in the
package insert and then hybridized to membrane in RapidHyb Buffer
(Amersham) and washed in sequentially more stringent SSC (1× SSC is
0.15 M NaCl plus 0.015 M sodium citrate) washes. Results were
visualized by autoradiographyand quantified by densitometry using the
Ambis software package.
Adenoviral transduction.
Peripheral blood lymphocytes were
obtained from buffy coats using a Ficoll-Hypaque density gradient.
Mononuclear cells were washed extensively and incubated with
neuraminadase-treated sheep red blood cells to rosette T cells.
CD14+ monocytes were then isolated from the resulting
mononulear cell population by negative depletion using the StemSep
(StemCell) system according to the manufacturer's instructions.
Enriched CD14+ cells were typically found to be 80 to 86%
pure by FACS analysis. An aliquot of cells was lysed with whole-cell
lysis buffer as above. CD14+ cells were maintained in RPMI
1640 supplemented with 10% heat-inactivated human AB serum. Cells were
allowed to plastic adhere for 30 h, and then washed gently with
fresh media. Cells were fed with one-half conditioned medium plus fresh
AB media. Five days after isolation, The cells were washed to remove
nonadherent cells and transduced with adenovirus (100 PFU/cell)
expressing HA-IB Ser 32/36 Ala or alkaline phosphatase. The
transduction efficiency was monitored by nitroblue tetrazolium-BCIP
(5-bromo-4-chloro-3-indolylphosphate) staining (Roche) and typically
was 90 to 95% efficient. Two days after harvest, cells were lysed in
whole-cell lysis buffer as described above.
| |
RESULTS |
Persistent activation of NF-B correlates with cell
differentiation and is dependent on the IKK complex kinase
activity.
To address the role of NF-B on monocyte
differentiation, we first sought to characterize a model of monocyte
differentiation in which NF-B activation is observed in the context
of differentiation (24, 25) and which is amenable to
genetic inhibition following identification of the mechanism regulating
NF-B nuclear translocation. Using the human promonocytic U937 cell
line, it is demonstrated that these cells become adherent to plastic
and express CD11c integrin, a cell surface marker of differentiated
macrophages, by 24 h following exposure to low-dose (2 ng/ml) PMA
(Fig. 1A). As has been previously
reported (24, 25), the differentiation program initiated
by PMA correlates with the progressive nuclear localization of NF-B
by 24 h posttreatment (compare lanes 1 and 2 in Fig. 1B with lanes
4 and 5 in Fig. 1C). EMSA analysis demonstrates that the nuclear
complex of NF-B is composed mainly of RelA-p50 dimers (Fig. 1B,
lanes 4 and 5). EMSA analysis shows that c-Rel or RelB antibodies fail
to appreciably shift either NF-B activity, while the lower activity
can be completely shifted with p50 antibodies (data not shown). Nuclear
localization of NF-B correlates with a marked reduction in IB
steady-state protein levels (Fig. 1C), as well as of IB and p105
(data not shown). Therefore, PMA-induced differentiation of the U937
promonocytic cell line to a macrophage phenotype is accompanied by a
progressive and persistent nuclear translocation of NF-B resulting
from constitutively decreased IB protein.
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FIG. 1.
U937 promonocytic cells differentiate into a
macrophage-like phenotype following exposure to phorbol esters. (A)
U937 cells grown in suspension become adherent following exposure to
PMA (2 ng/ml) by 24 h with persistence of the phenotype for up to
3 days after the first appearance the PMA differentiation signal. FACS
profiles of CD11c expression following treatment with PMA or DMSO
vehicle are inset. (B) Differentiation leads to the nuclear
translocation of prototypic NF-B heterodimers at 24 h post-PMA
exposure. c-Rel and RelB antibodies had minimal effect on the
mobilities of either NF-B complex (data not shown). TNF-
treatment lasted 8 min and served as a positive control. (C) IB
steady-state levels are markedly reduced at 24 h posttreatment
with PMA and correlate with the presence of nuclear RelA. TNF-
treatment was for 8 min and served as a positive control.
|
|
The decreased steady-state protein level of IB observed in
PMA-differentiated U937 cells is secondary to a decrease in the protein's half-life (Fig. 2A). The
decrease in IB protein half-life could potentially arise from
activation signals targeting the SRD. Alternately, the C-terminus PEST
domain, previously shown to confer instability to a host of proteins,
could be targeted during differentiation in order to further decrease
the IB half-life. Using U937 cells stably expressing either a
wild-type FLAG-tagged IB (WT) or FLAG-tagged IB in which
either the SRD or the C-terminal PEST domain are mutated (32/36 or 4C,
respectively), we observed that PMA-induced cell differentiation
targets the SRD but not the PEST domain of IB, as demonstrated by
a decrease in the steady-state protein levels of FLAG-IB WT and
FLAG-IB 4C (Fig. 2B, compare lanes 1 to 2 and lanes 5 to 6) but
not of FLAG-IB 32/36 (Fig. 2B, lanes 3 and 4). The functional
relevance of this observation is demonstrated by the fact that
RelA-associated IB is targeted by the PMA-induced differentiation
process (Fig. 2C) and by the lack of nuclear translocation of NF-B
in PMA-stimulated FLAG-tagged 32/36 IB cells (Fig. 2D, compare
lanes 2 and 5).
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FIG. 2.
IB in differentiated U937 cells has a shorter
half-life than in undifferentiated cells, and the half-life is
dependent upon the SRD. (A) U937 cells differentiated by exposure to
PMA (2 ng/ml) for 24 h were lysed at different points following
treatment with cycloheximide and analyzed for IB levels by
Western blot. -Actin was probed as a gel loading control. (B) To
determine which region of IB mediates reduced IB
steady-state levels, U937 clones stably expressing FLAG-tagged IB
transgenes were induced to differentiate by PMA (2 ng/ml) and lysed at
6 h into the differentiation program. WT, FLAG-IB; 32/36,
FLAG-IB 32/36Ser-to-Ala; 4C, FLAG-IB
283/288/293/291Ser/Thr-to-Ala. IB levels were
determined by Western blot. Similar results were seen at 12 and 24 h postdifferentiation (data not shown). (C) In order to address the
direct role of IB in cytosolic retention of NF-B, clones
expressing either the FLAG-tagged WT IB or FLAG-IB 32/36
were differentiated for 24 h and then treated with cycloheximide (50 µg/ml). Treated cells were lysed and immunoprecipitated with
anti-RelA. Immunoprecipitates were then analyzed for RelA-associated
IB levels by Western blot. Gels were analyzed by densitometry,
and the half-life was plotted as a function of IB levels over
time. (D) Gel shift analysis demonstrates that mutation of the SRD of
IB abrogates differentiation-induced activation of NF-B by
PMA. WT or 32/36 cells were treated with PMA for 24 h. TNF
treatment was for 4 h and serves as a control. The identity of the
upper band (filled circle) was confirmed by supershift analysis as
RelA/p50. The lower band (arrow) was confirmed as p50 homodimers (data
not shown). The results are representative of three independent
clones.
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The SRD of IB is targeted by a number of kinases, including the
IKK complex, pp90rsk, and PK-CK2 (23, 31, 52).
While IKK is well characterized to mediate IB phosphorylation by
punctual and rapid stimuli such as TNF or IL-1, we questioned whether
it could also be involved in targeting the IB SRD as a result of
monocyte differentiation. The kinase activity of the IKK complex was
analyzed at different time points along the cell differentiation
program. As seen in Fig. 3, an increase
in IKK activity toward the SRD of the IB substrate is detected as
early as 2 h following PMA, with further increases at 24 and 48 h.
The persistent and heightened IKK activity further correlates with
reduced steady-state levels of IB (Fig. 3, compare panels 1 and
5). In contrast, the kinase activity of PK-CK2, previously shown to
directly phosphorylate the PEST (39) and SRD domain of
IB (52), is not observed during the same time
points. Therefore, the persistent decrease in IB levels observed
during monocyte differentiation correlates with persistent and
increased activity of the IKK complex. This is notable in demonstrating
that the IKK complex is not only punctually activated by defined
inflammatory stimuli such as TNF or IL-1 but also in a persistent
manner by processes such as cell differentiation.
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FIG. 3.
IKK activity is increased in differentiated U937 cells.
The differentiation program of U937 cells was initiated by treatment
with PMA (2 ng/ml). Cells were collected and lysed at 2, 24, or 48 h after PMA treatment. Lysates were immunoprecipitated with antibody to
either PK-CK2 or the IKK complex. The resulting immunocomplex was used
to radiolabel GST-IB1-53 in an immunokinase assay.
IKK or PK-CK2 levels were detected by immunoblotting to ensure that
there were equal levels of kinase in each sample. Membrane was
Coomassie blue stained to ensure the presence of equal amounts of
substrate in each reaction (data not shown). IB levels at the
indicated time points were detected by Western blot. -Actin was
probed as a gel loading control. TNF- treatment was for 8 min and
served as a positive control.
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Activation of NF-B is necessary for surviving the monocyte
differentiation program.
Although it is well established that
NF-B is activated during monocyte differentiation, little is known
about the functional relevance of this activation. Identification of
the IKK complex and the SRD domain of IB as targets of signaling
pathways triggered by PMA-induced monocyte differentiation enables us
to test the role of NF-B in this process by inhibiting its
activation using genetic approaches. U937 clones stably expressing
FLAG-tagged IB containing mutations of the key SRD serines
(32/36) or empty vector (SFFV) were treated with PMA and
evaluated thereafter for differentiation markers as in Fig. 1. As early
as 24 h and as late as 72 h post-PMA treatment, a marked reduction
in the number of U937 cells expressing the SRD IB mutant (32/36)
which become plastic adherent is observed. Following the initiation of
the differentiation program, both SFFV and 32/36 cells can complete at
least one turn of the cell cycle, as evidenced by the cell cycle
profile in Fig. 4C and the increase in
cell numbers for SFFV cells (Fig. 4B). However, there is a marked
decrease in viable 32/36 cells following PMA-induced monocyte
differentiation compared to SFFV control cells (Fig. 4B). That the
decreased cell survival observed in the PMA-treated 32/36 U937 cells is
apoptotic in nature is demonstrated by the increase of subdiploid DNA
in the PI cell cycle profile shown in Fig. 4C. From these data it is
inferred that the persistent nuclear translocation of NF-B confers a
protection to promonocytic cells undergoing the differentiation
process.
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FIG. 4.
Activation of NF-B is necessary for surviving the
differentiation program in U937 cells. (A) U937 cells induced to
differentiate with PMA (2 ng/ml) were scored for adherence at 24 or
72 h post-PMA treatment, and values are expressed as in Fig. 1.
(B) Vector control (SFFV) or cells stably expressing an SRD mutant
IB (32/36) were treated with either PMA or DMSO and harvested at
2, 6, 12, 24, or 72 h posttreatment. Only viable cells were
scored. No differences were seen in vehicle control SFFV or 32/36
cells. Only PMA-treated points are shown (*, P < 0.05;  P < 0.10; Student's t
test). (C) SFFV and 32/36 cells treated for 0, 24, or 72 h with
PMA were collected, fixed, and DNA stained with PI. The Cell cycle
distribution is shown. The apoptotic population was scored as the
sub-G1 DNA fraction by FACS analysis.
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|
Differentiation-induced cell death of monocytes is triggered by
TNF.
The novel observation that NF-B is necessary to survive
the differentiation process led us to examine what NF-B opposed factors may be initiating differentiation-induced apoptosis. It is well
established that NF-B protects a number of cells from TNF-induced
apoptosis (3, 6, 53, 59). In addition, several studies
have shown that TNF is involved in a number of hematopoeitic cell
differentiation models (16, 18, 38, 62). We asked, therefore, whether endogenous production of TNF is mediating the apoptosis seen during differentiation. U937 clones stably expressing mutant FLAG-tagged IB (32/36) or empty vector (SFFV) were treated with PMA and evaluated for apoptosis using PI staining as described above. Selected wells were also treated with a TNFR-Fc protein to block
binding of endogenous TNF to native TNF receptors. IL4R-Fc was used as
a control. As seen in Fig. 5, a
relatively low dose of TNFR-Fc blocked nearly half of the
differentiation-induced cell death in 32/36 U937 cells, while the
IL4R-Fc fusion protein had no effect. From these data, it can be
inferred that endogenously produced TNF by differentiating monocytes
triggers apoptosis. Whether this TNF elicited during differentiation
(following PMA treatment) itself is a differentiation signal is
unknown.
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FIG. 5.
TNF--mediated differentiation-induced apoptosis. U937
cells stably transfected with either empty vector (SFFV) or the
IB SRD mutant (32/36) were induced to differentiate with PMA (2 ng/ml) for 24 h. DMSO served as a vehicle control. At the
initiation of differentiation, cells were treated with TNFR-Fc or
IL4R-Fc. Apoptotic cells were scored by FACS analysis as the
sub-G1, DNA fraction following PI staining as in Fig. 4.
columns:  , DMSO;
 , PMA;
 , PMA plus
TNFR-Fc;  , PMA
plus IL4r-Fc.
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NF-B regulates p21WAF1/Cip1 expression which is
required for monocyte survival during the differentiation process.
The antiapoptotic characteristics of NF-B are presumed to be
mediated by a number of known and yet-to-be-identified survival genes
(3, 10, 12, 19). Several studies have previously reported
a link between NF-B activation and p21 expression in a number of
cell systems (see the introduction). Because of the p21WAF1/Cip1 upregulation observed during monocyte
differentiation and the ability of this protein to confer resistance to
apoptosis in cells of monocytic origin (1, 9, 29, 60), we
hypothesized that the requirement of NF-B to protect the cell from
death during monocyte differentiation may be through the upregulation
of p21WAF1/Cip1. To test this, we first analyzed whether
there exists a direct correlation between levels of
p21WAF1/Cip1 and nuclear translocation of NF-B during
the process of macrophage differentiation. PMA-induced differentiation
of SFFV vector control U937 cells correlated with the upregulation of
cytosolic p21WAF1/Cip1 in the absence of cell death. By
contrast, PMA treatment of 32/36 U937 cells did not result in the
upregulation of p21WAF1/Cip1 expression and, as expected,
resulted in cell death in the absence of NF-B activation (Fig. 6A
and B). Northern analysis of mRNA from
SFFV vector control and 32/36 U937 cells verifies the role of NF-B
in regulating p21WAF1/Cip1 transcription. Interestingly,
TNF is unable to induce p21WAF1/Cip1 expression, despite
being a potent inducer of NF-B activity, in direct contrast to
observations in Ewing sarcoma cells (29). Therefore, the
inability of 32/36 U937 cells to survive the differentiation program
may be due to their inability to induce expression of p21WAF1/Cip1, which we infer is an NF-B-dependent
process. To formally address this issue, we utilized U937 cells stably
expressing or not antisense constructs for p21WAF1/Cip1.
While PMA-induced differentiation of p21(AS) and control U937 cells
induces NF-B nuclear translocation in both cells types, p21WAF1/Cip1 expression is, as expected, not observed in
the p21(AS) U937 cells, indicating that NF-B is upstream of
p21WAF1/Cip1. Moreover, the fact that p21(AS) U937 cells
die following PMA-induced cell differentiation despite the induction of
NF-B nuclear translocation (Fig. 7),
supports the role of p21WAF1/Cip1 as an NF-B-dependent
survival gene required for macrophage differentiation.
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FIG. 6.
Activation of NF-B is necessary for expression of
p21WAF1/Cip1. (A) U937 cells induced to differentiate were
lysed at the indicated times after PMA treatment, and cytosolic and
nuclear extracts were collected. Cytosols were probed for
p21WAF1/Cip1 expression by Western blot (small arrow).
-Actin was blotted as a loading control. Similar results were seen
with whole-cell extracts (data not shown). Nuclear NF-B activity was
detected by EMSA, and the identity of the NF-B components was
confirmed by supershift assay as for Fig. 1 (data not shown). RelA-p50
and p50-p50 complexes are indicated by filled circles and arrows,
respectively. (B) Vector control (SFFV) or cells stably expressing an
SRD mutant IB (32/36) were treated with PMA and harvested at
various times posttreatment. The cell cycle distribution is shown. The
apoptotic cells were scored by FACS analysis as the sub-G1
DNA fraction following fixation and PI staining as for Fig. 4. (C) U937
cells stably transfected with either empty vector (SFFV) or the
IB SRD mutant (32/36) were treated with PMA (2 ng/ml) or TNF-
(10 ng/ml) for 6 h, and the total RNA was harvested.
p21WAF1/Cip1 mRNA levels was determined by Northern
analysis, as were the GADPH levels as a loading control.
p21WAF1/Cip1 mRNA levels were enhanced only following PMA
treatment despite the activation of NF-B with both TNF- and PMA
treatments (data not shown).
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FIG. 7.
Ablation of p21WAF1/Cip1 expression results
in failure to survive the differentiation program, even in the presence
of NF-B activation. U937 cells stably transfected with either empty
vector (pREP4) or an antisense p21WAF1/Cip1 construct (F4)
were treated with 2 ng of PMA per ml to initiate the differentiation
program. pREP4 or F4 cells were collected at the indicated time points
post-PMA treatment, and nuclear and cytosolic fractions were collected
as for Fig. 6. F4 cells demonstrate a marked reduction in
p21WAF1/Cip1 expression. -Actin detection was included
as a control for loading. EMSA analysis reveals roughly equivalent
levels of nuclear NF-B dimers. RelA-p50 and p50-p50 complexes are
indicated by filled circles and arrows, respectively. Viable cells were
detected by PI staining as for Fig. 5 and are expressed as the
percent survival over time. A representative experiment is shown.
Similar results were obtained with a second p21(AS) clone.
|
|
Inhibition of NF-B reduces p21WAF1/Cip1 expression
in primary macrophages.
To investigate whether the
NF-B-dependent upregulation of p21WAF1/Cip1 in monocytic
cells undergoing macrophage differentiation is also present in primary
human cells, we optimized an adenovirus transduction model through
which NF-B could be inhibited by genetic approaches during the
process of differentiation form monocytes to macrophages. Highly
purified primary CD14+ peripheral human blood monocytes
were isolated by negative selection and separated into two aliquots,
one of which was lysed. The remaining cells were allowed to
differentiate in tissue culture to macrophages for 5 days. At this time
point adherent macrophages were transduced with adenovirus expressing
either alkaline phosphatase (control) or HA-IB 32/36A. After
48 h the cells were harvested, lysed, and analyzed by SDS-PAGE in
parallel with lysates obtained upon initial monocyte isolation for
p21WAF1/Cip1 levels. As shown in Fig.
8, a small amount of
p21WAF1/Cip1 is present in freshly isolated monocytes
compared to that observed following their differentiation to
macrophages. More importantly, transduction and expression of
HA-IB 32/36A but not of alkaline phosphatase reduces the level of
p21WAF1/Cip1 in differentiated macrophages, once more
supporting the regulatory role of NF-B on p21WAF1/Cip1
during macrophage differentiation.
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FIG. 8.
NF-B activation is necessary for
p21WAF1/Cip1 expression in differentiated human monocytes.
CD14+ cells were isolated from the buffy coat by negative
depletion. FACS analysis indicated 84% enrichment. Cells were either
lysed immediately after isolation or differentiated by plastic
adherence. At day 5 postadherence, cells were transduced with
adenovirus (Adv) expressing either HA-IB 32/36 (32/36) or
alkaline phosphatase (AP). Cells were lysed 48 h posttransduction
and analyzed for p21WAF1/Cip1 by immunoblotting. IB
levels are included to show the relative expression of endogenous
versus transduced protein. -Actin was included as a loading
control.
|
|
| |
DISCUSSION |
The present study provides a number of novel observations
pertaining to the regulation and functional role of NF-B in cell differentiation. First, we have documented that the persistent activation of NF-B that ensues following the initiation of monocyte differentiation is dependent on the continuous activation of the IKK
complex and hence The phosphorylation and/or degradation of IB
molecules. Second, we have demonstrated the necessary role of NF-B
in allowing the cell to survive the differentiation process. Finally,
we have shown that p21WAF1/Cip1 is indeed regulated by
IKK-dependent NF-B activation of p21WAF1/Cip1 in
differentiating monocytes and may be the effector protein required to
protect the cell from differentiation-induced cell death.
Several lines of evidence support the hypothesis that persistent
NF-B activation in a defined cell in the absence of punctual stimuli
is in part due to a continued degradation of IB molecules (35,
55). B cells at different stages of differentiation exhibit a
structured sequence of NF-B activation as they progress through the
various steps of B-cell differentiation, ultimately leading to mature B
cells and plasma cells which contain significant levels of NF-B in
the nucleus in a constitutive manner (3, 22). Our results
suggest a key role for the IKK complex in mediating this continuous
level of NF-B translocation. Therefore, the IKK complex can be in a
continuous state of activation, separate from the well-characterized
punctual inducibility and rapid regression of activity seen with
activators such as TNF or IL-1. In addition to the process of cell
differentiation, pathological conditions such as cell transformation
are also associated with a continuous and persistent level of NF-B
in the nucleus (12, 19, 36, 41). Whether the IKK complex
is persistently activated in cells undergoing transformation such as
shown in this study is unknown and deserves to be addressed in future
studies since the persistent levels of NF-B in the nuclei of cancer
cells impact the response to chemotherapeutic and ionizing agents.
It should be noted that it is unlikely in this model that PMA itself
acts directly upon signal transduction pathways converging on the IKK
complex. First, in contrast to the case of TNF, IKK activity is not
upregulated immediately following the treatment of monocytic cells with
relatively low doses (2 ng/ml) of PMA. Second, IKK activation, as well
as differentiation, is seen within 24 h and peaks at 3 to 5 days
following a single treatment dose of PMA, time points in which it is
unlikely that significant levels of PMA remain in the culture media.
Lastly, cells extensively washed after the initial PMA treatment and
recultured in PMA free media proceed with the differentiation program
(data not shown). The identity of the PMA-dependent stimulus or stimuli
which drives chronic IKK activation allowing macrophage differentiation
is unknown. However, it is possible that different cytokines produced as a result of PMA treatment may act in an autocrine fashion on the
cells to reinforce and perpetuate the differentiation program. This
possibility is supported by the observation that monocyte differentiation induces TNF production which, in turn, drives apoptosis
in the absence of NF-B. However, it should be noted that a single
dose of TNF in the absence of PMA minimally upregulates p21WAF1/Cip1 expression (Fig. 6). Therefore, it is likely
that additional differentiation-inducing signals are initiated by PMA
independent of TNF production. Although not exclusive of autocrine
factors, it is also possible that internal perturbations initiated by
PMA treatment may increase IKK activity. For example, treatment of U937
cells with okadaic acid, an inhibitor of phosphatases 1 and 2A, induces
differentiation, cell cycle arrest, and eventual cell death (28,
42, 50). It has previously been suggested that phosphatase 2A
may play a role in inhibiting basal IKK activity (13).
The necessary role of NF-B in macrophage differentiation is relevant
and justifies at a functional level the long-term observations that
NF-B is activated during the differentiation of macrophages and that
it is present at very high levels in the nuclei of fully differentiated
macrophages (24). While we cannot exclude that NF-B is
also influencing specific steps of the cell differentiation process,
such as promoting exit from the cell cycle, its role in protecting the
cell from undergoing death is clearly highlighted. Overexpression of
certain transcription factors alone can be sufficient to drive cell
differentiation in a number of cell models (21, 26, 32,
33). During the differentiation of promonocytic cells, NF-B
is potently and persistently activated. Whether this activation is
driving differentiation or merely a consequence thereof was unknown.
Our data suggest that NF-B may not be driving differentiation but
rather is necessary for surviving the differentiation program. Abundant
information supports the role of NF-B as an antiapoptotic molecule
following cell stimulation with apoptosis-inducing ligands such as TNF
or chemotherapeutic and ionizing agents (6, 10, 54, 58,
59). However, to our knowledge this study provides the first
evidence that the cell differentiation process triggered by chemical
agents such as phorbol esters is a significant cell injury that leads
to cell death unless NF-B is concomitantly activated. It remains to
be seen whether constitutive, forced activation of NF-B in
promonocytes, in the absence of PMA, alone is sufficient to either
force differentiation or confer resistance to subsequent apoptotic
stimuli. These experiments are currently under way.
It is unclear whether NF-B knockouts have an impairment in
macrophage differentiation since RelA animals are nonviable. Further, double knockouts of the p50 and p52 NF-B components, which
theoretically would render RelA inactive, have other deficiencies which
could impose upon a dificiency in macrophage development
(27). Our data, however, suggest that NF-B activity is
important for macrophage development and raise the question as to
whether NF-B activity is involved in differentiation of other
specific cell lineages. Knockout experiments, although a valuable tool,
may be unable to completely address this issue (26, 33).
In vivo, it is possible that other members of the Rel family may
compensate for the differentiation of specific cell lineages. Overlying
mortalities and morbidities such as seen with the nonviable RelA
knockout could make observations difficult and confusing. Finally,
NF-B activity may be important for discrete steps in
cell-lineage-specific development. For example, PU.1 knockout mice lack
both mature macrophages and granulocytes since PU.1 expression is
necessary for myeloid lineage commitment of pluripotent progenitors
(47). However, overexpression of PU.1 in cells cannot
drive further differentiation of myeloblasts to monocytes (43,
48), suggesting that PU.1 has a distinct role at a specific
stage of the differentiation program of pluripotent progenitors.
Knockout experiments may therefore mask the specific cell type and cell
stage requirements of a gene because of underlying pathologies.
A most intriguing observation is the fact that
p21WAF1/Cip1, generally considered a protein
restricted to the nuclear compartment in which it exerts its inhibition
of CDK and thus regulates cell cycle progression, is in the unique case
of macrophages, predominantly located in the cytosol, where it exerts
antiapoptotic functions (1). Based on our results
demonstrating that NF-B is required for cell survival, the link
confirmed in this study between NF-B and p21WAF1/Cip1
during the process of macrophage differentiation is noteworthy for several reasons. First and foremost, our results support that, in
cells of monocytic lineage, including primary macrophages, p21WAF1/Cip1 is an NF-B-dependent gene and
thus a potential effector of the antiapoptotic effects of NF-B. The
mechanism whereby NF-B ultimately leads to
p21WAF1/Cip1 upregulation is unknown. NF-B may
directly drive the transcription of p21WAF1/Cip1,
as suggested by Fig. 6, or have other effects on
p21WAF1/Cip1 expression, such as on protein or
mRNA stability. Experiments are currently under way to explore the link
of NF-B to p21WAF1/Cip1. Likewise, it will be
of interest to investigate whether cells with constitutive levels of
NF-B in the nuclei, such as mature B lymphocytes or transformed
cancer cells, also demonstrate upregulation of
p21WAF1/Cip1. If this were the case, it
could be postulated that under certain conditions, beyond those of
macrophage differentiation, p21WAF1/Cip1 may
exert important apoptotic functions relevant to, e.g., oncogenic processes.
The observation that endogenously produced TNF triggers the apoptosis
seen during monocyte differentiation (Fig. 5), coupled with the need
for p21WAF1/Cip1 expression for survival,
suggests that NF-B-dependent p21 expression directly opposes
TNF-induced apoptosis. This observation agrees with a number of
previous reports demonstrating that p21WAF1/Cip1
opposes TNF-induced apoptosis (15, 29, 30). How this
mechanism occurs is unknown and warrants further investigation. It
should be noted that PMA fails to induce apoptosis in fully
differentiated primary human macrophages even when NF-B is inhibited
(data not shown). This implies that the sensitivity to TNF-induced
apoptosis is unique to cells actively undergoing differentiation and is not seen in cells that have completed the maturational process. Alternatively, fully differentiated macrophages may have additional antiapoptotic mechanisms such as the upregulation of FLIP
(46). Previous studies have suggested that TNF plays an
active role in the differentiation of a number of hematopoietic cells.
Whether this is true also in monocytes remains unclear. One possibility is that it is the careful balance of TNF-induced apoptosis versus direct differentiation signals which mediate the final maturational outcome.
In summary, we have shown that constitutive activation of NF-B is
required for surviving the monocyte differentiation program and is
dependent on the chronic activation of IKK targeting the IB SRD.
Based on our data with cells stably expressing transdominant IB,
we propose that the maturation of monocytes involves two distinct
signals. The first signal drives the differentiation of the cell and
may be independent of NF-B activity. The second signal, mediated by
TNF-, initiates a death signal in the differentiation monocyte. This
death signal is directly opposed by NF-B activity. Finally,
persistent NF-B activation induces the expression of p21WAF1/Cip1, which may be the critical protein which
protects the monocyte from differentiation-induced cell death.
We thank Steven Grant for the p21(AS) U937 cell lines, Christian
Rust and Robert Simari for the HA-IB 32/36 and AP adenoviruses, Susana Asin for generation of the IB U937 cell lines, Hiroko Miyoshi for RNA preparation, David Lynch (Immunex Corporation) for the
TNFR-Fc and IL4R-Fc reagents, and Teresa Hoff for assistance in
preparing the manuscript. In addition, we thank members of C. V. Paya's laboratory for many thoughtful and stimulating discussions.
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B Kinase-Dependent Chronic Activation of NF-
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Abstract
Introduction
