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B and AP-1 Activation That Promotes Cyclooxygenase-2 Expression1
Department of Medicine IV, Experimental Division, Faculty of Medicine, University of Erlangen-Nürnberg, Erlangen, Germany
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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B and AP-1 mediated.
NF-B activation was confirmed by p50/p65-heterodimer formation,
IB-
degradation, and stimulation of a NF-B luciferase reporter
construct. Furthermore, a NF-B decoy approach abrogated
cyclooxygenase-2 (Cox-2) expression as well as inducible protection.
The importance of AP-1 for superoxide-mediated Cox-2 expression and
cell protection was substantiated by using the extracellular
signal-regulated kinase-inhibitor PD98059 and the p38-inhibitor
SB203580, which blocked Cox-2 expression. In corroboration, Cox-2
expression was hindered by a dominant-negative c-jun
mutant (TAM67). Protection from apoptosis was verified in human
macrophages with the notion that superoxide promoted Cox-2 expression,
which in turn attenuated nitric oxide-evoked caspase activation. We
conclude that the sublethal generation of oxygen radicals reprograms
macrophages by NF-B and AP-1 activation. The resulting
hyporesponsiveness reveals an attenuated apoptotic program in
association with Cox-2 expression. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Free radicals were studied within the context of a direct, destructive
role in biology. Although this is true for high radical concentrations,
it appears that some reactive oxygen (ROS) or nitrogen species play
physiologically important cellular messenger roles. ROS are
increasingly recognized to control signal transduction via activation
of mitogen-activated protein kinases (MAPK) or being implicated in the
regulation of transcription factors such as NF-B or AP-1
(13). Activation of NF-B is an important component for
cytokine-induced Cox-2 expression (14, 15), and more
generally is assumed as a classical redox-sensitive, i.e.,
ROS-responsive target system (16, 17). NF-B activation
promotes the expression of various stress genes (18), some
of which may contribute to cell survival in close association with an
antiapoptotic role of NF-B-regulated genes (19). Among
the potential protein kinases identified in phosphorylating the NF-B
inhibitor IB, both ERK- and c-Jun N-terminal kinase/stress-activated
protein kinase (JNK/SAPK)-signaling pathways were identified (20, 21). Moreover, superoxide-mediated activation of MAPK may
promote phosporylation of other substrates such as c-Jun, ATF2, and
Elk-1, all of which are associated with a transcriptional activity of
AP-1 (22, 23). AP-1 is a protein dimer composed of the
protooncogene products Fos and Jun (24). Its activation is
achieved, among others, by growth factors, cytokines, UV irradiation,
superoxide, as well as the phorbol ester
12-O-tetradecanoylphorbol-13-acetate (TPA) (25, 26), and hence the name TRE (TPA-responsive element) is used for
its recognition site. A role of AP-1 in Cox-2 induction has been
established in v-src-transformed cells (27, 28).
Radicals, i.e., ROS, can no longer be regarded solely as damaging
species. Its appears important to characterize their contribution to
the control of gene transcription and protein expression. The striking
possibility to reprogram the apoptotic behavior of macrophages by using
sublethal concentrations of superoxide prompted us to explore the
molecular mechanisms in detail. We established NF-B and AP-1
activation by the O2--generator
DMNQ in close association with Cox-2 expression. For murine and human
macrophages, we envision how ROS circumvent cell death, i.e.,
apoptosis, supporting the notion that expression of Cox-2 attenuates
programmed cell death.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Diphenylamine and LPS (Escherichia coli serotype
0127:B8) were purchased from Sigma (Deisenhofen, Germany). The Cox-2 Ab
was bought from Transduction Laboratories (Lexington, KY). The p50- and
p65-supershift Abs as well as the IB- Ab were obtained from Santa
Cruz Biotechnology (Heidelberg, Germany). Murine rIFN-
was provided
by Boehringer Mannheim (Mannheim, Germany). RPMI 1640, cell culture
supplements, and FCS were ordered from Biochrom (Berlin, Germany). The
luciferase-assay kit was obtained from Promega (Mannheim, Germany), and
the ß-galactosidase (ß-gal) detection kit came from Tropix
(Mannheim, Germany). Oligonucleotides (+/- fluorescein labeled) were
provided by Eurogentec (Seraing, Belgium). DMNQ was kindly provided by
Dr. Nicotera, University of Konstanz (Konstanz, Germany). PD98059,
SB203580, and Ac-DEVD-AMC were from Biomol (Hamburg, Germany). All
other chemicals were of the highest grade of purity and commercially
available.
Cell culture
The mouse monocyte/macrophage cell line RAW 264.7 was maintained in RPMI 1640 supplemented with 100 U/ml penicillin, 100 µg/ml streptomycin, and 10% heat-inactivated FCS (complete RPMI). All experiments were performed using complete RPMI. GSNO and LPS were dissolved in water and added as indicated. PD98059 and SB203580 were dissolved in DMSO. Appropriate solvent controls were performed. Preliminary experiments were performed with variable doses of all agents, and results are shown for optimal nontoxic concentrations of each agent.
Cell survival
The number of alive RAW 264.7 macrophages, following treatment with different agents, was determined by the trypan blue dye exclusion assay.
Monocyte isolation and culture
For each experiment, cells were isolated from 50-ml buffy coats (Transfusionsmedizin, Erlangen, Germany). Blood was diluted 1/2 with PBS and layered on a Ficoll-Isopaque gradient (p = 1077 g ml-1). The interphase containing PBMC was obtained following centrifugation (800 x g, 20 min). Cells were recovered, washed twice in PBS, and were allowed to adhere on culture dishes (Primaria 3072; Becton Dickinson, Heidelberg, Germany) for 90 min at 37°C. Nonadherent cells were removed. The medium was exchanged to fresh RPMI 1640 containing 10% heat-inactivated human AB serum (Sigma) and antibiotics (100 U/ml penicillin, 100 µg/ml streptomycin). Monocytes (5 x 106) were cultured in a volume of 10 ml per plate. Medium was changed every 2 to 3 days. After 6 days of culture, monocytes acquired a macrophage-like phenotype (29) and were used for the experiments. Flow cytometry confirmed that the macrophage-like population was 90–95% pure (CD14+ vs CD14-).
RNA extraction and semiquantitative RT-PCR
RNA was extracted using RNAClean (AGS, Heidelberg, Germany) according to the distributor’s manual. Reverse-transcription reactions and PCR for murine and human Cox-2 and GAPDH were performed using the SuperScript RNase H- Reverse Transcriptase (Life Technologies, Karlsruhe, Germany) and recombinant Taq DNA polymerase (Life Technologies). The sequence of the primers was as follows: human Cox-2 574–878(574–878) (30), TA = 52°C: 5'>3'-TTC AAA TGA GAT TGT GGG AAA AT; 3'>5'-TTC TAT GAG TCC GTC TCT ACT AGA; and GAPDH (human 155–759) (31), TA = 60°C: 5'>3'-GAA GGC CAT GCC AGT GAG CTT CC; 3'>5'-CCA TCA ACG ACC CCT TCA TTG ACC.
The number of amplification cycles (25 for GAPDH; 30 for human and murine Cox-2) was necessary to achieve exponential amplification in which product formation is proportional to starting cDNA. Products were run on 1.5% agarose gels and visualized by ethidium bromide staining.
Nuclear protein extracts
Preparation of crude nuclear extracts was basically as described (32). Briefly, following cell activation for the times indicated, 4 x 106 RAW 264.7 macrophages were washed in 1 ml of ice-cold PBS, centrifuged at 1,000 x g for 5 min, resuspended in 400 µl ice-cold hypotonic buffer (10 mM HEPES/KOH, 2 mM MgCl2, 0.1 mM EDTA, 10 mM KCl, 1 mM DTT, 0.5 mM PMSF, pH 7.9), left on ice for 10 min, vortex mixed, and centrifuged at 15,000 x g for 30 s. Pelleted nuclei were gently resuspended in 50 µl ice-cold saline buffer (50 mM HEPES/KOH, 50 mM KCl, 300 mM NaCl, 0.1 mM EDTA, 10% glycerol, 1 mM DTT, 0.5 mM PMSF, pH 7.9), left on ice for 20 min, vortex mixed, and centrifuged at 15,000 x g for 5 min at 4°C. Aliquots of the supernatant that contain nuclear proteins were frozen in liquid nitrogen and stored at -70°C. Protein was determined using a Bio-Rad II Kit (Richmond, CA).
Fluorogenic caspase-3-like activity determination
Cells (2 x 106) were incubated as indicated, recovered from cultured plates, and centrifuged (1,200 x g, 4°C, 5 min). Cell pellets were resuspended in lysis buffer (100 mM HEPES, pH 7.5, 10% sucrose, 0.1% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), 1 mM PMSF, 10 µg/ml pepstatin, 10 µg/ml leupeptin, and 1 mM EDTA) and left on ice for 30 min. Following sonification (Branson sonifier, 10 s, duty cycle 100%, output control 1) and centrifugation (10,000 x g, 10 min, 4°C), protein was determined with the DC Protein Assay. Cell supernatants (30 µg protein) were incubated in 100 mM HEPES, pH 7.5, 10% sucrose, 0.1% CHAPS, 1 mM EDTA, 1 mM PMSF, and 10 µg/ml leupeptin at 30°C with 12 µM of the caspase-3-like substrate Ac-DEVD-AMC. Substrate cleavage and accumulation of AMC were followed fluorometrically with excitation at 360 nm and emission at 460 nm during a 120-min incubation period. Substrate cleavage during the linear phase of the reaction was quantitated by internal AMC standards. Enzyme activity was expressed as nM AMC per minute per milligram protein (nM/min x mg).
Electrophoretic mobility shift assays
An established EMSA method, with slight modifications, was used
(33). Nuclear protein (5 µg) was incubated for 20 min at
room temperature with 20 µg BSA, 2 µg poly(dI-dC) from Pharmacia
(Piscataway, NJ), 2 µl buffer D (20 mM HEPES/KOH, 20% glycerol, 100
mM KCl, 0.5 mM EDTA, 0.25% Nonidet P-40, 2 mM DTT, 0.5 mM PMSF, pH
7.9), 4 µl buffer F (20% Ficoll-400, 100 mM HEPES/KOH, 300 mM KCl,
10 mM DTT, 0.5 mM PMSF, pH 7.9), and 20,000 cpm of a
32P-labeled oligonucleotide in a final volume of
20 µl. Supershift Abs (2 µg) were added as indicated. DNA-protein
complexes were resolved at 180 V for 4 h in a taurine-buffered,
native 6% polyacrylamide gel (4% for supershifts), dried, and
visualized (autoradiography using a Fuji x-ray film). Oligonucleotide
probes were labeled by a filling reaction using the Klenow fragment
(Boehringer Mannheim, Mannheim, Germany). Oligonucleotide (1 pmol) was
labeled with 50 µCi of [-32P]dCTP (3000
Ci/mmol; Amersham, Braunschweig, Germany), cold nucleotides (dATP,
dTTP, dGTP from Life Technologies, Eggenstein, Germany), purified on a
CHROMA SPIN-10 column (Clontech, Heidelberg, Germany), and stored at
-20°C until use. The following oligonucleotide sequences were used:
the NF-B site from the mouse Cox-2 promoter (34),
5'-GAG GTG AGG GGA TTC CCT TAG-3' and 3'-AC TCC CCT AAG GGA ATC
AATC-5', and a mutated NF-B-site, 5'-GAG GTG AGG GCC TTC CCT TAG-3'
and 3'-AC TCC CGG AAG GGA ATC AATC-5'; the AP-1 site from
the human collagenase gene (35), 5'-AGC TAA AGC ATG AGT
CAG ACA GCC T-3' and 3'-TT TCG TAC TCA GTC TGT CGG ATC GA-5' (the
oligonucleotide was kindly provided by Dr. P. Angel, Deutsches
Krebsforschungszentrum, Heidelberg, Germany); and the specific p53
binding site (36), 5'-GGG CAT GTC CGG GCA TGT-3' and
3'-GTA CAG GCC CGT ACA GG-5'.
Immunoblot analysis
Cell lysis was achieved with lysis buffer (50 mM Tris, 5 mM
EDTA, 150 mM NaCl, 0.5% Nonidet P-40, 1 mM PMSF, pH 8) and sonication
(Branson sonifier; 20 s, duty cycle 100%, output control 60%).
Following centrifugation (14,000 x g, 5 min), protein
was determined. Proteins (100 µg) were resolved on 10%
polyacrylamide gels and blotted onto nitrocellulose. Equal loading was
confirmed by Ponceau S staining. Filters were incubated overnight at
4°C with the Cox-2 Ab (1:250; Dianova, Hamburg, Germany), the p53
antiserum (hybridoma supernatant, clone PAb122, 1:5; kindly provided by
Dr. H. Stahl, Homburg/Saar, Germany), or the IB- Ab (1:500).
Proteins were detected by a HRP-conjugated polyclonal Ab (1:10,000)
using the ECL method (Amersham, Braunschweig, Germany).
Quantitative DNA fragmentation analysis
DNA fragmentation was measured with the diphenylamine assay, as reported elsewhere (37). Briefly, following incubations, cells were scraped off the culture plates; resuspended in 250 µl 10 mM Tris, 1 mM EDTA, pH 8 (TE buffer); and incubated with additional 250 µl lysis buffer (5 mM Tris, 20 mM EDTA, pH 8, 0.5% Triton X-100) for 30 min at 4°C. After lysis, intact chromatin (pellet) was separated from DNA fragments (supernatant) by centrifugation for 15 min at 13,000 x g. Pellets were resuspended in 500 µl TE buffer, and samples were precipitated overnight by adding 500 µl 10% TCA at 4°C. DNA was pelleted by centrifugation (4,000 x g, 10 min) and the supernatant was removed. After addition of 300 µl 5% TCA, samples were boiled for 15 min. DNA contents were quantitated using the diphenylamine reagent (38). The percentage of fragmented DNA was calculated as the ratio of the DNA content in the supernatant to the amount in the pellet.
Transient transfection of a dominant-negative c-jun mutant (TAM67)
To target transcription factor activation by transient transfection of upstream signaling components requires high transfection efficiency and/or selection of cells expressing the mutant protein. One day before transfection, cells were seeded at a density of 1 x 106 cells/ml into 10-cm non-cell culture plates. RAW 264.7 macrophages were transiently transfected with 15 µg of the expression vector (TAM67) that contains the sequence of a dominant-negative c-jun mutant (kindly provided by Dr. E. Gulbins, Tübingen, Germany) (39, 40). For positive selection of TAM67-positive cells, the vector pMACS4, designed to express a truncated human CD4 molecule, was cotransfected (ratio 1:10 compared with the TAM-67 expression vector). Transfection was achieved using a Pro Gentor II electroporator (Hoefer Scientific Instruments, San Francisco, CA). A total of 3 x 106 cells was resuspended in 400 µl complete medium, transferred to a cuvette, and pulsed (260 V, 1080 µF, 26 ms). Transfected cells were pooled and seeded in 10 ml complete medium into a 10-cm non-cell culture petri dish. Overnight (15-h) cultured cells were harvested and CD4-positive clones were enriched using a MiniMACS system (Miltenyi Biotech, Bergisch-Gladbach, Germany), according to the manufacturer’s instructions. Briefly, transfected cells were harvested and washed with PBS supplemented with 5 mM EDTA. A total of 1 x 107 cells was resuspended in 320 µl PBS, 0.5% BSA, 5 mM EDTA (PBE), and 80 µl MACSelect 4 Microbeads to achieve magnetic labeling of transfected cells. After 15 min on ice, the volume was adjusted to 2 ml with PBE. Cells were applied to a positive selection column (MS+), which was placed in the magnetic field of a Mini MACS separator. Unbound cells were washed out (2 ml PBE); the column was removed from the separator; and positive cells were collected, pooled, and seeded. In control examinations, 15 h has been determined as the most effective period for allowing the CD4 surface marker expression in RAW 264.7 macrophages.
Luciferase plasmid expression containing the NF-B site of the
mouse Cox-2 promoter
NF-B reporter constructs, cloned into the pGL3-enhancer
plasmid (Promega), contained four copies of the NF-B element taken
from the murine Cox-2 promoter (NF-B-sense) or its mutated form
(NF-B-mut) (see EMSA). Corresponding sequences were verified by DNA
sequencing. RAW 264.7 macrophages were transiently transfected using
the DEAE-dextran method, as previously described (41).
Cell selection was unnecessary because the synthesis of two
macrophage-unrelated proteins was analyzed. Briefly, one day before
transfection, cells were seeded in suspension at a density of 1 x
106 cells/ml. A total of 1 x
107 cells was harvested, washed twice with PBS,
and incubated for 3 h at 37°C in 1 ml RPMI 1640 supplemented
with 50 mM Tris-HCl (pH 7.3), 400 µg DEAE-dextran, 20 µg
luciferase-reporter construct (NF-B-sense or NF-B-mut), and 5
µg CMV-ß-gal plasmid as an internal control. To discard the
DNA/DEAE-dextran mixture, cells were washed twice with PBS and seeded
at a density of 1 x 106 cells/ml and
cultured for 24 h. Afterward, cells were stimulated for 12 h
with 5 µM DMNQ. Cell extracts were assayed for luciferase and ß-gal
activity. For calculations, luciferase activity was normalized for
ß-gal by using the formula: luciferase activity/ß-gal activity.
Decoy approach
RAW 264.7 cells were exposed to a NF-B or mutated NF-B
decoy oligonucleotides. One day before transfection, cells were seeded
at a density of 1 x 106 cells/well into
six-well plates. Decoy oligonucleotides (3 µM) were added 24 h
before cell stimulation. After changing the medium, cell stimulation
was performed as indicated. Decoy-oligonucleotide sequences were
identical with those used for EMSA.
Statistical analysis
Each experiment was performed at least three times, and statistical analysis was performed using the two-tailed Student’s t test. Otherwise, representative data are shown.
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Results |
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Incubation of macrophages with a high dose of the NO donor GSNO (1 mM) led to accumulation of the tumor suppressor p53 after 4 h and promoted 30–35% DNA fragmentation after 8 h. This is in corroboration with other studies and substantiates NO·-initiated apoptotic cell death (42, 43). Assuming a gene-regulatory potency of superoxide (O2-), we analyzed, in a first set of experiments, the influence of a nontoxic dose of the superoxide-generating agent DMNQ on subsequent NO·-mediated DNA fragmentation and p53 accumulation. GSNO was used as the NO donor because high doses were shown to generate high amounts of NO leading to apoptosis in macrophages (42). DMNQ was described to continuously generate O2- through redox cycling (44), thereby stimulating growth, triggering apoptosis, or causing necrosis depending on the dose and the duration of exposure.
Previous studies (5, 45) pointed to Cox-2 induction and
subsequent cAMP-evoked gene activation as the underlying mechanisms to
confer resistance against high dose NO-mediated toxicity. As a result
of these examinations, we incubated RAW 264.7 macrophages with 5 µM
DMNQ for 15 h. Prestimulation promoted Cox-2 expression (Fig. 1
A, lane 2) and
attenuated the p53 response following GSNO addition (Fig. 1A, lane 4 compared with lane 3). DNA
fragmentation detected by the diphenylamine assay was significantly
increased in response to GSNO, but preactivation with 5 µM DMNQ for
15 h reduced GSNO-elicited DNA fragmentation to control values
(Fig. 1C, lanes 3 and 4). The impact
of Cox-2 in promoting protection was assured by using the
Cox-2-specific inhibitor NS398 (Fig. 1B). NS398 has been
described to be a specific competitive inhibitor for Cox-2
(4). NS398 did not affect DMNQ-elicited Cox-2 expression,
but restored the NO-evoked p53 response, thus pointing to an active
Cox-2 in conveying cell protection. In analogy, inhibition of Cox-2 by
NS398 restored DNA fragmentation in response to 1 mM GSNO (Fig. 1C, last three columns) despite DMNQ prestimulation. This is
in line with our previous notion that enforced Cox-2 overexpression
protected macrophages from apoptosis (5). Our results
point to an inverse expression of Cox-2 and p53 that obviously is
closely correlated to initiation of apoptosis or its inhibition.
Superoxide-evoked protection encouraged us to identify molecular
mechanisms leading to Cox-2 expression.
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B activation by low dose DMNQ
With the notion that Cox-2 expression is regulated at least in
part by NF-B (14, 34), we analyzed NF-B activation
in response to the
O2--generating compound DMNQ.
Based on gel-shift analysis, we proved dose-dependent NF-B
activation by DMNQ (Fig. 2A).
Activation was minor in response to 0.5 µM DMNQ, was stronger
following the addition of 1 µM DMNQ, and was strongest with 5 µM of
the O2-
generator.
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B was absent in unstimulated cells, but was noticed
following the addition of LPS/IFN-, well-established
NF-B-activating agents (Fig. 2
A, second lane).
The response to 5 µM DMNQ and LPS/IFN- was comparable. Higher
concentrations of DMNQ have not been analyzed because of their
proapoptotic and/or necrotic behavior (data not shown). Gel-shift
examinations with an oligonucleotide that contained a mutated NF-B
sequence (replacement of two bases) demonstrated no shift and therefore
referred to the specificity of the NF-B/oligonucleotide binding
(data not shown).
The identity of the NF-B complex was further analyzed by supershift
experiments. Addition of either an anti-p50 or an anti-p65 Ab
shifted the existing NF-B complex to a higher m.w. or diminished
binding completely (Fig. 2B), which is in accordance with
previous studies (46). Therefore, it can be concluded that
NF-B consists of the p50 and p65 (Rel A) subunits. This complex has
been shown to include a DNA binding domain (p50) as well as
transactivation domain (p65), therefore enhancing or promoting gene
induction.
In additional experiments, we paid attention to the time course of
NF-B activation by following the degradation/disappearance of the
NF-B-specific inhibitor IB- (Fig. 2C). Western blot
analysis revealed a time-dependent decrease of IB- expression,
leading to almost complete disappearance of the inhibitor after a 4-h
treatment with 5 µM DMNQ. These results substantiate
O2--mediated NF-B
activation.
NF-B activation and subsequent gene transcription were verified in a
transactivation assay following transient transfection of a luciferase
reporter assay (Fig. 2D). A luciferase reporter construct
containing four copies of the NF-B site derived from the murine
Cox-2 promoter revealed a 3-fold induction of luciferase activity in
response to 5 µM DMNQ. Corresponding plasmids that contained a
mutated NF-B site showed no luciferase transactivation. Luciferase
activity that indicated NF-B activation in response to DMNQ was
attenuated by the addition of NF-B decoy oligonucleotides. Decoy
oligonucleotides, by competing with promoter regions in target genes
for the activated transcription factor, revealed effective suppression
of NF-B-mediated gene activation. This was verified by abrogating
DMNQ-induced Cox-2 expression following NF-B decoy-oligonucleotide
addition (Fig. 2E).
Our studies substantiate superoxide-mediated NF-B activation leading
to an enhanced expression of Cox-2, a NF-B-responsive gene.
AP-1 activation by low dose DMNQ
In another set of experiments, we wished to study activation of
the redox active transcription factor AP-1, which is composed of c-Jun
homodimers or c-Jun/c-Fos heterodimers. Initial examinations considered
two MAPK-specific inhibitors such as PD98059 (MEK-specific kinase
inhibitor) and SB203580 (p38-kinase inhibitor) on DMNQ-elicited Cox-2
expression (Fig. 3A). Western
blot analysis revealed a dramatically decreased DMNQ-stimulated Cox-2
expression in the presence of PD98059 (20 µM). SB203580 (5 µM)
showed a less potent, albeit significant inhibition (70 ± 8% SD
vs control). PD98059 and SB203580 could not be used at higher
concentration because of toxic side effects, as judged by trypan blue
uptake. The used inhibitors completely abrogated ERK-specific kinase
and p38-kinase activity (data not shown).
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A, lane 5). These results
encouraged us to explore expression of c-Jun, which resembles one
component of AP-1, in response to low level of the
O2--generator DMNQ (Fig. 3B). Western blot analysis showed up-regulation of c-Jun in
response to DMNQ. Expression was low at 1 h, increased after
2 h, and revealed highest expression after a 4-h incubation
period. Additional experiments such as electrophoretic mobility shift
analysis were undertaken to demonstrate a transcriptionally active AP-1
in response to DMNQ (Fig. 3C). Activation was low in
response to 0.5 µM DMNQ, became stronger after the addition of 1 µM
DMNQ, and showed maximal activation after a 5 µM challenge. With
respect to the time dependency, AP-1 activation in response to 5 µM
DMNQ peaked at 4 h, with a lower gel-shift response at 2 and
6 h, respectively (data not shown).
To correlate AP-1 activation to the inhibitory action of MAPK
inhibitors on Cox-2 expression, we sought to explore the potency of
PD98059 and SB203580 in AP-1 gel shifts (Fig. 3D). Addition
of PD98059 completely attenuated AP-1 activation in
response to DMNQ. SB203580 showed a minor, but still significant
inhibitory action, while the combination of both MAPK inhibitors was
again fully preventive. Our data indicate that DMNQ promotes AP-1
activation in RAW 264.7 macrophages. AP-1 activation and Cox-2
expression are partially blocked by SB203580 and completely attenuated
by PD98059, thus suggesting the involvement of at least two
MAPK-signaling pathways.
To verify these results and to link AP-1 activation to Cox-2
expression, we transiently transfected RAW 264.7 macrophages with a
dominant-negative c-jun mutant (TAM67) (Fig. 3E).
Transfection with TAM67 abrogated DMNQ-induced Cox-2 expression (Fig. 3E, upper panel, lane 4 compared with
2) and restored a functional p53 response following the
addition of GSNO (Fig. 3E, lower panel,
lane 6 compared with 5). To examine the
DNA-binding capacity of p53, its accumulation was determined using the
EMSA. There were no differences in the amount of accumulated p53
detected by Western blot analysis (data not shown) compared with the
response noticed in the DNA-binding assay (EMSA) (Fig. 3E,
lower panel). Control transfections with an empty vector
were without any influence on Cox-2 or p53 levels (data not shown).
These results underscore the involvement of AP-1 in superoxide-mediated
Cox-2 expression and protection from apoptosis and further point to the
inverse relation of Cox-2 and p53.
NF-B and AP-1 in protection from apoptosis
In a final set of experiments, we examined the contribution of
DMNQ-evoked NF-B and AP-1 activation in protection from
GSNO-mediated apoptosis (Fig. 4).
Therefore, NF-B-elicited gene activation was antagonized by decoy
oligonucleotides, whereas AP-1 activation was attenuated in
TAM67-transfected cells. Apoptosis was assessed by the diphenylamine
assay. For control reasons, a mutated decoy oligonucleotide was used,
and TAM67-unrelated transfections (control transfections) were
conducted in parallel.
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B
decoy-oligonucleotide approach. However, eliminating NF-B by the use
of specific decoy oligonucleotides abrogated DMNQ-evoked protection.
Also, protection from apoptosis was eliminated in TAM67-transfected
cells, whereas protective principles remained intact when control
transfections were performed.
Conclusively, our data imply that low level superoxide confers
protection from apoptosis via AP-1 and NF-B activation, thus leading
to Cox-2 induction.
Cox-2 expression in primary human macrophages attenuated NO-mediated apoptosis
To verify our results obtained with the murine macrophage-like
cell line RAW264.7 in primary macrophages, we isolated human monocytes,
followed by their differentiation to macrophages. In a first set of
experiments, we explored expression of Cox-2 in response to DMNQ (5
µM). Semiquantitative PCR showed low expression at 1 h,
increased levels after 2 h, and revealed highest mRNA amounts
after a 4- or 6-h incubation period (Fig. 5A). Relative appearance of
Cox-2 fragments was verified in proportion to the occurrence of GAPDH
PCR fragments (data not shown). As a positive control known to affect
Cox-2 transcription, we analyzed LPS-evoked Cox-2 mRNA accumulation
during a 2- to 6-h incubation period.
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B). Prestimulation
of human macrophages for 15 h with the
O2--generating compound DMNQ (5
µM) blocked NO-initiated caspase activation (Fig. 5B),
while the further addition of the Cox-2-specific inhibitor NS398
completely restored caspase activation. We conclude that preactivation
of murine or human macrophages for an extended time with DMNQ
attenuated NO-induced apoptosis by a pathway demanding an active Cox-2
enzyme. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B and AP-1, which both contribute to induction of Cox-2. The
active enzyme has been shown to convey resistance to apoptosis in
different cell types (5, 47, 48). This is achieved by the
production of prostaglandins (PGE2) that led to
an increase of intracellular cAMP, which in turn promoted cAMP-evoked
gene induction. We further provide evidence for an inverse expression
of Cox-2 and p53, which reflect protection from cell death and
apoptosis-related pathways, respectively. Our data support the notion
that ROS are efficient regulators of gene activation, clearly allowing
to dissect this signaling component from oxygen radical-mediated
toxicity. The generation of ROS is an established response during inflammation or infection and is implicated, among others, in TNF- or platelet-derived growth factor-evoked signal transmission (49, 50, 51). Oxygen- or nitrogen-based radicals are produced in part by specialized cells such as macrophages and neutrophils with the implication of immunological host defense (52, 53, 54). A delicate balance between formation and detoxification of radicals exists and is regarded an important determinant of cell survival and cell death. Protection of cells involved in immunological host defense such as macrophages against naturally occurring apoptotic cell death must be considered to have a major impact on immunoregulation.
Activation of NF-B and AP-1 by ROS and reactive nitrogen species is
established and, especially in the case of TNF--mediated apoptosis
activation of NF-B, seems to convey protection, i.e., prevention of
apoptosis (19). In response to DMNQ, we established
activation of a functional p50/p65 heterodimer by supershift analysis,
observed IB- degradation, substantiated NF-B activation in a
luciferase transactivation assay, and provided evidence that inducible
protection as well as Cox-2 expression were sensitive to NF-B decoy
oligonucleotides. This is in line with the work of Wang et al.
(55) and Quillet-Mary et al. (56), who
noticed NF-B activation in response to
H2O2-elicited oxidative
stress. Although we have been working with a
O2--generating compound, we
cannot exclude the possibility that disproportionation of
O2- by superoxide dismutase
with the resulting formation of hydrogen peroxide actually causes
NF-B activation. Decoy-oligonucleotide approaches were used to
scavenge active transcription factors, thereby blocking their binding
to the promoter regions in target genes (57, 58, 59). Our
results are in line with the report of Schmedtje et al.
(60), who eliminated hypoxia-induced Cox-2 expression by
NF-B p65 decoy oligonucleotides in human endothelial cells.
In addition to NF-B, we noticed activation of AP-1 in response to
DMNQ. Activation of AP-1 by ROS has been described by Zwacka and
coworkers (13), who reduced ischemia/reperfusion-based
redox activation of AP-1 by adenoviral-enforced overexpression of
mitochondrial superoxide dismutase. Moreover, Manna et al.
(61) showed that manganese superoxide dismutase
overexpression attenuated TNF-induced AP-1 activation by quenching
reactive oxygen intermediates. Several potential oxygen
radical-generating sources such as NAD(P)H oxidases, cyclooxygenases,
the mitochondrial respiratory chain, or xanthine oxidase are known that
may initiate radical signal transmission (1, 4, 62, 63).
One possible mechanism for Cox-2 in attenuating NO-induced apoptosis
may stem from its peroxidase function that generates peroxyl radicals
(1, 62). These radicals may directly scavenge
NO·, thereby eliminating potential damaging
species. However, based on observations that Cox-derived prostanoids
that provoke an intracellular cAMP increase reproduce protection make
the radical interaction theory less favorable.
Activation of NF-B and AP-1 is inevitably related to macrophage
protection. Attenuating NF-B or AP-1 activation by decoy
oligonucleotides or transfection of a dominant-negative
c-jun mutant resulted in a parallel block of inducible
protection and Cox-2 expression. Our results substantiate the
obligatory role of NF-B in promoting Cox-2 expression, which is
fully compatible with the observation of a NF-B binding site in the
promoter region of the murine Cox-2 enzyme (34). Binding
of AP-1 to the murine Cox-2 promoter is rationalized and most probably
achieved via a CRE site because AP-1-enforced Cox-2 expression through
this promoter site has been noticed (28). This is verified
when we showed a time- and concentration-dependent AP-1 activation by
DMNQ that was blocked by the ERK-specific kinase inhibitor PD98059 and
in part by the p38-kinase inhibitor SB203580. Both inhibitors were
described to be selective at the concentrations used (64, 65). Our results are in line with the report of Hwang and
coworkers, in which LPS-induced Cox-2 expression was inhibited by the
two MAPK kinase inhibitors PD98059 and SB203580 (14).
The potential antiapoptotic role of Cox-2 is in analogy to examinations
in which Cox-2 blocked butyrate-mediated apoptosis (47).
Further proof for a survival-promoting function of Cox-2 came from
genetic studies that point to the early involvement of the protein in
the progression leading to colon cancer or from correlative
investigations showing a high incidence of Cox-2 expression in human
tumors (4). For macrophages, it seems conceivable to
assume PGE2 formation as a result of Cox-2
expression that, in a self-regulatory feedback loop, will enhance
intracellular cAMP formation. Interestingly, intervention in the
macrophage cyclic nucleotide system, i.e., supplementation of
lipophilic cAMP analogues (59) or the addition of
PGE2 will attenuate apoptosis (66).
In some analogy, preactivation of macrophages with
LPS/IFN-/NG-monomethyl-L-arginine
(NAE) promoted protection from NO·-elicited
apoptosis, which was Cox-2 mediated (5). Attenuating
macrophage apoptosis reminds of the unique property of endotoxin to
achieve low responsiveness, a phenomenon known as endotoxin tolerance
(67, 68, 69). Control of endotoxin responsiveness may limit
proinflammatory macrophage responses. Low level ROS formation may be
used as an indicator for oxidative stress and may signal gene
expression and thus protective protein synthesis. This rescue system
allows macrophages to evade apoptosis. We show that NF-B and AP-1
activation promotes Cox-2 expression and protects RAW 264.7 macrophages
against NO-induced apoptosis. Protection is reversed by AP-1 or NF-B
inhibition, thus restoring a functional p53 response, which is
indicative for NO-mediated apoptosis. Murine and human macrophages may
use ROS as an autocrine-regulatory pathway to maintain cell viability,
a process that contributes and fulfills a fundamental role during
immunologic homeostasis.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Bernhard Brüne, Faculty of Medicine, University of Erlangen-Nürnberg, Loschgestrasse 8, 91054 Erlangen, Germany. E-mail address:
3 Abbreviations used in this paper: Cox-2, cyclooxygenase-2; Ac-DEVD-AMC, N-acetyl-Asp-Glu-Val-Asp-7-amino-4-methylcoumarin; DMNQ, 2,3-dimethoxy-1,4-naphtoquinone; ERK, extracellular signal-regulated kinase; GSNO, S-nitrosoglutathione; MAPK, mitogen-activated protein kinases; ROS, reactive oxygen species; ß-gal, ß-galactosidase; IB, inhibitory protein that dissociates from NF-B.
Received for publication November 2, 1998. Accepted for publication June 11, 1999.
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0.005 vs GSNO-treated
samples).
