Overexpression of Protein Kinase C Isoforms Protects RAW 264.7 Macrophages from Nitric Oxide-Induced Apoptosis: Involvement of c-Jun N-Terminal Kinase/Stress-Activated Protein Kinase, p38 Kinase, and CPP-32 Protease Pathways

Chang-Duk Jun, Chun-Do Oh, Hyun-Jeong Kwak, Hyun-Ock Pae, Ji-Chang Yoo, Byung-Min Choi, Jang-Soo Chun, Rae-Kil Park and Hun-Taeg Chung
J Immunol March 15, 1999, 162 (6) 3395-3401;

Abstract

Nitric oxide (NO) induces apoptotic cell death in murine RAW 264.7 macrophages. To elucidate the inhibitory effects of protein kinase C (PKC) on NO-induced apoptosis, we generated clones of RAW 264.7 cells that overexpress one of the PKC isoforms and explored the possible interactions between PKC and three structurally related mitogen-activated protein (MAP) kinases in NO actions. Treatment of RAW 264.7 cells with sodium nitroprusside (SNP), a NO-generating agent, activated both c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) and p38 kinase, but did not activate extracellular signal-regulated kinase (ERK)-1 and ERK-2. In addition, SNP-induced apoptosis was slightly blocked by the selective p38 kinase inhibitor (SB203580) but not by the MAP/ERK1 kinase inhibitor (PD098059). PKC transfectants (PKC-βII, -δ, and -η) 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.

Nitric oxide (NO),3 a radical produced in mammalian cells from arginine in a reaction catalyzed by NO synthase (NOS), has pleiotropic biologic activities 1, 2, 3 . NO is produced during inflammatory reactions and has been implicated as a signaling molecule 4, 5, 6 as well as a toxic effector 7, 8, 9 . NO mediates activation or inhibition of various enzyme systems 6, 10 , DNA damage 11 , and oxidative reactions 12, 13, 14 , with a variety of biologic effects, including killing of microorganisms 15 , antiviral activity 16 , and cytostasis and cell death 2, 3, 7, 8, 9 .

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., βII, δ, and η) could block apoptosis along with concomitant inhibition of MAP kinase activation after NO addition.

Materials and Methods

Materials

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-βII, -δ, and -η) 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 δ (Transduction Laboratories, Lexington, KY) or with polyclonal Abs for βII (Santa Cruz Biotechnology, Santa Cruz, CA) and η (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 × 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 [γ-32P]ATP, and 20 mM HEPES, pH 7.4. The reaction was terminated by adding 5 μl of 5× 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 × 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 × 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.

Results

NO activates JNK/SAPK and p38 kinase but does not affect ERK-1 and -2

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. 1⇓A, 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. 1⇓B). However, SNP induced neither phosphorylation of ERK-1 and -2 nor alteration of total amounts of ERK protein (Fig. 1⇓C). 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.

FIGURE 1.
FIGURE 1.

NO activates JNK/SAPK and p38 kinase but does not affect ERK-1 and -2. A, RAW 264.7 cells were incubated for various times (0–2 h) with SNP (1 mM). Then, the cells were harvested, lysed, and immunoprecipitated with a specific Ab against JNK-1 and analyzed for their catalytic activities to phosphorylate GST-c-Jun-NT, as described in Materials and Methods. The reaction mixtures were separated by SDS/PAGE, and phosphorylated GST-c-Jun protein was visualized by autoradiography. B, RAW 264.7 cells were incubated for various times (0–4 h) with SNP (1 mM). Cell lysates were blotted with Abs specific for the tyrosine-phosphorylated form of p38 kinase and were visualized using a peroxidase-conjugated secondary Ab and the enhanced chemiluminescence system. C, RAW 264.7 cells were incubated for various times (0–4 h) with SNP (1 mM). Cell lysates were blotted with Abs specific for the tyrosine-phosphorylated form of ERK or Abs for ERK-1 and -2 protein and visualized as in B.

FIGURE 2.
FIGURE 2.

Induction of apoptosis by SNP and its inhibition by SB203580. RAW 264.7 cells were incubated for 8 h with SNP (0.5–1 mM) in the presence or absence of SB203580 or PD098059. For the combination treatment, cells were incubated with SB203580 or PD098059 for 2 h before SNP addition. Quantitative analysis of apoptosis was determined by in situ TdT-apoptosis assay method. Results are expressed means ± SE of three independent experiments. ∗, p ≤ 0.001 vs the value obtained in the control.

Overexpression of PKC protects against NO-induced apoptosis and reduces both JNK/SAPK and p38 kinase activity

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 βII, δ, and η. Parental RAW 264.7 cells expressed α, δ, ε, λ/ι, μ, and η isoforms of PKC, while PKC-βII 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 (βII, δ, and η) expressed substantial amounts of the appropriate isoforms (Fig. 3⇓B).

FIGURE 3.
FIGURE 3.

Overexpression of PKC isoforms (βII, δ, and η) in RAW 264.7 cells. A, Cell lysates from parental RAW 264.7 cells were blotted with Abs specific for the individual PKC isoforms (α, βII, δ, ε, λ/ι, and μ) and visualized using a peroxidase-conjugated secondary Ab and the enhanced chemiluminescence system. B, Total cell lysates extracted from clones of RAW 264.7 cells transfected with EV-4 or vector containing cDNA for individual PKC isoforms (βII, δ, and η) was used to detect PKC isoforms by Western blotting.

With the use of SNP and GSNO, we elicited DNA cleavage in RAW 264.7 macrophages (Fig. 4⇓, A–C). Internucleosomal DNA degradation determined qualitatively by agarose gel electrophoresis (Fig. 4⇓A) or quantitatively by in situ TdT-apoptosis assay method (Fig. 4⇓C) 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. 4⇓C). Similar results were obtained with EV-transformed cells (EV-4). In contrast, exposure of PKC transfectants (PKC-βII, -δ, and -η) to SNP or GSNO resulted in substantially less DNA cleavage. Clones PKC-βII-4, δ-5, and η-6, which contained higher levels of PKC isoforms, remained viable with little evidence of apoptosis within the 12-h incubation period. Clone PKC-δ-3, which expressed the lowest amount of PKC-δ 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. 4⇓A).

FIGURE 4.
FIGURE 4.

Overexpression of PKC isoforms (βII, δ, and η) blocks DNA fragmentation in response to NO donor exposure. A, Analysis of NO-induced DNA fragmentation of RAW 264.7 cells by agarose gel electrophoresis. RAW 264.7 parent (WT), EV-transformed (EV-4), or PKC-overexpressed cells (PKC-βII-4, -δ-3, -δ-5, or -η-6) were incubated for 12 h with SNP (0.5 mM), PFC (0.5 mM), or GSNO (0.5 mM). Then, genomic DNA was purified and subjected to gel electrophoresis (M; molecular size marker). B, RAW 264.7 parent and PKC-overexpressed cells (PKC-δ-5 clone) were incubated for 12 h with SNP (0.5 mM). Then, the cells were stained with Hoechst dye 33342 and examined by fluorescence microscopy. RAW 264.7 parent cells (a) exhibited apoptotic chromatin condensation, while PKC-overexpressed cells (b) showed normal nuclear morphology by diffuse chromatin structure. C, RAW 264.7 parent (WT), EV-transformed (EV-4), or PKC-overexpressed cells (PCK-βII-4, -βII-5, -δ-3, -δ-5, -η-5, or -η-6) were incubated for 12 h with SNP (0.5 mM) or GSNO (0.5 mM). Quantitative analysis of apoptosis was determined by in situ TdT-apoptosis assay method. Results are expressed means ± SE of four independent experiments.

To test the regulatory role of PKC on NO-induced apoptosis, we observed the effect of STSN, a potent PKC inhibitor, in RAW 264.7 parent or PKC transfectants. STSN (20 nM) significantly increased NO-induced apoptosis in both RAW 264.7 parent and PKC transfectants (Fig. 5⇓).

FIGURE 5.
FIGURE 5.

Effect of STSN on NO-induced apoptosis in RAW 264.7 parent and PKC transfectants. RAW 264.7 parent (WT), EV-transformed (EV-4), or PKC-overexpressed cells were incubated for 12 h with SNP (0.5 mM) in the presence or absence of STSN (20 nM). Quantitative analysis of apoptosis was determined by in situ TdT-apoptosis assay method. Results are expressed means ± SE of three independent experiments.

To elucidate the mechanism that caused the resistance of PKC transfectants against death induced by the exposure to NO donors, the activities of three MAP kinase subfamilies were investigated in RAW 264.7 parent and PKC transfectants. Treatment of PKC-δ-5 cells with SNP (1 mM) significantly suppressed JNK/SAPK activity as compared with parental RAW 264.7 cells (Fig. 6⇓A). 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. 6⇓B). Phosphorylation of p38 kinase was also decreased in PKC-δ-5 cells as compared with RAW 264.7 parent cells (Fig. 6⇓C, top and Fig. 1⇑B). However, phosphorylation of ERK-1 and -2 was not changed (Fig. 6⇓C, bottom and Fig. 1⇑C). Similar results were obtained from other PKC-overexpressed cells including PKC-βII-4 and PKC-η-6 clones (data not shown).

FIGURE 6.
FIGURE 6.

Overexpression of PKC suppresses NO-induced JNK/SAPK and p38 kinase activation but does not affect ERK-1 and -2. A, RAW 264.7 parent (WT) or PKC-δ-5 cells were cultured as in Fig. 1⇑A. Then, JNK/SAPK activity was examined, as described in Materials and Methods. B, Up- or down-regulation of NO-induced JNK/SAPK activation by PKC modulator. RAW 264.7 cells were incubated for 2 h with SNP (1 mM), PMA (200 nM), STSN (100 nM), SNP plus PMA, or SNP plus STSN. Then, JNK/SAPK activity was examined as in A. C, PKC-overexpressed cells (PKC-δ-5) were incubated for various times (0–4 h) with SNP (1 mM). Cell lysates were blotted with Abs specific for the tyrosine-phosphorylated form of p38 kinase (top) or Abs for ERK (bottom) and were visualized using a peroxidase-conjugated secondary Ab and the enhanced chemiluminesence system. Note: both Fig. 6⇓C and Fig. 1⇑, B and C are representative of the same blot.

Previously, we reported that endogenously generated or exogenously applied NO markedly inhibits expression of PKC isoforms, such as PKC-δ, 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⇓).

FIGURE 7.
FIGURE 7.

Down-regulation of PKC isoforms after NO addition. PKC-overexpressed cells were incubated for various times (0–12 h) with SNP (0.5 mM). Total cell lysates extracted from clones of RAW 264.7 cells transfected with individual PKC isoforms (βII, δ, and η) were used to detect PKC isoforms by Western blotting.

Overexpression of PKC isoforms blocks activation of CPP32-like protease

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-δ-5 cells. Interestingly, SNP significantly activated CPP32-like protease in the parent and EV-transformed cells but not in PKC-δ-5 cells (Fig. 8⇓A). 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. 8⇓B).

FIGURE 8.
FIGURE 8.

Overexpression of PKC suppresses NO-induced CPP32-like protease activation. A, Cytosolic extract was prepared from RAW 264.7 parent (WT), EV-transformed (EV-4), or PKC-overexpressed cells (PKC-δ-5 clone) cultured for various times (0–12 h) with SNP (1 mM). CPP32-like protease activity was measured with Ac-DEVE-pNA using a colorimetric assay. Data represented the result from one of the three similar experiments. B, Effect of Ac-DEVD-CHO on NO-induced apoptosis. RAW 264.7 cells were cultured for 3 h in medium that contained Ac-DEVD-CHO (100 μM). Then, the cells were treated with SNP (0.5 mM) and cultured for 12 h. Quantitative analysis of apoptosis was determined by in situ TdT-apoptosis assay method. Results are expressed means ± SE of three independent experiments.

Discussion

Our present results show that NO affects differentially the activation of the three known MAP kinase subfamilies; it strongly activates both JNK/SAPK and p38 kinase, but does not activate ERK-1 and -2 in RAW 264.7 macrophages. Activation of both JNK/SAPK and p38 kinase can be effectively antagonized by the transfer of various PKC isoforms (PKC-βII, -δ, and -η). 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-ε suppresses apoptosis and induces bcl-2 expression in human IL-3-dependent cells 53 . It also reported that overexpression of atypical PKC-ι 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-βII, -δ, and -η) neither blocked IFN-γ/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-ε isotype but not with PKC-α, -βI, or -δ isotypes resulted in the expression of NOS 56 . Obviously, therefore, our results further suggest that PKC (at least in such isotypes as PKC-βII, -δ, and -η) 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-δ-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.

Footnotes

  • ↵1 This work was supported by grants from Korea Science and Engineering Foundation (981-0504-021-2), Korean National Cancer Control Program, Ministry of Health and Welfare (1998), and a grant-in-aid from Korea Research Foundation (1997).

  • ↵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: cdjun{at}wonnms.wonkwang.ac.kr

  • ↵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 July 2, 1998.
  • Accepted December 7, 1998.

References

  1. ↵
    Moncada, S., A. Higgs. 1993. The l-arginine-nitric oxide pathway. N. Engl. J. Med. 329: 2002
    OpenUrlCrossRefPubMed
  2. ↵
    Moncada, S., R. M. J. Palmer, E. A. Higgs. 1989. Biosynthesis of nitric oxide from l-arginine: a pathway for the regulation of cell function and communication. Biochem. Pharmacol. 38: 1709
    OpenUrlCrossRefPubMed
  3. ↵
    Nathan, C.. 1992. Nitric oxide as a secretory product of mammalian cells. FASEB J. 6: 3051
    OpenUrlAbstract
  4. ↵
    Berdeaux, A.. 1993. Nitric oxide: an ubiquitous messenger. Fundam. Clin. Pharmacol. 7: 401
    OpenUrlCrossRefPubMed
  5. ↵
    Lowenstein, C. J., S. H. Snyder. 1992. Nitric oxide, a novel biologic messenger. Cell 70: 705
    OpenUrlCrossRefPubMed
  6. ↵
    Schmidt, H. H. H. W., S. M. Lohmann, U. Walter. 1993. The nitric oxide and cGMP signal transduction system:regulation and mechanism of action. Biochem. Biophys. Acta 1178: 153
    OpenUrlPubMed
  7. ↵
    Corbett, J. A., J. R. Lancaster, Jr, M. A. Sweetland, M. L. McDaniel. 1991. Interleukin-1β-induced formation of EPR-detectable iron-nitrosyl complexes in islets of Langerhans: role of nitric oxide in interleukin-1β-induced inhibition of insulin secretion. J. Biol. Chem. 266: 21351
    OpenUrlAbstract/FREE Full Text
  8. ↵
    McCartney-Francis, N., J. B. Allen, D. E. Mizel, J. E. Albina, Q.-W. Xie, C. F. Nathan, S. M. Wahl. 1993. Suppression of arthritis by an inhibitor of nitric oxide synthase. J. Exp. Med. 178: 749
    OpenUrlAbstract/FREE Full Text
  9. ↵
    Weinberg, J. B., D. L. Granger, D. S. Pisetsky, M. F. Seldin, M. A. Misukonis, S. N. Mason, A. M. Pippen, P. Ruiz, E. R. Wood, G. S. Gilkeson. 1994. The role of nitric oxide in the pathogenesis of spontaneous murine autoimmume disease: increased nitric oxide production and nitric oxide synthase expression in MRL-lpr/lpr mice, and reduction of spontaneous glomerulonephritis and arthritis by orally administered NG-monomethyl-l-arginine. J. Exp. Med. 179: 651
    OpenUrlAbstract/FREE Full Text
  10. ↵
    Brüne, B., E. G. Lapetina. 1990. Properties of a novel nitric oxide-stimulated ADP-ribosyl transferase. Arch. Biochem. Biophys. 279: 286
    OpenUrlCrossRefPubMed
  11. ↵
    Wink, D. A., K. S. Kasprzak, C. M. Maragos, R. K. Elespuru, M. Misra, T. M. Dunams, T. A. Cebula, W. H. Koch, A. W. Andrews, J. S. Allen, L. K. Keefer. 1991. DNA deaminating ability and genotoxicity of nitric oxide and its progenitors. Science 254: 1001
    OpenUrlAbstract/FREE Full Text
  12. ↵
    Stamler, J. S., D. J. Singel, J. Loscalzo. 1992. Biochemistry of nitric oxide and its redox activated forms. Science 258: 1898
    OpenUrlAbstract/FREE Full Text
  13. ↵
    Stamler., J. S.. 1994. Redox signalling: nitrosylation and related target interactions of nitric oxide. Cell 78: 931
    OpenUrlCrossRefPubMed
  14. ↵
    Stamler, J. S., D. I. Simon, J. A. Osborne, M. E. Mullins, O. Jaraki, T. Michel, D. J. Singel, J. Loscalzo. 1992. S-nitrosylation of protein with nitric oxide: synthesis and characterization of biologically active compounds. Proc. Natl. Acad. Sci. USA 89: 444
  15. ↵
    Nathan, C. F., J. B. Hibbs. 1991. Role of nitric oxide synthesis in macrophage antimicrobial activity. Curr. Opin. Immunol. 3: 65
    OpenUrlCrossRefPubMed
  16. ↵
    Karupiah, G., Q.-W. Xie, R. M. L. Buller, C. Nathan, C. Duarte, J. D. MacMicking. 1993. Inhibition of viral replication by interferon-γ-induced nitric oxide synthase. Science 261: 1445
    OpenUrlAbstract/FREE Full Text
  17. ↵
    Albina, J. E., S. Cui, R. B. Mateo, J. S. Reichner. 1993. Nitric oxide-mediated apoptosis in murine peritoneal macrophages. J. Immunol. 150: 5080
    OpenUrlAbstract/FREE Full Text
  18. ↵
    Cui, S., J. S. Reichner, R. B. Mateo, J. E. Albina. 1994. Activated murine macrophages induced apoptosis in tumor cells through nitric oxide-dependent or-independent mechanisms. Cancer Res. 54: 2462
    OpenUrlAbstract/FREE Full Text
  19. ↵
    Messmer, U. K., E. G. Lapetina, B. Brüne. 1995. Nitric oxide-induced apoptosis in RAW 264.7 macrophages in antagonized by protein kinase C- and protein kinase A-activating compounds. Mol. Pharmacol. 47: 757
    OpenUrlAbstract
  20. ↵
    Arends, M. J., A. H. Wyllie. 1991. Apoptosis: mechanisms and roles in pathology. Int. Rev. Exp. Pathol. 32: 223
    OpenUrlCrossRefPubMed
  21. ↵
    Ignarro, L. J.. 1990. Biosynthesis and metabolism of endothelium-derived nitric oxide. Annu. Rev. Pharmacol. Toxicol. 30: 535
    OpenUrlCrossRefPubMed
  22. ↵
    Mohr, S., J. S. Stamler, B. Brüne. 1996. Posttranslational modification of glyceraldehyde-3-phosphate dehydrogenase by S-nitrosylation and subsequent NADH attachment. J. Biol. Chem. 271: 4209
    OpenUrlAbstract/FREE Full Text
  23. ↵
    Karin, M.. 1995. The regulation of AP-1 activity by mitogen-activated protein kinases. J. Biol. Chem. 270: 16483
    OpenUrlFREE Full Text
  24. ↵
    Cobb, M. H., E. J. Goldsmith. 1995. How MAP kinases are regulated. J. Biol. Chem. 270: 14843
    OpenUrlFREE Full Text
  25. ↵
    Hunter, T.. 1997. Oncoprotein networks. Cell 88: 333
    OpenUrlCrossRefPubMed
  26. ↵
    Boulton, T. G., G. D. Yancopoulos, J. S. Gregory, C. Slaughter, C. Moomaw, J. Hsu, M. H. Cobb. 1990. An insulin-stimulated protein kinase similar to yeast kinases involved in cell cycle control. Science 249: 64
    OpenUrlAbstract/FREE Full Text
  27. ↵
    Zhou, G., Z. Q. Bao, J. E. Dixon. 1995. Components of a new human protein kinase signal transduction pathway. J. Biol. Chem. 270: 12665
    OpenUrlAbstract/FREE Full Text
  28. ↵
    Hibi, M., A. Lin, T. Smeal, A. Minden, M. Karin. 1993. Identification of an oncoprotein- and UV-responsive protein kinasse that binds and potentiates the c-Jun activation domain. Genes Dev. 7: 2135
    OpenUrlAbstract/FREE Full Text
  29. ↵
    Derijard, B., M. Hibi, I. H. Wu, T. Barret, B. Su, T. Deng, M. Karin, R. J. Davis. 1994. JNK1: a protein kinase stimulated by UV light and Ha-ras that binds and phosphorylates the c-Jun activation domain. Cell 76: 1025
    OpenUrlCrossRefPubMed
  30. ↵
    Kyriakis, J. M., P. Banerjee, E. Nikolakaki, T. Dai, E. A. Rubie, M. F. Ahmad, J. Avruch, J. R. Woodgett. 1994. The stress-activated protein kinase subfamily of c-Jun kinases. Nature 369: 156
    OpenUrlCrossRefPubMed
  31. ↵
    Han, J., J. D. Lee, L. Bibbs, R. J. Ulevitch. 1994. A MAP kinase targeted by endotoxin and hyperosmorality in mammalian cells. Science 265: 808
    OpenUrlAbstract/FREE Full Text
  32. ↵
    Rouse, J., P. Cohen, S. Trigon, M. Morange, A. Alonso-Llamazares, D. Zamanilo, T. Hunt, A. R. Nebreda. 1994. A novel kinase cascade triggered by stress and heat shock that stimulates MAPKAP kinase-2 and phosphorylation of the small heat shock protein. Cell 78: 1027
    OpenUrlCrossRefPubMed
  33. ↵
    Lee, J. C., J. T. Laydon, P. C. McDonnell, T. F. Gallagher, S. Kumar, D. Green, D. McNulty, M. J. Blumenthal, J. R. Heys, S. W. Landvatter, et al 1994. A protein kinase involved in the regulation of inflammatory cytokine biosynthesis. Nature 372: 739
    OpenUrlCrossRefPubMed
  34. ↵
    Cano, E., L. C. Mahadevan. 1995. Parallel signal processing among mammalian MAPKs. Trends Biochem. Sci. 20: 117
    OpenUrlCrossRefPubMed
  35. ↵
    Lander, H. M., A. T. Jacovina, R. J. Davis, J. M. Tauras. 1996. Differential activation of mitogen-activated protein kinase by nitric oxide-related species. J. Biol. Chem. 271: 19705
    OpenUrlAbstract/FREE Full Text
  36. ↵
    Park, D. S., L. Stefanis, C. Y. I. Yan, S. E. Farinelli, L. A. Greene. 1996. Ordering the cell death pathway: differential effects of BCL2, an interleukin-1-converting enzyme family protease inhibitor, and other survival agents on JNK activation in serum/nerve growth factor-deprived PC12 cells. J. Biol. Chem. 271: 21898
    OpenUrlAbstract/FREE Full Text
  37. ↵
    Messmer, U. K., B. Brüne. 1997. Attenuation of p53 expression and Bax down-regulation during phorbol ester mediated inhibition of apoptosis. Br. J. Pharmacol. 121: 625
    OpenUrlCrossRefPubMed
  38. ↵
    Goodnight, A., H. Mischack, W. Kolch, J. F. Mushinski. 1995. Immunocytochemical localization of eight protein kinase C isozymes overexpressed in NIH3T3 fibroblasts: isoform-specific association with microfilaments, Golgi, endoplasmic reticulum, and nuclear and cell membranes. J. Biol. Chem. 270: 9910
    OpenUrl
  39. ↵
    Chun, J.-S., M.-J. Ha, B. S. Jacobson. 1996. Differential translocation of protein kinase C-δ and -ε during HeLa cell adhesion to a gelatin substratum. J. Biol. Chem. 271: 13008
    OpenUrlAbstract/FREE Full Text
  40. ↵
    Mischack, H., J. Goodnight, W. Kolch, G. Martiny-Baron, C. Schaechtle, M. G. Kazanietz, P. M. Blumberg, J. H. Pierce, J. F. Mushinski. 1993. Overexpression of protein kinase C-δ and -ε in NIH3T3 cells induces opposite effects on growth, morphology, anchorage dependence, and tumorigenicity. J. Biol. Chem. 268: 6090
    OpenUrlAbstract/FREE Full Text
  41. ↵
    Sutherland, C. L., A. W. Heath, S. L. Pelech, P. R. Young, M. R. Gold. 1996. Differential activation of the ERK, JNK, and p38 mitogen-activated protein kinases by CD40 and the B cell antigen receptor. J. Immunol. 157: 3381
    OpenUrlAbstract
  42. ↵
    Wright, S. C., U. Schellenberger, H. Wang, D. H. Kinder, J. W. Talhouk, J. W. Larrick. 1997. Activation of CPP32-like proteases is not sufficient to trigger apoptosis: inhibition of apoptosis by agents that suppress activation of AP24, but not CPP32-like activity. J. Exp. Med. 186: 1107
    OpenUrlAbstract/FREE Full Text
  43. ↵
    Maciejewski, J. P., C. Selleri, T. Sato, H. J. Cho, H. J. Keefer, C. F. Nathan. 1995. Nitric oxide supression of human hematopoiesis in vitro. contribution to inhibitory action of interferon-γ and tumor necrosis factor-α. J. Clin. Invest. 96: 1085
  44. ↵
    Schwenger, P., P. Bellosta, I. Vietor, C. Basilico, E. Y. Skolink, J. Vilcek. 1997. Sodium salicylate induces apoptosis via p38 mitogen-activated protein kinase but inhibits tumor necrosis factor-induced c-Jun N-terminal kinase/stress-activated protein kinase activation. Proc. Natl. Acad. Sci. USA 94: 2869
    OpenUrlAbstract/FREE Full Text
  45. ↵
    Bitangcol, J. C., A. S. Chau, E. Stadnick, M. J. Lohka, B. Dicken, E. K. Shibuya. 1998. Activation of the p42 mitogen-activated protein kinase pathway inhibit Cdc 2 activation and entry into M-phase in cycling Xenopus egg extracts. Mol. Biol. Cell. 9: 451
    OpenUrlAbstract/FREE Full Text
  46. ↵
    Jun, C. D., B. M. Choi, S. Y. Lee, S. S. Kang, H. M. Kim, H. T. Chung. 1994. Nitric oxide inhibits the expression of protein kinase C δ gene in the murine peritoneal macrophages. Biochem. Biophys. Res. Commun. 204: 105
    OpenUrlCrossRefPubMed
  47. ↵
    Nicholson, D. W., A. Ali, N. A. Thornberry, J. P. Vailancourt, C. K. Ding, M. Gallant, Y. Gereau, P. R. Griffin, M. Lavelle, Y. A. Lazebnik. 1995. Identification and inhibition of the ICE/CED-3 protease necessary for mammalian apoptosis. Nature 376: 37
    OpenUrlCrossRefPubMed
  48. ↵
    Schlegel, J., I. Peters, S. Orrenius, D. K. Miller, N. A. Thornberry, T. T. Yamin, D. W. Nicholson. 1996. CPP32/apopain is a key interleukin 1β converting enzyme-like protease involved in Fas-mediated apoptosis. J. Biol. Chem. 271: 1841
    OpenUrlAbstract/FREE Full Text
  49. ↵
    Jacobson, M. D., M. Weil, M. C. Raff. 1996. Role of Ced-3/ICE-family protease in staurosporine-induced programmed cell death. J. Cell. Biol. 133: 1041
    OpenUrlAbstract
  50. ↵
    McConkey, D. J., P. Hartzell, M. Jonda, S. Orrenius. 1989. Inhibition of DNA fragmentation in thymocytes and isolated thymocyte nuclei by agents that stimulate protein kinase C. J. Biol. Chem. 264: 13399
    OpenUrlAbstract/FREE Full Text
  51. ↵
    Haimovitz-Friedman, A., C. C. Kan, D. Ehleiter, R. S. Persaud, M. McLoughlin, Z. Fuks, R. N. Kolesnik. 1994. Ionizing radiation acts on cellular membranes to generate ceramide and initiate apoptosis. J. Exp. Med. 180: 525
    OpenUrlAbstract/FREE Full Text
  52. ↵
    Obeid, L. M., C. M. Linardic, L. A. Karolak, Y. A. Hannun. 1993. Programmed cell death induced by ceramide. Science 259: 1769
    OpenUrlAbstract/FREE Full Text
  53. ↵
    Gubina, E., M. S. Rinaudo, Z. Szallasi, P. M. Blumberg, R. A. Mufson. 1998. Overexpression of protein kinase C isoform epsilon but not delta in human interleukin-3-dependent cells: supresses apoptosis and induced bcl-2 expression. Blood 91: 823
    OpenUrlAbstract/FREE Full Text
  54. ↵
    Murray, N. R., A. P. Field. 1997. A typical protein kinase C ι protects human leukemic cells against drug-induced apoptosis. J. Biol. Chem. 272: 27521
    OpenUrlAbstract/FREE Full Text
  55. ↵
    Jun, C. D., S. J. Park, B. M. Choi, H. J. Kwak, Y. C. Park, M. S. Kim, R. K. Park, H. T. Chung. 1997. Potentiation of the activity of nitric oxide by the protein kinase C activator phorbol ester in human myeloid leukemic HL-60 cells: association with enhanced fragmentation of mature genomic DNA. Cell. Immunol. 176: 41
    OpenUrlCrossRefPubMed
  56. ↵
    Díaz-Guerra, M. J. M., O. G. Bodelón, M. Velasco, R. Whelan, P. J. Parker, L. Boscá. 1996. Up-regulation of protein kinase C-ε promotes the expression of cytokine-inducible nitric oxide synthase in RAW 264.7 cells. J. Biol. Chem. 271: 32028
    OpenUrlAbstract/FREE Full Text
Back to top

In this issue

The Journal of Immunology: 162 (6)
The Journal of Immunology
Vol. 162, Issue 6
15 Mar 1999
Print
Download PDF
Article Alerts
Email Article
Citation Tools

Jump to section