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-Mediated, Caspase-Dependent, Apoptotic Death Pathway in a Human Myelomonocyte Cell Line1
National Centre for Biological Sciences, Bangalore, Karnataka, India
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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transduces signals of survival or death via its two
receptors, R1/p55/p60 and RII/p80/p75. The role of caspases as
effectors of cell death is universally accepted, although caspase
inhibitors may potentiate TNF cytotoxicity in some instances. In
conditions when macromolecular synthesis is blocked, caspases are part
of the machinery that executes TNF-triggered apoptotic death in U937, a
human myelomonocyte cell line, and in the Jurkat T cell line. However,
inhibition of p38 mitogen-activated protein kinase (p38 MAPK) triggered
TNF cytotoxicity in U937 cells and murine splenic macrophages, but not
the Jurkat cell line. TNF induced expression of the antiapoptotic
protein c-IAP2 (cytoplasmic inhibitor of apoptosis protein 2), and was
blocked in the presence of a p38 MAPK inhibitor, which also induced
caspase-dependent, TNF-mediated apoptosis in U937 cells. Thus,
inhibition of p38 MAPK resulted in the activation of caspase 9 and
cleavage of the adaptor molecule BH3 interacting domain death
agonist, and blocked NF-
B-mediated transactivation, without
affecting the nuclear translocation of NF-B. Collectively, these
data show that activation of p38 MAPK is critical to cell survival by
TNF in U937 cells, and demonstrate lineage-specific regulation of
TNF-triggered signals of activation or
apoptosis. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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is a multifunctional cytokine that transduces
signals of survival, differentiation, and death via its receptors
TNFRI/p55/p60 and TNFRII/p75/p80 in diverse cell types
(1, 2, 3). TNF elicits diverse biological consequences by
inducing the expression of various genes, and in many systems the
expression of these is regulated by the transcription factor, NF-B
(4, 5). NF-B can regulate the expression of genes
involved in inflammation, cell cycle regulation, immune responses via
the expression of cytokines, and antiapoptotic molecules that protect
cells from the deleterious effects of TNF (5, 6). The
p50/p65 NF-B heterodimer normally exists in the cytosol, with the
DNA-binding dimer held as an inactive complex with the inhibitor
subunit I-B3
(inhibitory protein that dissociates from NF-B) (7).
Nuclear translocation and activation of NF-B occur on the
phosphorylation and subsequent degradation of I-B (8),
which releases NF-B to bind B response elements and initiate
transcription. Phosphorylation of I-B is achieved by sequential
steps involving multisubunit kinase complexes that belong to the
mitogen-activated protein kinase (MAPK) kinase family of protein
kinases (9).
TNF also activates the p38 MAPK pathway (3, 10, 11).
MAPK family members are required for signal transduction in response to
many stimuli including cytokines, and p38 MAPK have been implicated in
the transcription-activating potential of NF-B
(12, 13, 14). Transcription factors such as ATF-2 (activating
transcription factor-2), Elk-1, etc., are also activated via p38 MAPK,
which contributes to TNF-triggered gene expression (11, 15), and existing data support a role for p38 MAPK activation in
both death and survival pathways (3, 16, 17, 18, 19).
In situations in which TNF induces death, the cytoplasmic domains of
TNFR, primarily TNFRI, recruit the adapter proteins, TNFR-associated
death domain, and Fas-associated death domain, which in turn activate
the initiator caspase, Fas-associated death domain-like
IL-1
-converting enzyme/procaspase 8. Caspase 8 triggers apoptotic
damage by activating multiple effector caspases, each of which target a
limited set of cellular proteins (2). Similarly,
procaspase 9 also functions as an initiator/regulator caspase. The
N-terminal prodomain of this caspase interacts with Apaf-1 in the
presence of cytochrome c and dATP, resulting in the cleavage
and activation of caspase 9, which activates caspases 3, 6, and 7.
Thus, there are at least two major pathways, initiated by caspases 8
and 9, which can activate caspase cascades in cells. Effector caspases
are required for the execution phase of apoptosis and are responsible
for many of the morphological changes and cellular damage that occur
during cell death. The essential role of these proteases has been
demonstrated in diverse systems (20). TNF-mediated
apoptosis is also dependent on caspase activation (1, 2, 21), but paradoxically, a number of studies have shown that
inhibition of caspase activity can lead to necrotic death in many cell
types (22, 23, 24, 25).
In this study, we identify a novel role for p38 MAPK in the TNF-triggered antiapoptotic response in U937 cells. We find that p38 MAPK is necessary to TNF-triggered antiapoptotic gene expression, and inhibition of p38 MAPK triggers a TNF-mediated, caspase-dependent apoptotic death pathway in U937 cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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U937, a human myelomonocytic cell line, and Jurkat, a T
lymphoblastoid cell line of human origin, were used in all experiments.
Murine splenic macrophages were isolated by adherence on plastic petri
dishes. In each experiment, unfractionated cells (20 x
106 cells/ml) from three individual spleens were
incubated for 45 min in complete medium in tissue culture dishes. At
the end of incubation, floating cells were removed by gentle swirling
and decantation of dish contents. Plates were washed twice with
complete medium using a similar procedure. After the final wash, 2 ml
of cold medium was added to dishes, and adherent cells were removed
using a cell scraper and used in functional assays. rTNF and Abs to
TNFR1 and TNFRII were obtained from R&D Systems (Minneapolis, MN).
Cycloheximide (CHX), Hoechst 33342, and propidium iodide were obtained
from Sigma (St. Louis, MO). The p38 MAPK inhibitor PD 169316 was
purchased from Calbiochem (San Diego, CA). The peptide inhibitors
Z-Val-Ala-Asp(O-methyl)-fluoromethyl ketone (ZVAD-FMK),
Boc-Asp(O-methyl)-FMK (BD-FMK), Ile-Glu-Thr-Asp-FMK
(IETD-FMK), Val-Glu-Ile-Asp-FMK (VEID-FMK), Leu-Glu-His-Asp-FMK
(LEHD-FMK), and Z-Phe-Ala-FMK were obtained from Enzyme Systems
Products (Dublin, CA), dissolved as 20 mM stock solutions in DMSO, and
stored at -70°C. The
5,5',6,6'-tetrachloro-1,1',3,3'-tetraethyl-benzimidazoleyl-carbocyanine
iodide (JC-1) and 3,3'-diethyloxacarbocyanine
(DiOC6) were obtained from Molecular Probes
(Eugene, OR), made up as stock solutions in DMSO, and stored at
-70°C. Abs to cleaved caspase 9, phosphoisoforms of p38, and ATF-2
were from New England Biolabs (Beverly, MA); cytoplasmic inhibitor of
apoptosis protein 1 (c-IAP1), c-IAP2, TNFR-associated factor-2
(TRAF-2), BclxL, p38 MAPK, Fas, and NF-B were
from Santa Cruz Biotechnology (Santa Cruz, CA); and biotin- or
avidin-conjugated Abs were obtained from Jackson ImmunoResearch (West
Grove, PA). Ab to BH3 interacting death domain agonist (BID) was
obtained from R&D Systems.
Apoptotic nuclear damage and cell lysis
Cell lysis was quantitated by flow cytometry using propidium iodide. Apoptotic nuclear morphology was assessed using Hoechst 33342, as described previously (26). All assays of nuclear morphology were scored double blind. For imaging nuclei, cells were fixed with 0.1% paraformaldehyde for 10 min at room temperature, then washed and stained with 20 nM of the dye SYTOX Green (Molecular Probes) for 10 min at ambient temperature. At the end of incubation, the cells were washed and resuspended in PBS (106/ml) for analysis of nuclear morphology.
Mitochondrial transmembrane potential
For DiOC6 staining, cell pellets were incubated for 20 min in the dark at 37°C in 0.5 ml staining solution (PBS containing 40 nm DiOC6), as described previously (26). Cells were washed in excess PBS and resuspended in 0.5 ml PBS containing propidium iodide to identify dead cells, and the propidium iodide-negative subset was analyzed flow cytometrically. For staining with JC-1, cell pellets were resuspended in staining solution (200 µl PBS containing 5 µg/ml JC-1) and incubated at 37°C for 10 min. At the end of incubation, cells were washed in excess PBS and resuspended in 0.3 ml PBS and analyzed flow cytometrically, according to the manufacturer’s instructions. All flow cytometric analysis was performed on a Becton Dickinson FACS (Becton Dickinson, Mountain View, CA).
Immunofluorescence analysis by flow cytometry
Cells fixed with 1% paraformaldehyde at room temperature for 20 min were washed twice in chilled permeabilization buffer (PBS containing 0.3% saponin, 0.1% sodium azide, and 1% FCS). A total of 20 µl of relevant Ab (diluted in permeabilization buffer) was added directly to the cell pellets, and the cells were incubated on ice for 30 min. Primary Ab was followed by secondary biotin-conjugated Ab, and all samples were finally stained with PE-avidin. Each step of Ab staining was followed by two washes in permeabilization buffer to remove excess Ab. At the end of the incubation with PE-avidin, cells were washed once with permeabilization buffer, followed by a wash in FACS buffer (PBS containing 1% FCS and 0.1% sodium azide), and resuspended in 0.4 ml FACS buffer for flow cytometric analysis.
Immunofluorescence analysis by microscopy
Cells were fixed in 70% ethanol for 2 h, washed twice in saponin buffer, and stained as described for flow cytometric analysis, except that biotin-conjugated Ab was followed by avidin conjugated to Alexa-546 (Molecular Probes). After the last wash, cells were resuspended in a minimal volume of PBS, mounted on slides, and photographed using a Pro-Series 3, CCD camera (Media Cybernetics).
Western blot analysis
The 106 cells treated under various conditions were harvested, washed twice by centrifugation in chilled PBS, lysed by the addition of SDS sample buffer (62.5 mM Tris-HCl, 2% w/v SDS, 10% glycerol, 50 mM DTT, and 0.1% bromphenol blue), vortexed to reduce sample viscosity, denatured by boiling, and then cooled on ice. Samples were microcentrifuged for 5 min before loading on 10% SDS-PAGE gels. Proteins were transferred onto Hybond-ECL nitrocellulose membrane (Amersham, Little Chalfont U.K.). Membranes were blocked for 1 h in blocking buffer (5% Blotto, 0.1% Tween 20 in PBS) and incubated overnight with primary Ab in blocking buffer at 4°C, with gentle rocking. Membranes were washed three times for 5 min each with 10 ml of PBS containing 0.1% Tween-20, and then incubated for 1 h at 25°C with goat anti-rabbit HRP (Pierce, Rockford, IL), diluted 1/5000 in blocking buffer. After another three washes, proteins were detected by chemiluminescence, according to the manufacturer’s instructions (Pierce).
NF-B reporter assay
A total of 6–10 x 106 U937 cells
was transfected with 5 µg of thymidine kinase-6 NF-B luciferase by
electroporation at 250 mV and 960 µF capacitance. Twenty-four hours
after transfection, live cells were harvested by Ficoll density
centrifugation, and cells were treated with TNF (10 ng/ml), or TNF +
PD, or continued in medium alone. Six to 8 h after initiation of
treatment, cell lysates were assayed for luciferase activity using a
substrate provided by Promega (Madison, WI), according to the
manufacturer’s instructions, and read using a luminometer from Turner
Design (Palo Alto, CA). Promoter activity is expressed as fold
induction over readings obtained from mock-transfected cells cultured
in medium alone.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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U937 cells were cultured with 5 ng/ml TNF in the presence or
absence of a broad spectrum caspase inhibitor peptide, ZVAD-FMK or CHX.
Cells were scored for lysis using propidium iodide and apoptotic
nuclear morphology using Hoechst 33342 after 18 h in culture. As
seen in Fig. 1
A, U937 cultured
with TNF alone showed minimal levels of apoptosis (<10% apoptotic
nuclei at 18 h), but the presence of 200 ng/ml CHX resulted in a
sharp increase in apoptotic nuclear damage in these cells (Fig. 1, A and B). As previously reported
(25), the caspase inhibitor ZVAD-FMK also potentiated
TNF-induced death (Fig. 1A), but a large proportion of the
dead (propidium iodide-positive) cells in the TNF + ZVAD preparations
had a distorted, condensed morphology (Fig. 1, A and
C), and did not show distinct nuclear fragmentation.
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A). Under these
conditions, pretreatment with BD-FMK, a broad spectrum caspase
inhibitor; VEID-FMK, an inhibitor of caspase 6; or IETD-FMK, an
inhibitor of caspase 8, blocked TNF + CHX-induced apoptotic nuclear
damage in a dose-dependent manner (Fig. 2A). In the same
experiment, BD-FMK and VEID-FMK only partially inhibited the loss of
mitochondrial transmembrane potential (Fig. 2B), while
IETD-FMK had no effect, even at concentrations that block nuclear
damage. Concentrations of IETD-FMK higher than 50 µM have not been
tested, as the resultant toxicity masked the protective effects, if
any, of this inhibitor. In contrast, LEHD-FMK, an inhibitor of caspase
9, did not block either nuclear or mitochondrial damage in these cells
(Fig. 2, A and B). The partial inhibition of
mitochondrial damage by the caspase inhibitors (Fig. 2B)
suggests a role for other proapoptotic molecules such as reactive
oxygen species in this pathway (22, 25). ZVAD-FMK
expectedly did not block either nuclear (Fig. 2A) or
mitochondrial damage (Fig. 2B) triggered by TNF + CHX. The
peptides used as caspase inhibitors are based on optimal peptide
recognition motifs that have been described for the different caspases
(27). These have been used to design caspase-specific
substrates as well (28). Although both BD-FMK and ZVAD-FMK
are broad spectrum inhibitors of caspases, differences in their ability
to inhibit death have been observed in other systems as well
(29). A recent study has suggested that ZVAD-FMK acts at a
very early step in apoptotic cascades to block caspases or a hitherto
undefined, but related activity in cells (28).
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C), although ZVAD-FMK expectedly was most potent in
inhibiting apoptotic death at concentrations as low as 5 µM (data not
shown) in these cells. To address the possibility of nonspecific
effects of the peptides, we also tested an inhibitor of cathepsin,
Z-Phe-Ala-FMK, which did not block TNF + CHX-mediated death in these
cells (data not shown). TNF + CHX triggers the activation of caspase 9 in Jurkat cells
The differential induction of caspase 9 in Jurkat cells, suggested
in the previous experiments, was confirmed by assaying for the
expression of cleaved caspase 9 in intact, treated cells. Furthermore,
to aid comparison and normalize the kinetics of the apoptotic response
in the two cells, we treated Jurkat cells with TNF in the presence of a
high dose of CHX (5 µg/ml) so as to induce TNF + CHX-mediated
apoptosis with kinetics similar to U937 cells. Using a polyclonal Ab
that specifically recognized the cleaved form of the endogenous
protease, Jurkat cells treated with the higher dose of CHX and TNF were
stained after 2 or 4 h for expression of caspase 9. A small shift
indicative of cleaved caspase 9 was detected at 2 h (data not
shown); however, by 4 h all the cells stained positive with this
Ab (filled histogram, Fig. 2D). Specific fluorescence was
not detected in control, unstimulated cells, or in cells treated with
Ab isotype controls (dotted and shaded line open histograms, Fig. 2D). Under these conditions, TNF + CHX induced 60.5%
apoptotic nuclear damage by 6 h, which was reduced to 20.6% in
LEHD-FMK-treated cells. As expected from the functional assays, cleaved
caspase 9 could not be detected in U937 cells (Fig. 2E) at
either early or later time points in the death pathway, although U937
treated with TNF + CHX (0.5 µg/ml) showed a measurable increase in
nuclear damage by 2 h. Thus, TNF + CHX induced 73% apoptotic
damage compared with 2.3% apoptotic nuclei scored in control,
untreated cells 6 h after initiation of the assay. These data
indicate a role for caspase 9 in TNF-mediated apoptosis in Jurkat
cells, but not U937 cells, in conditions when TNF cytotoxicity is
induced by CHX.
Although CHX has been used routinely in many studies to analyze
TNF-mediated cytotoxicity, it can affect multiple events in cells.
Notwithstanding, the experiments with CHX in this study suggest the
contextual regulation of TNF-initiated signaling events in the two cell
types under study. In U937 cells, TNF induced necrosis in the presence
of a caspase inhibitor (Fig. 1 and 25), and activates
proapoptotic caspases in the presence of CHX (Fig. 1, B and
D, and Fig. 2). Thus, it was of interest to try and identify
the nature of the input signals that can regulate caspase activation in
TNF-treated U937 cells
Inhibition of p38 MAPK potentiates TNF cytotoxicity in U937 cells
TNF signaling leads to the activation of kinase signaling
cascades, and these can regulate caspase activation in various cell
death pathways. To further characterize the regulation of TNF-mediated
signaling in U937 cells, we tested for the effect of an inhibitor of
p38 MAPK, PD169316 (30, 31, 32). As shown in Fig. 3A, TNF, in the presence of
PD169316 (TNF + PD), induced apoptotic nuclear damage at concentrations
of PD that were nontoxic (Fig. 3A, open squares) to the
cells. Agonist Ab to TNFR1 (Fig. 3B) triggered apoptotic
death in the presence of 20 µM PD in a dose-dependent manner, but
there was no induction of cytotoxicity by an Ab to TNFRII (Fig. 3B). These results are comparable with the effects of TNFR1
and TNFRII Abs in the presence of CHX (Fig. 3C) in these
cells. PD169316 did not potentiate TNF-mediated apoptotic death in
Jurkat cells (Fig. 3D), suggesting a specific role for p38
MAPK in TNF-mediated survival in U937 cells.
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F shows an experiment that
demonstrates the induction of phosphorylated p38 MAPK in cells treated
with 10 ng/ml TNF for 30 min (lane 2) as compared
with untreated cells (lane 1). This experiment shows
that pretreatment of cells with PD169316 before the addition of TNF
blocks the phosphorylation of p38 MAPK (lane 3).
Lane 4 served as a positive control, and shows the induction
of phosphorylated p38 MAPK within 30 min in U937 cells treated with
hydrogen peroxide (19). Interestingly, in keeping with the
differences in TNF-triggered signaling observed in U937 and Jurkat
cells, a role for p38 MAPK as a possible effector of Fas-mediated death
has been recently suggested in Jurkat cells (33). In
subsequent experiments, we have further characterized the TNF +
PD-mediated death pathway in U937 cells. Inhibition of p38 MAPK triggers a caspase-dependent death pathway in U937 cells
Because PD169316 triggered TNF-mediated apoptosis, we tested the
effect of caspase inhibitors on TNF + PD-mediated apoptosis in the U937
cell line. In the experiment shown in Fig. 3E, 25 µM of
the caspase inhibitors BD-FMK, VEID-FMK, or IETD-FMK blocked TNF +
PD-mediated apoptosis, whereas ZVAD-FMK was unable to block apoptotic
nuclear damage (Fig. 3E). Unexpectedly, LEHD-FMK, the
inhibitor of caspase 9, also blocked nuclear damage in TNF + PD-treated
cells.
These experiments suggested a role for p38 MAPK in the modulation of
caspase 9 activation. Alternatively, it is also possible that caspase 9
activation or activity is directly inhibited by CHX, which may explain
the lack of effect of the peptide LEHD-FMK on TNF + CHX-mediated death
(Fig. 2). Therefore, we tested for inhibition of caspase 9 by CHX. As
shown in Fig. 4A, TNF +
PD-induced expression of activated caspase 9 (Fig. 4B, solid
line) was not blocked by 0.5 µg/ml CHX (filled histogram in Fig. 4, A or B) or 50 ng/ml CHX (data not shown). Caspase
9 expression in control, untreated cells is indicated by the solid line
histogram in Fig. 4A. In functional assays for caspase 9
activity, we tested whether 0.5 µg/ml (CHX) or lower concentrations
of CHX (50 ng/ml, CHXlo) would block the inhibitory effect of LEHD-FMK
on TNF + PD-mediated death. As shown in Fig. 4C, PD and CHX
have additive effects on TNF-mediated death. Apoptotic death cells
treated with LEHD-FMK + TNF + PD + CHX were comparable with that of
LEHD-FMK + TNF + CHX for each concentration of CHX. These data are
consistent with the possibility that LEHD-FMK blocks the TNF +
PD-triggered, caspase 9 component of the death pathway and does not
affect TNF + CHX-mediated cell death. Additionally, these experiments
suggest that caspase 9 activation is not regulated directly by
CHX.
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D, there is a loss of expression or immunoreactivity of the
death-adaptor molecule BID, as early as 2 h following treatment
with TNF + PD (lanes 3 and 5) as compared
with cells treated with TNF (lane 2) or left
untreated (lane 1). These conditions lead to
induction of apoptotic damage by 6 h. Furthermore, cells treated
with IETD-FMK (lane 3) at concentrations that block
cell death show restoration of BID expression as compared with cells
treated with TNF + PD (lane 2). All blots were
reprobed for expression of Fas, which showed that changes in BID
immunoreactivity were not owing to alterations in cell number. Thus
caspase 8-mediated cleavage of BID can lead to the activation of
caspase 9 in TNF + PD-treated cells. As shown in Fig. 4E–G,
IETD-FMK partially blocks the activation of caspase 9 (G)
induced by TNF + PD (F), suggesting that caspase 9
activation may, in part, be dependent on caspase 8-mediated cleavage of
BID in U937 cells treated with TNF + PD. Inhibition of p38 MAPK blocks antiapoptotic gene expression
To assess whether the effect of p38 MAPK on TNF signaling was
limited to U937 cells alone, we also tested for the effect of PD on
TNF-induced cytotoxicity in freshly isolated murine splenic
macrophages. As shown in Fig. 5A, adherent macrophages
isolated from murine spleens when cultured overnight showed minimal
induction of apoptotic damage. Cells treated with TNF for 18 h
also showed minimal cytotoxicity as compared with control, untreated
cells. However, as was seen with U937 cells, PD169316 induced a
striking increase in TNF-mediated apoptotic nuclear damage (solid bars)
and cell lysis (hatched bars) as well.
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B
(6), and is therefore sensitive to inhibition by agents
such as CHX. U937 cells were cultured with TNF for 2 h in the
presence or absence of 20 µM PD169316, and analyzed for the
cytoplasmic levels of c-IAP1, c-IAP2, BclxL,
Bcl-2, and TRAF-2. U937 cells express very low levels of c-IAP1 and
c-IAP2 (data not shown). TNF-induced expression of c-IAP2 (Fig. 5C, solid line) was markedly reduced by PD169316 (Fig. 5C, filled histogram); expression of c-IAP1 (Fig. 5B, solid line) was partially blocked (Fig. 5B,
filled histogram), but the high levels of constitutively expressed
TRAF-2 (Fig. 5D, shaded histogram) were unchanged by this
inhibitor (Fig. 5D). U937 cells do not express Bcl-2 (data
not shown). BclxL is expressed in U937 cells at
very low levels (Fig. 5E, filled histogram); its expression
is not enhanced further by TNF (Fig. 5E, heavy gray line),
nor altered in TNF + PD-treated cells. The role of p38 MAPK in
TNF-mediated up-regulation of c-IAP2 in U937 cells appears similar to
its role in CD40-mediated signaling in dendritic cells
(34).
p38 MAPK does not affect the nuclear translocation of NF-B
Activation of the transcription factor NF-B is central to the
survival pathway triggered by TNF in many systems. p38 MAPK appeared to
function as a TNF-triggered survival signal in U937 cells, and because
the effect of p38 kinase on NF-B-induced gene expression has been
shown in other systems (13, 14), we tested whether p38
MAPK can also modulate NF-B activation. NF-B is normally trapped
in the cytoplasm by its inhibitor I-B, but the association is
regulated by phosphorylation of I-B by members of the MAPK kinase
family. We wished to test whether p38 MAPK blocked the nuclear
translocation of NF-B. Thus, cells treated with TNF show an early
nuclear translocation (within 20 min of addition of TNF) of NF-B,
which can be visualized by staining with an Ab that recognizes the p65
subunit of NF-B (Fig. 5, F–H). As shown in Fig. 5F, the fluorescent signal with NF-B Ab in control,
untreated cells is confined principally to the cytoplasmic region of
the cells. The nucleus can be seen as a central, sparsely stained
region that is largely devoid of fluorescence in these cells. Exposure
to TNF for 20 min results in the translocation of NF-B to the
nucleus, which can be visualized as the bright fluorescence in the
nucleus (Fig. 5G). The fluorescence is sustained for at
least 45 min after addition of TNF (data not shown). The translocation
of NF-B is not inhibited in TNF + PD-treated cells, as these cells
also show bright nuclear localized fluorescence (Fig. 5H), comparable with that seen in cells treated with TNF
alone.
To examine the effect of PD on TNF-induced NF-B-dependent gene
transcription, we transfected a NF-B reporter construct in U937
cells. As shown in Fig. 5I, U937 cells treated with TNF for
6–8 h in three experiments showed a 10- to 15-fold increase in NF-B
reporter activity relative to control, untreated cells. This induction
was markedly blocked in cells treated with TNF + PD, but complete
inhibition was not consistently observed in these assays. As shown in
the immunoblots in panel J, concentration of PD that
results in a reduction in levels of phospho-p38 kinase in TNF
+PD-treated cells also blocked induction of expression of the
downstream transcription factor phospho-ATF-2 in these cells. Hydrogen
peroxide (H2O2)-treated
cells were used as a positive control in these experiments, as
described for Fig. 3.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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p38 MAPK has been implicated as a mediator of apoptosis (16, 17), but recent reports have also described a protective role for this kinase in TNF-mediated signaling (3, 19). A recent report has indicated a protective role for p38 MAPK in response to oxidative stress in U937 (19), although the role of antiapoptotic proteins, if any, in this system was not discussed. TNF-mediated death is blocked by cytokines in T cells (36), and because p38 MAPK regulates expression of cytokines in different cells (10, 12, 13), an analysis of cytokines produced in response to TNF signaling in U937 cells may reveal additional antiapoptotic signals generated via this pathway. The up-regulation of antiapoptotic proteins, such as c-IAP2, is one way by which p38 MAPK contributes to TNF-mediated survival, and thereby the determination of cell fate.
We have attempted to address the possible interaction of TNF-triggered
p38 MAPK and NF-B in this system. In cardiac myocytes, p38 MAPK
regulates the expression of IL-6, via interactions between MKK6 (MAPK
kinase), which lies directly upstream of p38, and I-B kinase, which
results in NF-B activation (13) in TNF-treated cells.
This model of a direct impact of p38 MAPK on NF-B activation is not
supported by our data, which are more consistent with the effect of p38
MAPK on the TNF-triggered transcription-activating potential of
NF-B. TNF-induced nuclear translocation of NF-B (Fig. 5G) was not modulated by PD169316 (Fig. 5H), but
affected NF-B transactivation (Fig. 5I) and expression of
the antiapoptotic gene inhibitor of apoptosis protein-2 (Fig. 5C) in these cells. A similar requirement for the p38 MAPK
pathway in NF-B function, independent of its DNA binding, has been
reported in response to CD40 and TNF in other systems (14, 37, 38). The mechanism by which p38 MAPK modulates NF-B
transcription is not known. One possibility consistent with the
experimental results in this study and others is that p38 MAPK or a
downstream kinase phosphorylates a transcriptional coactivator such as
CBP/p300, which may modulate NF-B-driven gene activation
(39). We also find that p38 MAPK-mediated phosphorylation
of the transcription factor ATF-2 is inhibited by PD169316 (Fig. 5J). c-IAP2, which has been functionally implicated in the
inhibition of TNF-mediated death, is transcriptionally regulated by
NF-B (40). Transcription factors such as ATF-2 provide
an additional layer of regulation wherein p38 MAPK may modulate
NF-B-mediated transcription of genes in response to TNF.
An earlier study has shown that inhibition of p38 MAPK does not
potentiate TNF cytotoxicity in L929 cells (38). We
attribute a role in cell survival to p38 MAPK, because inhibition of
p38 MAPK triggered death in U937 and splenic adherent cells (Figs. 3 and 5). This study shows that inhibition of p38 MAPK resulted in
activation of caspase 9 and the loss of full-length BID. Additionally,
caspase 8-dependent cleavage of the adaptor molecule BID and partial
inhibition of caspase 9 activation by an inhibitor of caspase 8 suggest
that caspase 9 may, in part, be activated by a caspase 8-dependent
signaling event.
Procaspase 9 is recruited to an apoptosome complex that comprises several Apaf-1 molecules, the oligomerization of which is dependent on cytochrome c and dATP. We do not detect an early release of cytochrome c in these cells (data not shown). Heat-shock protein (Hsp) 27, a small molecular mass protein that is a pleiotropic inhibitor of cell death, is another negative regulator of caspase 9 activation that binds cytochrome c and inhibits formation of the apoptosome complex in cells (41). Hsp27 can also bind Akt kinase/protein kinase B, which also has the capability of phosphorylating and inactivating caspase 9 (42). Hsp27 is phosphorylated by p38 MAPK (43, 44), and it is possible that in the TNF-mediated survival pathway, activated p38 MAPK phosphorylates Hsp27, which inactivates the apoptosome and contributes to the inhibition of caspase 9 activation in U937 cells.
Based on biochemical and functional assays, this study provides three
lines of evidence that p38 MAPK is a component of the survival response
triggered by TNF in U937, a myelomonocyte cell line. Inhibition of p38
MAPK triggers TNF cytotoxicity in functional assays of death in two
experimental systems, the U937 cell line and splenic macrophages.
Inhibition of p38 MAPK inhibits NF-B-mediated gene activation in
reporter assays, although nuclear translocation of NF-B is not
blocked (Fig. 5). NF-B is a well-defined mediator of the
TNF-triggered survival response, and among other proteins regulates
expression of the antiapoptotic protein c-IAP2 (6, 39).
The TNF-induced up-regulation of c-IAP2 is also blocked by the
inhibitor of p38 MAPK (Fig. 5). Thus, the experiments show that p38
MAPK can regulate NF-B-mediated gene transcription and also the
expression of the antiapoptotic protein c-IAP2, although it remains to
be established whether these two events are related in the present
system.
Cells use several mechanisms to regulate the activity of the caspase family of proteases. Activation of caspase-9 may be regulated by both transcription-dependent (c-IAP-mediated) (2, 6), and transcription-independent (phosphorylation of regulatory proteins) (41, 42) events. Although p38 MAPK may principally act via a phosphorylation-dependent event, in this study we present evidence that this kinase can also intersect a key transcription-dependent pathway and regulate TNF-mediated death in cells. Thus, these experiments provide additional evidence of multiple protective mechanisms influenced by this kinase. Collectively, the data indicate that the p38 MAPK pathway is critical to the TNF-triggered antiapoptotic response and functions as a key cell fate determinant in U937 cells.
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Acknowledgments |
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B, the
thymidine kinase-6 NF-B luciferase reporter construct, and V.
Karthik for assistance with the reporter assays. We are grateful to Dr.
Gaiti Hasan and V. Karthik for their comments on the manuscript, and
Khursheed Wani for his assistance with preparation of the
manuscript. |
Footnotes |
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2 Address correspondence and reprint requests to Dr. Apurva Sarin, National Centre for Biological Sciences, University of Agricultural Sciences, Gandhi Krishi Vigyan Kendra Campus, New Bellary Road, Bangalore 560065, Karnataka, India. E-mail address: sarina{at}ncbs.res.in
3 Abbreviations used in this paper: I-B, inhibitory protein that dissociates from NF-B; ATF-2, activating transcription factor-2; BD-FMK, Boc-Asp(O-methyl)-FMK; BID, BH3 interacting domain death agonist; c-IAP, cytoplasmic inhibitor of apoptosis protein; CHX, cycloheximide; DiOC6, 3,3'-diethyloxacarbocyanine; Hsp27, heat-shock protein 27; IETD-FMK, Ile-Glu-Thr-Asp-FMK; JC-1, 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethyl-benzimidazoleyl-carbocyanine iodide; LEHD-FMK, Leu-Glu-His-Asp-FMK; MAPK, mitogen-activated protein kinase; TRAF-2, TNFR-associated factor-2; VEID-FMK, Val-Glu-Ile-Asp-FMK; ZVAD-FMK, Z-Val-Ala-Asp(O-methyl)-fluoromethyl ketone.
Received for publication October 13, 2000. Accepted for publication March 22, 2001.
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) or with 5 ng/ml TNF (
) and increasing concentrations of
PD169316, and in B, without (
) or with (
).
B–E, Expression of c-IAP1 (B) and c-IAP2
(C) in U937 cells treated with TNF (solid line, open
histogram) or TNF + PD (filled histogram), 2.5 h after addition of
5 ng/ml TNF. Cells stained with TRAF-2 (filled histogram) are shown in
D for both TNF and TNF + PD-treated cells. The isotype
controls for TNF or TNF + PD-treated cells are indicated by the fine or
dotted line histogram, respectively, in each figure. U937 cells were
stained for expression of BclxL (E) either
in untreated cells (filled histogram, med) or after treatment with 5
ng/ml TNF (gray thick line, TNF) for 3 h. The dotted line and dark
line represent isotype controls for untreated and TNF-treated cells,
respectively. F–H, U937 cells were incubated for 20 min
in the presence (G and H) or absence
(F) of 5 ng/ml TNF. Cells in H were
preincubated with 20 µM PD169316 for 30 min before the addition of
TNF. Cells were harvested, fixed, and stained with an Ab that
recognizes the p65 subunit of NF-