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| 2007 JASN IMPACT FACTOR 7.111 | HOME AUTHOR INFO EDITORIAL BOARD SUBSCRIBE FEEDBACK ALERTS HELP | |||
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Pharmazentrum Frankfurt, Klinikum der Johann Wolfgang Goethe-Universität, Frankfurt, Germany.
Correspondence to Dr. Josef Pfeilschifter, Klinikum der Johann Wolfang Goethe-Universität, Pharmazentrum Frankfurt, Theodor-Stern-Kai 7, 60590 Frankfurt, Germany. Phone: +49-69-6301-6951; Fax: +49-69-6301-7942; E-mail: Pfeilschifter{at}em.uni-frankfurt.de
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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induced cellular inhibitor of
apoptosis 1 (cIAP1) mRNA expression within 3 h. In contrast, X
chromosome-linked inhibitor of apoptosis (XIAP) mRNA levels remained
unaffected by cytokines. Although coincubation of cells with IL-1ß and
tumor necrosis factor- or IL-1ß and basic fibroblast growth factor
resulted in synergistic induction of inducible NO synthase, comparable
potentiating effects on cIAP1 induction were absent. Exogenously released NO
from NO donors promoted cIAP1 mRNA upregulation in mesangial cells, whereas
XIAP mRNA was downregulated. However, the changes observed on the mRNA level
were not adequately translated to the protein level, and corresponding values
for cIAP1 and XIAP were only slightly affected. In contrast, in
lipopolysaccharide/interferon-
-stimulated RAW 264.7 macrophages,
massive NO-dependent downregulation of cIAP1 and XIAP protein levels, which
correlated temporally with the induction of apoptosis, was observed. This
effect was at least partially reversed by
NG-monomethyl-L-arginine, an inhibitor of NO synthase
activity. In summary, a direct correlation between the downregulation of IAP
protein levels and the induction of apoptosis by endogenous NO was observed in
macrophages. In contrast, a stable level of IAP protein in mesangial cells
might represent a mechanism for the resistance of the cells to endogenously
produced NO. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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NO is a highly versatile and short-lived messenger that mediates a number of diverse physiologic and pathophysiologic processes. One of the most intensively investigated features of NO signaling is its effect on cell survival and cell death (4). Several years ago, NO derived from an iNOS isoform was initially reported to induce apoptotic cell death in macrophages; the involvement of NO was demonstrated with the use of specific NO synthase inhibitors (5). Apoptosis is distinguished from necrosis by specific biochemical and structural events, such as DNA ladder formation, chromatin condensation, and finally fragmentation into apoptotic bodies. NO has emerged as a bifunctional regulator of apoptosis (6), whose function depends on the cell type and the amount of NO released within a specific time period. Large amounts of NO released exogenously by NO donors or endogenously via iNOS induction result in apoptotic alterations in, for example, RAW 264.7 macrophages (7). Apoptotic death signaling involves, at least in part, accumulation of the tumor suppressor p53 (8,9), activation of caspases (10,11), and altered expression of Bcl-2 family proteins (12). Moreover, copper/zinc superoxide dismutase protein and mRNA downregulation (13) and stress-activated protein kinase activation (14) have been described. In addition to RAW 264.7 macrophages, which have been extensively characterized in terms of NO-induced apoptotic signal transduction, numerous studies have confirmed the ability of NO to initiate apoptosis in several other cell types, including pancreatic ß-cells, neuronal cells, mast cells, chondrocytes, thymocytes, and renal mesangial cells (15). In contrast, many reports indicated that NO or related molecules of endogenous or exogenous origin could inhibit programmed cell death in a variety of cells and tissues (16). Initially, protective effects of NO were observed in Epstein-Barr virus-infected B lymphoma cell lines that constitutively expressed iNOS and exhibited an increased spontaneous apoptosis rate when exposed to NG-monomethyl-L-arginine (L-NMMA) (17). Antiapoptotic effects of NO were subsequently demonstrated in several other cell types, such as hepatocytes, eosinophils, endothelial cells, thymocytes, and embryonic kidney cells (16). The general protective function of NO seems to be largely independent of the apoptotic stimulus (4). As one potential protective mechanism, NO-dependent S-nitrosylation of several caspase isoenzymes, such as caspases 3 and 8, and concomitant inhibition of caspase enzyme activity were demonstrated mainly in in vitro studies (18).
One family of proteins that may extensively modulate apoptosis signal
transduction is the inhibitor of apoptosis (IAP) family of proteins
(19). IAP were first
identified in insect viruses and seem to be evolutionarily conserved proteins
that are characterized by baculovirus inhibitory repeat (BIR) domains and in
part by a carboxyl-terminal, zinc-binding, RING motif
(20). In human cells, several
IAP relatives have been identified, i.e., cellular inhibitor of
apoptosis 1 (cIAP1), cIAP2, X chromosome-linked inhibitor of apoptosis (XIAP),
neuronal apoptosis inhibitory protein, survivin, and apollon. Mouse orthologs
of most human IAP have also been identified, whereas IAP family members in rat
cells are only poorly characterized [cIAP1 correlates with rat inhibitor of
apoptosis 1 (RIAP1), cIAP2 correlates with RIAP2, and XIAP correlates with
RIAP3]. Functionally, cIAP1, cIAP2, XIAP, and survivin were demonstrated to
bind to and potently inhibit caspases 3, 7, and 9 but not caspases 1, 6, 8, or
10
(21,22).
In addition to directly affecting effector proteases, the IAP family of
proteins was linked to signal transduction pathways used by members of the
tumor necrosis factor (TNF) receptor family
(19). cIAP1 and cIAP2 are
recruited to the TNF receptor by binding to TNF receptor-associated
factor-1/TNF receptor-associated factor-2 heterocomplexes
(23), which seem to be
functionally implicated in TNF induction of nuclear factor 
B
(NF-B) and protection from apoptosis
(24).
In this work, we focused on the role of IAP family proteins during NO-induced apoptosis in rat mesangial cells and mouse RAW 264.7 macrophages. RAW 264.7 cells die by apoptosis in response to exogenously or endogenously released NO, and we observed a massive downregulation of cIAP1 and XIAP protein levels. In contrast, in rat mesangial cells, which are resistant to endogenously released NO and rarely die by apoptosis in response to NO donors (25), cIAP1 (RIAP1) and XIAP (RIAP3) proteins remained constitutively expressed. Therefore, we suggest that NO-mediated IAP regulation constitutes an additional mechanism for the positive or negative regulation of apoptosis by NO.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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(specific activity, 6.6 x 106 U/mg) was a generous gift from
Knoll AG (Ludwigshafen, Germany). RPMI 1640 medium, cell culture supplements,
and fetal calf serum were from Life Technologies (Eggenstein, Germany). All
other chemicals were of the highest grade of purity commercially
available.
Cell Culture and Cell Treatment
Rat renal mesangial cells were cultured and characterized as described
previously (26). In a second
step, single cells were cloned by limiting dilution in 96-well plates. Clones
with apparent mesangial cell morphologic features were characterized by
positive staining for the intermediate filaments desmin and vimentin (which
are considered to be specific for myogenic cells), positive staining for
Thy1.1 antigen, and negative staining for factor VIII-related antigen and
cytokeratin (to exclude endothelial and epithelial contaminations,
respectively). For the experiments in this study, passages 13 to 23 were
used.
Cells were cultured in RPMI 1640 medium containing 10% fetal calf serum, 100 U/ml penicillin, and 100 µg/ml streptomycin. For all experiments, subconfluent cells were starved for 24 h and exposed to the different substances in starvation medium, i.e., Dulbecco's modified Eagle's medium containing 0.1 mg/ml fatty acid-free bovine serum albumin, 100 U/ml penicillin, and 100 µg/ml streptomycin.
S-Nitrosoglutathione Synthesis
S-Nitrosoglutathione (GSNO) was synthesized as described
previously
(8,27).
Briefly, glutathione was dissolved in HCl at 4°C before the addition of
NaNO2. The mixture was stirred at 4°C for 40 min, followed by
the addition of 2.5 vol of acetone. Precipitates were filtered, washed with
acetone and diethyl ether, and dried under vacuum. GSNO was characterized by
HPLC analysis and ultraviolet spectroscopy.
Quantitation of DNA Fragmentation
DNA fragmentation was assayed essentially as reported previously
(12). Briefly, after
incubation, cells were scraped off the culture plates, resuspended in 250
µl of 10 mM Tris, 1 mM ethylenediaminetetraacetic acid (EDTA) (Tris-EDTA
buffer, pH 8.0), and incubated for 30 min at 4°C with an additional 1 vol
of lysis buffer (5 mM Tris, 20 mM EDTA, pH 8.0, 0.5% Triton X-100). 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 of Tris-EDTA buffer, and samples were
precipitated by addition of 500 µl of 10% TCA at 4°C. Samples were
pelleted at 4000 rpm for 10 min, and the supernatant was removed. After
addition of 300 µl of 5% TCA, samples were boiled for 15 min. DNA contents
were quantitated using the diphenylamine reagent
(28). The percentage of
fragmented DNA was calculated as the ratio of the DNA content in the
supernatant to the amount in the pellet. Alternatively, histone-associated DNA
fragments were assayed by using a cell death detection enzyme-linked
immunosorbent assay, according to the protocol described by the manufacturers
(Roche Molecular Biochemicals).
Probe DNA
All cDNA fragments used as probes for RNase protection assays were cloned
by PCR, using the following nucleotide primers (Life Technologies). For the
rat IAP1 probe, 5'-CTCATG(GC)A-CAAAACTG(CT)CTCC-3' was used as the
5'-primer and
5'-CT(AG)GG(AG)TA(GCT)A(AGC)CT(GT)T(CT)T(AG)TGC-3' was used as the
3'-primer. For the rat XIAP (RIAP3) probe,
5'-TGACTTTTAACAGTTTTGAAGG-3' was used as the 5'-primer and
5'-GTCTTCCAAC(TA)GCTGAGTCTCC-3' was used as the 3'-primer.
For the rat ß-actin probe, 5'-ATGGATGA(CT)GATATCGC(TC)GCG-3'
was used as the 5'-primer and 5'-ATGGGGTACTTCAGGGT(CG)AGG-3'
was used as 3'-primer. A cDNA fragment of rat glyceraldehyde-3-phosphate
dehydrogenase was obtained by using 5'-CATCACCATCTTCCAGGAGCGAG-3'
as the 5'-primer and 5'-GTTGTCATA(CT)TTCTC(AG)TGGTTC-3' as
the 3'-primer.
Primer sequences for the mouse cIAP1 probe were 5'-TCGGCCAAGGTACCTTACACC-3' as the 5'-primer and 5'-AGATGGAGACTGCAGACTGGC-3' as the 3'-primer. For the mouse cIAP2 probe, 5'-GACTTGGGTCAGTCTGCTTC-3' was used as the 5'-primer and 5'-TGGCCTTTGATTCTGAGCAAG-3' was used as the 3'-primer. For the mouse XIAP probe, 5'-GCTAACTTCCCAAGTAGTAG-3' was used as the 5'-primer and 5'-CCACACAGTTTTCAGATTTG-3' was used as the 3'-primer. For mouse ß-actin, 5'-ATGGATGA(CT)GATATCGC(TC)GCG-3' was used as the 5'-primer and 5'-ATGGGGTACTTCAGGGT(CG)AGG-3' was used as the 3'-primer. For mouse glyceraldehyde-3-phosphate dehydrogenase, 5'-CATCACCATCTTCCAGGAGCGAG-3' was used as the 5'-primer and 5'-GTTGTCATA(CT)TTCTC(AG)TGGTTC-3' was used as the 3'-primer.
RNA Isolation and RNase Protection Analyses
RNA isolation was performed as described
(29). Twenty micrograms of
total RNA for the different experimental time points of the cell culture
experiments were used for RNase protection assays.
DNA probes were cloned into the transcription vector pBluescript II KS(+)
(Stratagene, Heidelberg, Germany) or pCR2.1-TOPO (Invitrogen, Leek,
Netherlands) and linearized. An antisense transcript was synthesized in
vitro by using RNA polymerase T3 or T7 and [-32P]UTP
(800 Ci/mmol; Amersham, Freiburg, Germany). RNA samples were hybridized
overnight at 42°C with 100,000 cpm of the labeled antisense transcript.
Hybrids were digested with RNase A and T1 for 1 h at 30°C. Under these
conditions, every single mismatch is recognized by the RNases. Protected
fragments were separated on 5% acrylamide/8 M urea gels and analyzed using a
PhosphorImager (Raytest, Straubenhardt, Germany). All protection assays were
performed with at least three different sets of RNA from independent
experiments.
Western Blot Analyses
Cells were cultured and incubated as described. Cell lysis was achieved
with lysis buffer (50 mM Tris, 5 mM EDTA, 150 mM NaCl, 0.5% Nonidet P-40, 1 mM
phenylmethylsulfonyl fluoride, pH 8.0) and sonication (Branson sonifier;
duration, 10 s; duty cycle, 100%; output control, 10%), followed by
centrifugation (4000 x g, 5 min) and protein determination
using the method described by Bradford
(30). Proteins were normalized
to 50 µg/lane or 10 µg/lane (RIAP3), resolved on 7.5% (cIAP1/RIAP1) or
10% (XIAP/RIAP3) polyacrylamide gels, and blotted onto polyvinylidene
difluoride sheets. Sheets were washed twice with Tris-buffered saline (TBS)
(140 mM NaCl, 50 mM Tris, pH 7.2) containing 0.1% Tween-20 before nonspecific
binding was blocked with TBS with 5% skim milk. Filters were incubated
overnight at 4°C with either mouse anti-human/mouse human IAP-like protein
antibody (clone 28, 1:250 in TBS with 0.5% skim milk; Transduction
Laboratories) to assay mouse XIAP expression, mouse anti-human/mouse human
IAP-like protein antibody (clone 48, 1:12,500 in TBS with 0.5% skim milk;
Transduction Laboratories) to assay RIAP3 expression, or rabbit anti-cIAP1
anti-body (1 µg/ml in TBS with 0.5% skim milk; Santa Cruz Biochemicals,
Santa Cruz, CA) to assay mouse cIAP1 and RIAP1 expression. Sheets were washed
five times, and nonspecific binding was blocked as described. Detection was
performed for 1.5 h at room temperature with horseradish peroxidase-conjugated
goat anti-mouse monoclonal antibodies (1:5000) or goat anti-rabbit monoclonal
antibodies (1:5000), using the enhanced chemiluminescence method
(Amersham).
Metabolic Labeling
For the detection of new RIAP1 protein synthesis, confluent rat mesangial
cells were starved for 20 h with methionine-free minimal essential medium
containing 0.1 mg/ml bovine serum albumin and the appropriate stimuli. Sixteen
hours after starvation, 0.14 mCi of [35S]methionine (Amersham) was
added to each plate for an additional period of 4 h. After incubation and
labeling, cells were lysed in 1 ml of lysis buffer [50 mM Tris, pH 7.4, 150 mM
NaCl, 10% glycerol, 2 mM EDTA, 2 mM ethylene glycol bis(ß-aminoethyl
ether)-N,N,N',N'-tetraacetic acid (EGTA), 40 mM
ß-glycerophosphate, 50 mM NaF, 10 mM sodium pyrophosphate, 1 mM
phenylmethylsulfonyl fluoride, 100 nM okadaic acid, 200 µM sodium
orthovanadate, 2 mM dithiothreitol] and homogenized with 10 strokes of a
26-gauge needle. After centrifugation (13,000 x g, 10 min),
RIAP1 was immunoprecipitated overnight at 4°C in lysis buffer containing
5% fetal calf serum, 0.5 mM iodoacetamide, 1 µg/ml anti-RIAP1 antibody
(Santa Cruz), and protein A/G-Sepharose. Beads were washed twice with low-salt
buffer [50 mM Tris, pH 7.4, 150 mM NaCl, 0.2% Triton X-100, 2 mM EDTA, 2 mM
EGTA, 0.1% sodium dodecyl sulfate (SDS)] and three times with high-salt buffer
(50 mM Tris, pH 7.4, 500 mM NaCl, 0.2% Triton X-100, 2 mM EDTA, 2 mM EGTA,
0.1% SDS) before a 5-min heating period at 95°C in 50 µl of LD buffer
(100 mM Tris, pH 7.4, 10% glycerol, 5% SDS, 10% ß-mercaptoethanol). After
gel electrophoresis, labeled proteins were detected by PhosphorImager
analysis.
Statistical Analyses
Each experiment was performed at least three times. Statistical analyses
were performed using the two-tailed t test or ANOVA. For multiple
comparisons, the data were corrected using Dunn's method.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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(IFN-) caused apoptotic cell death in
approximately 40 to 50% of the stimulated cells within 24 h
(10). Similar to findings for
RAW 264.7 macrophages but with different kinetics, NO donors such as
spermine/NO, GSNO, S-nitroso-N-acetylpenicillamine, and a
NO-saturated solution caused apoptosis in rat mesangial cells
(25). We confirmed these
results and demonstrated apoptotic DNA fragmentation within 24 h, which
amounted to approximately 22% with 375 µM spermine/NO, approximately 22%
with 750 µM GSNO, and approximately 15% with 1 mM DETA/NO
(Figure 1). Apoptosis was
confirmed by morphologic investigations using Hoechst dye 33258 and
fluorescence microscopy, which demonstrated that all NO-releasing compounds
induced chromatin condensation and all other signs of nuclear pyknosis
(25).
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Next, we investigated whether endogenously released NO also affects mesangial cell viability. IL-1ß elicited iNOS induction and long-lasting release of NO but only weakly elicited apoptotic cell death (Figure 2). Only iNOS superinduction by IL-1ß and bFGF significantly potentiated NO release and led to approximately 9% apoptotic DNA fragmentation (Figure 2A) (32) and, correspondingly, approximately 8 to 9% apoptotic cells (data not shown). The data obtained with the quantitative diphenylamine assay were qualitatively confirmed with a DNA/histone-based enzyme-linked immunosorbent assay (Figure 2B) and with morphologic investigations using Hoechst dye 33258 staining (data not shown).
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Effects of Cytokines and NO on RIAP1 and RIAP3 Expression in
Mesangial Cells
Gene expression of IAP, which are essential regulators of apoptosis signal
transduction (19), is known to
be affected by TNF- via NF-B activation
(33). Therefore, we first
investigated whether there was a direct correlation between resistance against
NO-induced apoptosis and IAP gene expression in rat mesangial cells. As shown
in Figure 3B, TNF-
potently and rapidly (within 3 h after TNF- stimulation) elicited RIAP1
gene expression; elevated RIAP1 expression was detectable for >24 h
(control levels of RIAP1 mRNA were sustained at the 3-h level during the
entire incubation period; data not shown). Like TNF-, IL-1ß was
able to induce RIAP1 gene expression, beginning 3 h after treatment, and
continuously increasing mRNA induction was detectable up to 24 h
(Figure 3A). We next
investigated whether bFGF, which synergistically potentiated
IL-1ß-induced iNOS expression, also affected IL-1ß-induced RIAP1
gene expression. As demonstrated in Figure
3A, bFGF had no effect alone on RIAP1 expression and was not able
to modulate IL-1ß-mediated RIAP1 expression. Also, in contrast to iNOS
expression, the combination of IL-1ß and TNF- had no potentiating
effect on RIAP1 gene expression (Figure
3B). None of the tested cytokines affected RIAP3 mRNA expression
(Figure 3).
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In the following set of experiments, we examined a potential effect of endogenously released NO on IL-1ß- or IL-1ß/bFGF-mediated RIAP1/RIAP3 gene expression. For these experiments, we coincubated rat mesangial cells with the appropriate cytokines in the absence or presence of 2 mM L-NMMA, which potently blocked endogenous NO release (34). As shown in Figure 4, RIAP1 mRNA induction by IL-1ß or IL-1ß/bFGF was not affected by the presence of L-NMMA during the first 8 h. In contrast, the maximal response obtained after 24 h of cytokine stimulation was significantly lower in the presence of L-NMMA. With the delayed induction of iNOS in mesangial cells (34), this clearly demonstrates a contribution of endogenous NO to RIAP1 mRNA expression in mesangial cells. As observed with exogenous NO, RIAP3 expression was not altered by endogenous NO (Figure 4).
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To confirm a direct modulatory effect of NO on RIAP1 gene expression, we
exposed mesangial cells to spermine/NO and DETA/NO. Both NO donors rapidly
elicited RIAP1 gene upregulation, within 3 h
(Figure 5). However, continuous
exposure of cells to NO donors for up to 24 h revealed that RIAP1 gene
upregulation by NO was only transient. This was true for spermine/NO as well
as for DETA/NO. In contrast to RIAP1, RIAP3 mRNA expression was downregulated
by both NO donors, beginning 8 h after NO donor addition. Interestingly,
costimulation by NO donors and TNF- led to superinduction of RIAP1 and
enhanced RIAP3 downregulation. Therefore, the exogenously added NO may
compensate for the poor NO-inducing capability of TNF- and may result
in a synergistic effect on RIAP1 expression comparable to that observed with
IL-1ß, which potently triggered endogenous NO release
(Figure 4).
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In the next step, we investigated whether cytokine- or NO-induced RIAP1
mRNA expression was translated into changes in RIAP1 protein expression. As
documented in Figure 6A,
TNF- caused a slight but significant increase in RIAP1 protein
expression within 24 h. In contrast, IL-1ß and IL-1ß/bFGF only
weakly affected RIAP1 protein expression. Similarly, NO donors such as
spermine/NO and DETA/NO only marginally affected RIAP1 protein levels
(Figure 6A). We detected no
significant change in RIAP3 protein expression within 24 h with either
cytokines or NO donors, although RIAP3 mRNA was downregulated by NO
(Figure 6B).
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To mechanistically address the apparently discrepant behavior of the RIAP1
protein and mRNA levels, we used the translational inhibitor cycloheximide and
performed metabolic labeling experiments. Incubation of rat mesangial cells
with [35S]methionine revealed an approximately fivefold increase in
de novo synthesis of RIAP1 after TNF- stimulation
(Figure 7A). This increased
RIAP1 biosynthesis, which probably reflects the increased RIAP1 mRNA levels,
must be counterbalanced by a concomitant increase in RIAP1 protein degradation
to explain the unaltered steady-state levels of RIAP1 protein. Experiments
using cycloheximide revealed that RIAP1 was degraded by approximately 30%.
Coincubation of cells with cycloheximide and TNF- moderately enhanced
RIAP1 degradation (Figure 7B).
These experiments indeed indicate the regulation of RIAP1 protein at the
levels of both protein synthesis and protein degradation.
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Effects of NO on cIAP1 and XIAP Expression in Mouse RAW 264.7
Macrophages
Because the effect of NO on RIAP1 expression in mesangial cells was limited
to mRNA regulation, with only marginal changes in RIAP1 and RIAP3 protein
levels, we next investigated the effects of NO on cIAP1 and XIAP mRNA and
protein levels in mouse RAW 264.7 macrophages. In contrast to mesangial cells,
RAW 264.7 macrophages rapidly die after exposure to NO, either exogenously
released or endogenously produced in response to LPS/IFN- stimulation.
As in mesangial cells, NO donors such as GSNO and spermine/NO produced an
increase in cIAP1 mRNA levels within 3 to 8 h
(Figure 8, A and B). In
contrast, XIAP mRNA levels remained unaffected.
Figure 8, C and D, demonstrates
that LPS/IFN- temporally upregulate cIAP1 mRNA within 2 to 4 h,
followed by a decrease back to control levels at 18 h and further
downregulation at 24 h. Surprisingly, blockade of NO release with the
application of 1 mM L-NMMA left cIAP1 mRNA regulation unaffected except at the
24-h time point, when L-NMMA blocked cIAP1 mRNA downregulation. This indicates
that NO, via eventual activation of proapoptotic pathways, led to cIAP1 mRNA
downregulation concomitantly with apoptosis induction. Moreover,
LPS/IFN- elicited rapid and NO-independent downregulation of XIAP mRNA
levels within 8 to 24 h (Figure 8, C and
D).
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Next, cIAP1 and XIAP protein expression was investigated by Western blot
analysis. As demonstrated in Figure
9, GSNO and spermine/NO potently induced cIAP1 protein
downregulation beginning 2 to 3 h after addition of the NO donor. cIAP1
protein downregulation/degradation temporally preceded the first signs of
apoptosis induction, such as DNA laddering and apoptotic morphologic features
(which first occur approximately 4 to 5 h after NO donor addition)
(7), and appeared together with
p53 upregulation (10). In
contrast, XIAP protein levels were not altered by NO donor exposure. Similarly
to cIAP1 mRNA regulation, cytokines elicited a biphasic effect: 3 to 6 h after
LPS/IFN- addition; cIAP1 protein expression was downregulated, followed
by upregulation at 10 to 14 h and finally degradation at 24 h. Coexposure to
L-NMMA demonstrated that the final downregulation event at 24 h depended on NO
release. Similarly, XIAP protein levels were downregulated by LPS/IFN-
within 18 to 24 h, which correlates with the time course of apoptosis
induction. Again, 1 mM L-NMMA blocked LPS/IFN--induced XIAP protein
downregulation (Figure 9).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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; the
amounts of NO were comparable to those produced by RAW 264.7 macrophages
stimulated with LPS/IFN-. Whereas RAW 264.7 macrophages die
NO-dependently within 24 h, rat mesangial cells were highly resistant to
endogenously produced NO (Figure
2) (25). Moreover,
RAW 264.7 macrophages exhibited homogeneous iNOS expression and apoptotic
nuclei in the iNOS-containing cells after stimulation with IFN- and
LPS, whereas mesangial cells that expressed iNOS did not display signs of
apoptosis and, conversely, cells that exhibited characteristic features of
apoptosis did not stain for iNOS
(35). One possible explanation
is that IL-1ß not only induces the expression of iNOS but also stimulates
a protecting mediator in mesangial cells. In line with this hypothesis are
experiments demonstrating that preincubation of mesangial cells with
IL-1ß attenuated DNA damage attributable to exogenous NO by approximately
50% (35). One such protective
factor may be a member of the Bcl-2 family of proteins, such as Bcl-2 itself,
which is upregulated in response to simultaneous generation of NO and
O2-
(36). Similar to findings with
balanced cogeneration of NO and O2-, preactivation of
RAW 264.7 macrophages with nontoxic concentrations of NO donors or the redox
cycler 2,3-dimethoxy-1,4-naphthoquinone for 15 h attenuated GSNO-initiated
apoptotic cell death and averted accumulation of p53, which is indicative of
macrophage apoptosis (37).
Mechanistically, the promotion of cyclooxygenase-2 expression by NF-B
and activator protein-1 activation seems to mediate protection against
NO-induced cytotoxicity (38).
Other known mechanisms that have been suggested to provide resistance to NO
include the activation of MEK kinases, because the MEK inhibitor PD98059
reverses resistance (39), and
the upregulation of superoxide dismutase expression
(13). Moreover, nontoxic
concentrations of NO were reported to block the activity of certain caspases,
such as caspase 3, via S-nitrosylation
(18,40).
However, the in vivo relevance of the latter observation remains to
be proven. In this work, we describe another family of proteins that are
regulated by NO and may be involved in resistance to NO, namely the IAP family
of proteins.
Evidence that NO Is a Direct Inducer of cIAP1 mRNA
At least five different mammalian IAP family members (XIAP, cIAP1, cIAP2,
neuronal apoptosis inhibitory protein, and survivin) exhibit antiapoptotic
activity in cell culture. IAP are able to block a broad spectrum of apoptotic
stimuli, including members of the TNF receptor family, proapoptotic members of
the Bcl-2 family, cytochrome c, and chemotherapeutic agents. XIAP
seems to have the broadest and strongest anti-apoptotic activity
(41). For at least some IAP,
the antiapoptotic potential can be explained by the inhibition of certain
caspases. The IAP/caspase ratio might be modulated by transcriptional or
translational regulation of both groups of proteins, or IAP might be regulated
by localization, post-translational modifications, binding by other proteins,
or degradation (42). Here we
provide evidence that NO acts as a potent transcriptional regulator of cIAP1
in different cells, such as rat mesangial cells and RAW 264.7 macrophages. In
both cell systems, exogenous application of NO produced selective upregulation
of cIAP1 mRNA, whereas the message for XIAP was downregulated in rat mesangial
cells and remained unaffected in RAW 264.7 macrophages. In addition to NO,
cytokines such as IL-1ß and TNF- were proven to upregulate cIAP1
mRNA in rat mesangial cells and LPS/IFN- transiently induced cIAP1 mRNA
in mouse macrophages. These data are in line with several other observations
concerning IAP regulation, such as strong upregulation of cIAP1, cIAP2, and
XIAP in human endothelial cells by IL-1ß, TNF-, or LPS
(33) and marked induction of
the IAP family proteins survivin and XIAP by vascular endothelial growth
factor (43). Concerning
signaling pathways leading to IAP upregulation, several studies demonstrated a
dependence on NF-B transcription factor activity by using transient or
stable transfection with inhibitor B
(33,44,45,46).
Because NO and NO-releasing compounds have been reported to activate
NF-B (47), it can
speculated that NO-induced IAP mRNA induction is mediated by NF-B
activation. However, the effect of NO on NF-B activation is
controversial. NF-B activation by NO was reported for lymphocytes
(48) and for liver and lung
during hemorrhagic shock (49);
however, NO inhibits NF-B activation in part by hindering DNA binding
at least in vitro
(50). Similar to NO modulation
of cell death, the NF-B-activating activities may be attributable to
small concentrations of NO and the NF-B-blocking effects may result
from high concentrations of NO. However, because toxic NO donor concentrations
also result in cIAP1 mRNA upregulation, alternative mechanisms, such as
activator protein-1 activation by NO
(47,51),
must be considered. Further work is needed to investigate this matter.
NO, Cell Death, and IAP Protein Levels
One of our major findings is that mesangial cells, which are highly
resistant to endogenously released NO and die with slow kinetics in response
to high doses of NO donors, maintain cIAP1 and XIAP protein expression at high
levels, whereas RAW 264.7 macrophages, which rapidly die in response to NO,
demonstrate massive rapid cIAP1 and XIAP downregulation in response to
LPS/IFN-. Consistently, there was a direct correlation between cIAP1
downregulation and rapid induction of apoptotic cell death by NO.
Surprisingly, in our experiments cIAP1 mRNA upregulation by cytokines or NO did not result in cIAP1 protein upregulation in mesangial cells, indicating that changes in IAP mRNA levels did not necessarily translate into changes in protein levels. In vivo labeling experiments and protein stability studies using cycloheximide revealed an increase in new RIAP1 protein biosynthesis that was accompanied by an increase in protein degradation, thus explaining the constant RIAP1 protein levels in rat mesangial cells after cytokine or NO stimulation. Similar findings were recently reported for different tumor cell lines, in which XIAP, cIAP1, and cIAP2 mRNA levels did not correlate with protein levels, indicating post-transcriptional regulation of expression (52). Post-transcriptional regulation of IAP protein levels was previously described for cIAP1 and XIAP, levels of which were substantially decreased during thymocyte apoptosis elicited by dexamethasone (53), and for XIAP, which is cleaved into two fragments during Fas-induced apoptosis (42). In the former report, IAP were degraded in a proteasome-dependent manner before cell death, which indicates that IAP degradation is a prerequisite for thymocyte apoptosis; in the latter report, XIAP was cleaved into two fragments, comprising either the BIR-1 and -2 domains or the BIR-3 domain and the RING domain and exhibiting abilities to block caspase 3 and 7 or caspase 9, respectively. We now report on cIAP1 downregulation during NO-induced apoptosis of RAW 264.7 macrophages. It is tempting to speculate that, as in thymocytes, efficient NO-induced apoptosis requires IAP downregulation/degradation. Differences in cIAP1 protein levels between rat mesangial cells and RAW 264.7 macrophages, resulting from differences in specific degradation pathways, may explain the different sensitivities to NO-induced programmed cell death. For mechanistic evaluation of the role of IAP downregulation in RAW 264.7 macrophages, IAP overexpression and IAP antisense experiments will be required in future studies. IAP degradation may also explain the lack of correlation between IAP mRNA and protein levels in rat mesangial cells. Future work must shed light on these regulatory events, to increase our understanding of the different susceptibilities of cells to NO and the role of IAP regulation in glomerulonephritis and mesangial cell function.
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), or S-nitrosoglutathione
(GSNO; 
). DNA fragmentation was assessed using the diphenylamine
reaction. Values are means ± SD of four individual experiments. At all
time points depicted, the data are statistically different from the value at
time zero (P < 0.05, t test).
