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*
Department of Microbiology and Immunology, Wonkwang University School of Medicine, Iksan, Chonbuk, Korea;
Medicinal Resources Research Center of Wonkwang University, Iksan, Chonbuk, Korea; and
Department of Biology, College of Natural Science, Kyung-Pook National University, Taegu, Korea
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, and -
) showed substantial
protection from cell death induced by the exposure to NO donors such as
SNP and S-nitrosoglutathione (GSNO). In contrast, in RAW
264.7 parent or in empty vector-transformed cells, these NO donors
induced internucleosomal DNA cleavage. Moreover, overexpression of PKC
isoforms significantly suppressed SNP-induced JNK/SAPK and p38 kinase
activation, but did not affect ERK-1 and -2. We also explored the
involvement of CPP32-like protease in the NO-induced apoptosis.
Inhibition of CPP32-like protease prevented apoptosis in RAW 264.7
parent cells. In addition, SNP dramatically activated CPP32 in the
parent or in empty vector-transformed cells, while slightly activated
CPP32 in PKC transfectants. Therefore, we conclude that PKC protects
NO-induced apoptotic cell death, presumably nullifying the NO-mediated
activation of JNK/SAPK, p38 kinase, and CPP32-like protease in RAW
264.7 macrophages. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Recently, the action of NO has been related to induction of programmed cell death, or apoptosis, in various cells including murine RAW 264.7 macrophages 17, 18, 19 . Apoptosis is an active, energy-dependent mode of cell death of typical morphologic changes, such as nucleoplasmic and cytoplasmic condensation, and the formation of extensive membrane blebs and novel membranous structures known as apoptotic bodies 20 . Although activation of soluble guanylyl cyclase, followed by 3':5'-cGMP generation, has been known as a prime physiological NO action 21 , toxic or apoptotic NO-signaling is still an enigma. Possible mechanisms include interactions between NO and iron-sulfur enzymes or protein thiol groups 3 , the NAD(H)-dependent modification of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) 22 , or direct DNA damage 11 .
The mitogen-activated protein (MAP) kinase is an essential part of the signal transduction machinery and occupies a central position in cell growth, differentiation, and programmed cell death 23, 24, 25 . To date, several mammalian MAP kinases have been identified, including the p42 and p44 (extracellular signal-regulated kinase (ERK)) 26, 27 , c-Jun N-terminal protein kinase/stress-activated protein kinase (JNK/SAPK) 28, 29, 30 , and p38 kinase 31, 32, 33 . All three MAP kinases phosphorylate substrates on serine and threonine residues located adjacent to proline residues, and members of all MAP kinases are activated as a results of simultaneous phosphorylation on threonine and tyrosine residues by upstream dual-specificity kinases. However, the three MAP kinases are activated in response to different extracellular stimuli, have different downstream targets, and, therefore, perform different functions. ERKs are characteristically activated by growth factors, usually by means of a Ras-Raf-1-dependent cascade 24, 34 , whereas JNK/SAPK 30 and p38 kinase 31, 32, 33 are strongly activated by UV irradiation, osmotic stress, and the inflammatory cytokines TNF and IL-1.
While several studies have demonstrated the significant role of MAP kinases in apoptotic signaling, there is limited information concerning the roles of MAP kinases in NO actions. A previous report indicated that NO and related chemical species (NOx) activate the ERK, p38, and JNK/SAPK subgroups of MAP kinases in human Jurkat T cells 35 . In contrast, another report demonstrated that NO promotes PC12 cell survival and blocks JNK activation caused by trophic factor withdrawal 36 .
Recently, several reports demonstrated that NO-induced apoptosis is
inhibited by exposure to phorbol ester and protein kinase C (PKC)
activation in murine RAW 264.7 cells 19, 37 . Despite the evidence
that PKC mediates down-regulation of p53 and Bax expression 37 ,
questions regarding its inhibitory action on apoptotic cell death in
RAW 264.7 remain unanswered. Therefore, the first objective of this
study was to determine which MAP kinases were tyrosine-phosphorylated
and activated in response to NO in murine RAW 264.7 macrophages. The
second objective was to determine whether activation of PKC by phorbol
ester or up-regulation of PKC isoforms (i.e., ßII, , and )
could block apoptosis along with concomitant inhibition of MAP kinase
activation after NO addition.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Glutathione S-transferase (GST)-c-Jun N-terminal protein, JNK/SAPK, and anti-JNK1 Abs were purchased from Stratagene (La Jolla, CA). Anti-ERK-1 or -2 mAbs were purchased from Transduction Laboratories (Lexington, KY). Abs specific to phosphorylated ERK-1, -2, and p38 kinase were obtained from New England Biolab (Beverly, MA). SB203580, PD098059, N-acetyl-Asp-Glu-Val-p-nitroanilide (Ac-DEVD-pNA), N-acetyl-Asp-Glu-Val-Asp-aldehyde (Ac-DEVD-CHO), and propidium iodide (PI) were purchased from Calbiochem-Behring (La Jolla, CA). Sodium nitroprusside (SNP), potassium ferricyanide (PFC), Hoechst 33342, PMA, and staurosporine (STSN) were purchased from Sigma (St, Louis, MO). S-nitrosoglutathione (GSNO) was purchased from Alexis (San Diego, CA). Molecular size marker of DNA was purchased from Bethesda Research Laboratories (Bethesda, MD). Genomic DNA purification kit was obtained from Promega (Madison, WI). All reagents used for in situ nick translation were obtained from Oncor (Gaithersburg, MD).
Generation of PKC overexpressing cell lines
The expression vector (i.e., MTH vector containing cDNA
for PKC-ßII, -, and -) that was used has been described
previously 38 . Clones of RAW 264.7 cells that overexpress one of the
PKC isoforms or a control plasmid lacking the PKC genes (empty vector
(EV)-4) were generated by the transfection of expression vector using
lipofectamine (Life Technologies, Gaithersburg, MD) with the procedure
recommended by the manufacturer. The transfected cells were
subsequently grown in selection medium (G418 at 800 µg/ml of complete
medium). Following 10–20 days in selection medium, single colonies
were picked and subsequently examined for the presence of PKC proteins
by Western blotting. The cells were maintained in RPMI 1640 medium
supplemented with 10% FCS and antibiotics.
Western blot analysis of PKC isoforms
Cell lysates (50 µg) from RAW 264.7 cells and PKC
overexpressed cells were separated by SDS-PAGE and transferred to a
nitrocellulose membrane. PKC isoforms were detected with
isoform-specific anti-PKC mAb for (Transduction Laboratories,
Lexington, KY) or with polyclonal Abs for ßII (Santa Cruz
Biotechnology, Santa Cruz, CA) and (Biomol, Plymouth Meeting, PA).
PKC isoforms were visualized using a peroxidase-conjugated secondary Ab
and the enhanced chemiluminescence system 39, 40 .
Protein kinase assay for JNK/SAPK
JNK/SAPK activity was assayed as described previously 41 .
Cells were stimulated according to experimental protocols and lysed
using buffer A containing 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM
PMSF, 1% Nonidet P-40, 0.5% deoxycholate, and 0.1% SDS. Cell lysates
were subjected to centrifugation at 12,000 x g for 10
min at 4°C. The soluble fraction was incubated 1 h at 4°C with
Abs against JNK/SAPK. After the addition of protein G-agarose, the
reaction mixtures were incubated for 1 h at 4°C and then
subjected to microcentrifugation. The immunopellets were rinsed three
times with buffer A, then twice with 20 mM HEPES, pH 7.4. Immunocomplex
kinase assays were performed by incubating the immunopellets for 30 min
at 30°C with a GST-c-Jun protein (2 µg) in 20 µl of the reaction
buffer containing 0.2 mM sodium orthovanadate, 2 mM DTT, 10 mM
MgCl2, 2 µCi [
-32P]ATP, and 20 mM HEPES,
pH 7.4. The reaction was terminated by adding 5 µl of 5x sample
buffer and heating the solution at 80°C for 3 min. The reaction
mixture was subjected to electrophoresis on 12% polyacrylamide gel.
The phosphorylated substrates were visualized by autoradiography.
Assay of ERK-1, -2, or p38 kinase
Cells were stimulated according to experimental protocols. Proteins were extracted with a buffer (50 mM Tris-HCl, pH 7.4, containing 150 mM NaCl, 1% Nonidet P-40, 0.1% SDS, 0.1% deoxycholate, 5 mM sodium fluoride, 1 mM sodium orthovanadate, and 1 mM 4-nitrophenyl phosphate, 10 µg/ml of leupeptin, 10 µg/ml of pepstatin A, 1 mM of 4-(2-aminoethyl) benzenesulfonyl fluoride). The activation of MAP kinase was examined by determining its phosphorylation state using Ab specific to phosphorylated ERK-1, -2, and p38 kinase.
CPP32-like protease activity assay
CPP32-like protease activity was measured as described in detail previously 42 . RAW 264.7 cells (5 x 106 cells) were harvested from the cultured 6-well plates, washed with ice-cold PBS, and resuspended in 200 µl of buffer A' (100 mM HEPES, pH 7.4, 140 mM NaCl, and the protease inhibitors, including 0.5 mM PMSF, 5 µg/ml pepstatin, and 10 µg/ml leupeptin). The cell suspension was lysed by three cycles of freezing and thawing. The crude cytosol was obtained as the supernatant from centrifugation at 12,000 x g for 20 min at 4°C. Assays were set up in flat-bottom 96-well plates containing 400 µM Ac-DEVD-pNA in buffer B' (100 mM HEPES, pH 7.4, 20% glycerol, and protease inhibitors) and 200 µg of cytosol in a total volume of 200 µl. The CPP32-like protease activity was determined by measuring absorbance at 405 nm for 3 h. The reaction mixture without substrate was used as a control.
Cell viability and apoptosis assay
Cell viability was determined by PI exclusion test. Cells were treated with different inducers and/or inhibitors, washed with PBS twice, resuspended in PBS containing 20 µg/ml PI, and then immediately analyzed on a FACStar (Becton Dickinson, Rutherford, NJ). Cells that permitted PI uptake, interpreted as nonviable, were expressed as a percentage of the total cell number. For apoptosis assay, cells after agent treatment were fixed in 4% neutral-buffered paraformaldehyde, permeabilized with PBS/0.5% Triton X-100, and nuclei were stained for 20 min with the chromatin-staining Hoechst dye 33342. The coverslips were then washed, mounted onto slides, and viewed with a fluorescence microscope.
Quantitation assay of apoptosis
To quantitate the number of cells undergoing apoptosis, cells were fixed with 4% neutral-buffered formalin. Apoptotic cells were stained using the terminal deoxynucleotidyl transferase (TdT) method (Apotag). Endogenous peroxidase was first quenched with 2% hydrogen peroxide, and the cells were permeabilized using company-supplied equilibration buffer. The 3' OH ends of degraded DNA were reacted with TdT and digoxygenin-labeled ATP for 30 min. After washing with PBS, slides were reacted with an anti-digoxygenin mAb conjugated to peroxidase, washed, and developed with 3,3'-diaminobenzidine tetrahydrochloride. Stained cells were then counted using a light microscope.
DNA extraction and electrophoresis
The pattern of DNA cleavage was analyzed by agarose gel electrophoresis as described 43 . Briefly, genomic DNA was purified by Wizard Genomic DNA purification kit. After ethanol precipitation, samples of 10 µg in each lane were subjected to electrophoresis on a 1.4% agarose at 50 V for 3 h. DNA was stained with ethidium bromide.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Initially we wished to determine which MAP kinases were
tyrosine-phosphorylated and activated in response to NO in murine RAW
264.7 macrophages. As shown in Fig. 1
A, SNP significantly elevated
JNK/SAPK activity within 30 min. This increase reached a maximum level
of approximately 10-fold by 1 h after SNP addition. However, the
total amounts of JNK/SAPK protein were not affected by SNP (Fig. 1A, bottom). We then attempted to determine
whether NO also activate other members of MAP kinase family, the p38
kinase and ERK-1 and -2. Cultures of RAW 264.7 cells were incubated for
various times (0–4 h) with SNP (1 mM), and then the cell lysates were
prepared and phosphotyrosine-containing bands were visualized by
immunoblot analysis (Fig. 1, B and C).
Phosphorylation of p38 kinase was detected after 4 h of incubation
with SNP, but not detected at the early time periods (0–2 h) (Fig. 1B). However, SNP induced neither phosphorylation of ERK-1
and -2 nor alteration of total amounts of ERK protein (Fig. 1C). In addition, although RAW 264.7 cells were treated with
SNP for longer time periods, there were no detectable changes of
phosphorylation of ERK-1 and -2 (data not shown). To determine whether
the selective inhibitors of p38 kinase and MAP/ERK1 kinase,
SB203580 44 and PD098059 45 , respectively, could affect NO-induced
cytotoxicity, RAW 264.7 cells were treated for 8 h with either
SB203580 (10 µM) or PD098059 (10 µM) in the presence of SNP (0.5–1
mM). We found that SB203580, which by itself had no effect on cell
morphology or viability, slightly reduced the cytotoxic action of SNP
while PD098059 had no effect (Fig. 2),
suggesting that p38 kinase is involved in the induction of programmed
cell death by NO in RAW 264.7 macrophages. Although we have not
currently determined the functional involvement of JNK/SAPK, activation
of JNK/SAPK might be important for NO-induced signaling in RAW 264.7
macrophages.
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NO releasing compounds permit the investigation of NO signaling
irrespective of NOS involvement. Within 8 h after exposure to NO
donors, RAW 264.7 macrophages responded with apoptotic cell death,
characterized by chromatin condensation and by DNA laddering 19, 37 .
To determine the specific down-modulatory role of PKC on NO-induced
apoptotic cell death in murine RAW 264.7 macrophages, we produced
clones of RAW 264.7 cells that overexpress individual PKC isoforms such
as ßII, , and . Parental RAW 264.7 cells expressed 
, ,
, 
/
, µ, and isoforms of PKC, while PKC-ßII was
basically not detectable by Western blotting (Fig. 3, A and B). As
expected, RAW 264.7 cells that had been stably transfected with
expression vectors containing cDNA for PKC isoforms (ßII, , and
) expressed substantial amounts of the appropriate isoforms (Fig. 3B).
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, A–C).
Internucleosomal DNA degradation determined qualitatively by agarose
gel electrophoresis (Fig. 4A) or quantitatively by in situ
TdT-apoptosis assay method (Fig. 4C) was selected as a
reliable apoptotic parameter. SNP or GSNO, exposed for 12 h,
elicited 45–50% DNA degradation in RAW 264.7 parent cells (Fig. 4C). Similar results were obtained with EV-transformed cells
(EV-4). In contrast, exposure of PKC transfectants (PKC-ßII, -,
and -) to SNP or GSNO resulted in substantially less DNA cleavage.
Clones PKC-ßII-4, -5, and -6, which contained higher levels of
PKC isoforms, remained viable with little evidence of apoptosis within
the 12-h incubation period. Clone PKC--3, which expressed the lowest
amount of PKC- among the stable transfectants, showed no protection
(Fig. 4, A and C). To verify the involvement of
NO on SNP-induced DNA fragmentation, we evaluated the effect of PFC,
which is structurally similar to SNP except for the absence of a
nitroso group. As expected, PFC (0.5 mM) alone did not induce DNA
fragmentation in RAW 264.7 parent or EV-transformed cells as determined
by gel electrophoresis (Fig. 4A).
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).
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-5 cells with SNP (1 mM) significantly suppressed JNK/SAPK
activity as compared with parental RAW 264.7 cells (Fig. 6A). In addition, activation
of JNK/SAPK by SNP was slightly reduced after treatment of parental RAW
264.7 cells with PMA (200 nM), which was already known to inhibit
NO-induced apoptosis 19 , but increased after treatment with STSN (100
nM), implying the regulatory roles of PKC on NO-induced JNK/SAPK
pathway (Fig. 6B). Phosphorylation of p38 kinase was also
decreased in PKC--5 cells as compared with RAW 264.7 parent cells
(Fig. 6C, top and Fig. 1
B). However,
phosphorylation of ERK-1 and -2 was not changed (Fig. 6C,
bottom and Fig. 1C). Similar results were
obtained from other PKC-overexpressed cells including PKC-ßII-4 and
PKC--6 clones (data not shown).
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, in murine macrophages 46 . To determine whether SNP has any
effect on PKC isoform expression in transfected RAW 264.7 cells, the
cells were incubated for various times (0–24 h) with SNP (0.5 mM). As
expected, treatment of PKC transfectants with SNP significantly
decreased the expression of PKC isoforms in a time-dependent manner
(Fig. 7).
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Recently, CPP32, a member of IL-1-converting enzyme (ICE) family
cysteine proteases, has emerged as one of the key proteases in
spontaneous 47 , anti-Fas- 48 , and STSN-mediated apoptosis in
various cell types 49 . To gain further insights for the protective
role of PKC on NO-induced apoptosis, we investigated the involvement of
CPP32-like protease in RAW 264.7 parent, EV-transformed
(EV-4), and PKC--5 cells. Interestingly, SNP significantly activated
CPP32-like protease in the parent and EV-transformed cells but not in
PKC--5 cells (Fig. 8A). In
addition, NO-induced apoptosis was significantly reduced in the
presence of CPP32-like protease inhibitor, Ac-DEVD-CHO, in RAW 264.7
parent cells (Fig. 8B).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, and -). Previous investigations established
that RAW 264.7 macrophages are highly susceptible to endogenously
generated or exogenously supplied NO 19, 37 . The cellular response to
NOS induction, with concomitant massive and sustained NO formation, is
compatible with apoptosis, as characterized by chromatin condensation
and DNA laddering. In those experiments all apoptotic alterations were
blocked by the addition of the NOS inhibitor,
NG-monomethyl-L-arginine, thereby
relating endogenous NO generation to macrophage apoptosis 19 .
Cytotoxic and/or cytostatic actions of NO are not only directed against
invading pathogens but also can affect susceptible host cells.
Therefore, the existence of cellular defense mechanisms that oppose the
damaging potential of these radicals and that account for differential
cellular susceptibilities to NO seem likely. Protective mechanisms may
be attributable to an altered NO-target interaction, scavenging of NO,
or efficient repair mechanisms. Our results strongly suggest that the
anti-apoptotic mechanism of PKC is closely related with the
inhibition of JNK/SAPK and p38 kinase, which play critical intermediary
roles in mediating signal transduction from external stimuli to
the nucleus 28, 29, 30, 31, 32, 33 . Previous investigations established that NO and related chemical species (NOx) activate all of the three MAP kinase subfamilies such as ERK, p38, and JNK/SAPK in human Jurkat T cells 35 . It is particularly interesting that NO does not affect ERK activity in RAW 264.7 macrophages. This result indicates that activation of MAP kinases are cell-type specific. For example, JNK/SAPK was not activated in response to stimulation of macrophages by the phorbol ester, in contrast to what has been seen in a number of other cell types including T lymphocytes 28, 29 . In addition, NO promoted PC12 cell survival and blocked JNK/SAPK activation caused by trophic factor withdrawal 36 .
Although the factors regulating apoptosis remain obscure, the
involvement of PKC has been implied by numerous lines of evidence. For
example, PKC activation blocks apoptotic cell death in rat thymocytes
exposed to Ca2+ ionophores and glucocorticoids 50 ,
suppresses radiation-induced sphingomyelin hydrolysis and apoptosis in
aortic endothelial cells 51 , and prevents ceramide-induced programmed
cell death in U937 monoblastic leukemic cells 52 . In addition, recent
report demonstrated that overexpression of PKC- suppresses apoptosis
and induces bcl-2 expression in human IL-3-dependent cells
53 . It also reported that overexpression of atypical PKC- protects
human leukemia cells against drug-induced apoptosis 54 . On the other
hand, we previously reported that phorbol ester, a PKC activator,
synergistically augments NO-induced apoptosis in human leukemic HL-60
cells 55 . Taken together, these findings suggest that PKC-dependent
signaling processes may, in some instances, depend on the diverse
stimuli and specific cell types. Our results provide evidence that PKC
overexpression completely suppressed NO-mediated apoptosis and DNA
laddering within the first few hours (
12 h) after NO donor
application in RAW 264.7 macrophages. PKC overexpression (PKC-ßII,
-, and -) neither blocked IFN-/LPS signaling pathways
resulting in inducible NOS expression nor endogenous NO-formation (data
not shown). Our results corroborate previous report that the
transfection of RAW 264.7 cells with plasmids harboring PKC- isotype
but not with PKC-, -ßI, or - isotypes resulted in the
expression of NOS 56 . Obviously, therefore, our results further
suggest that PKC (at least in such isotypes as PKC-ßII, -, and
-) blocks NO-mediated cell death events through direct or indirect
regulation of MAP kinase subfamilies in RAW 264.7 macrophages.
Recently, studies have implicated CPP32, a member of the ICE family
cysteine proteases, as an obligate component of the cell death pathway
in various cell types 47, 48, 49 . In this report, we showed that a
CPP32-like protease is also involved in NO-induced apoptosis as
assessed by colorimetric assay. In addition, overexpression of PKC
isotype (PKC--5) suppressed NO-induced activation of CPP32-like
protease. Although we do not know whether the suppression of CPP32-like
protease activity in PKC transfectants is due to the direct interaction
of PKC with CPP32-like protease, PKC function might be required at a
step before CPP32-like protease activation to protect NO-induced
apoptosis signaling.
Collectively, although the results of this study provide strong evidence that either activation of PKC or overexpression of PKC isoforms inhibit NO-mediated signaling pathways such as JNK/SAPK, p38 kinase, and CPP32-like protease and the resulting induction of apoptosis, the point in the pathway at which PKC is involved is not clear. Additional experiments will be required to establish whether of any mechanisms account for the inhibition of NO-mediated signaling pathways in PKC-overexpressing RAW 264.7 macrophages.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Hun-Taeg Chung, Department of Microbiology and Immunology, Wonkwang University School of Medicine, 344–2, Shin-Yong Dong, Iksan, Chonbuk 570–749, Korea. E-mail address:
3 Abbreviations used in this paper: NO, nitric oxide; NOS, NO synthase; MAP kinase, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal protein kinase; SAPK, stress-activated protein kinase; PKC, protein kinase C; SNP, sodium nitroprusside; PFC, potassium ferricyanide; STSN, staurosporine; PI, propidium iodide; GSNO, S-nitrosoglutathione; Ac-DEVD-pNA, N-acetyl-Asp-Glu-Val-Asp-p-nitroanilide; Ac-DEVD-CHO, N-acetyl-Asp-Glu-Val-Asp-aldehyde; TdT, terminal deoxynucleotidyl transferase; ICE, IL-1-converting enzyme; EV, empty vector; GST, glutathione S-transferase.
Received for publication July 2, 1998. Accepted for publication December 7, 1998.
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T. Matsuoka, H. Tabata, and S. Matsushita Monocytes Are Differentially Activated Through HLA-DR, -DQ, and -DP Molecules Via Mitogen-Activated Protein Kinases J. Immunol., February 15, 2001; 166(4): 2202 - 2208. [Abstract] [Full Text] [PDF]
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 J. P. Hall, E. Merithew, and R. J. Davis c-Jun N-terminal kinase (JNK) repression during the inflammatory response? Just say NO PNAS, December 19, 2000; 97(26): 14022 - 14024. [Full Text] [PDF]
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H.-S. Park, S.-H. Huh, M.-S. Kim, S. H. Lee, and E.-J. Choi Nitric oxide negatively regulates c-Jun N-terminal kinase/stress-activated protein kinase by means of S-nitrosylation PNAS, December 19, 2000; 97(26): 14382 - 14387. [Abstract] [Full Text] [PDF]
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 S. Ghatan, S. Larner, Y. Kinoshita, M. Hetman, L. Patel, Z. Xia, R. J. Youle, and R. S. Morrison p38 Map Kinase Mediates Bax Translocation in Nitric Oxide-Induced Apoptosis in Neurons J. Cell Biol., July 24, 2000; 150(2): 335 - 348. [Abstract] [Full Text] [PDF]
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0.001 vs
the value obtained in the control.
