Activation of p38 and c-Jun N-terminal kinase pathways and induction of apoptosis by chelerythrine do not require inhibition of protein kinase C.

Chelerythrine, a natural benzophenanthridine alkaloid, has been reported to mediate a variety of biological activities, including inhibition of protein kinase C (PKC). Here we report that chelerythrine induced time- and dose-dependent activation of JNK1 and p38 in HeLa cells, which was mediated the upstream kinases, MEKK1 and MKK4. However, treatment with two other potent and selective PKC inhibitors, GF-109203X and Gö6983, or down-regulation of PKC activity by prolonged treatment with phorbol 12-myristate 13-acetate had no effect on JNK1 and p38 activities. Furthermore, under the conditions where JNK1 and p38 were activated, we did not observe any significant inhibitory effect of chelerythrine on the activities of PKC isozymes present in HeLa cells. Interestingly, pretreatment with the antioxidants, N-acetyl-L-cysteine, dithiothreitol, and glutathione, impaired chelerythrine-induced JNK1 and p38 activation. In addition, chelerythrine induced apoptosis that was blocked by the antioxidants and the dominant-negative mutants of MEKK1, MKK4, JNK1, and p38. Together, these results uncover a novel biochemical property of chelerythrine, i.e. activation of MEKK1- and MKK4-dependent JNK1 and p38 pathways through an oxidative stress mechanism, which mediate the induction of apoptosis, but are independent of PKC inhibition.

activated by the dual phosphorylation on Tyr and Thr residues by a member of MEKs or MKKs, which, in turn, are activated by an upstream MEKK. To date, at least six MAPK members have been identified in mammalian cells. Three of them have been extensively studied: the extracellular signal-regulated protein kinases (ERKs) (5), the c-Jun N-terminal kinases (JNKs, also referred to as stress-activated protein kinases) (6,7), and the p38s (8). The ERK pathway is predominantly activated by mitogens through a Ras-dependent mechanism (9), and it is required for cell proliferation and differentiation (10). Unlike ERK, however, JNK and p38 are regulated by distinct MAPK modules and are preferentially activated by pro-inflammatory cytokines (11) and various environmental stresses such as UV light (6), ␥-irradiation (12), DNA-damaging agents (13), protein synthesis inhibitors (14), heat shock (15), osmotic shock (16), and oxidative stresses (17). Activation of JNK and p38 has been implicated in the regulation of a variety of cellular processes such as T cell activation (18), production of cytokines (19), cell differentiation (20), and apoptotic cell death (21,22).
Protein kinase C (PKC) represents a family of serine/threonine kinases, which presently consists of at least 11 isozymes (23,24). According to their structure and cofactor regulation, these isozymes have been classified into three groups as follows: (i) conventional PKCs (␣, ␤I, ␤II, and ␥) that are activated by calcium or diacylglycerol; (ii) novel PKCs (␦, ⑀, , , and ) that are independent of calcium but responsive to diacylglycerol; (iii) atypical PKCs ( and /) that do not require either calcium or diacylglycerol for activation. A large number of studies indicate that PKC serves as a central signaling molecule and regulates a variety of cellular processes either directly or through integration with other signaling pathways (23). PKC has been shown to activate Ras-MAPK pathway, leading to cell proliferation (25). Activation of a certain PKC isozyme has also been shown to regulate T cell activation through a JNK-dependent pathway (18). Recently, PKC has been demonstrated to prevent apoptosis in different cell lines, presumably by increasing phosphorylation of Bcl-2 (26,27). Consistent with this notion, inhibition of PKC has been shown to result in apoptosis (28,29).
Chelerythrine is a natural benzophenanthridine alkaloid that displays a wide range of biological activities such as antiplatelet (30), anti-inflammatory (31), and antibacterial effects (31). Most notably, chelerythrine exhibits anti-tumor property (32) and induces apoptosis in murine lymphoma (33) and human leukemia (28,29). Thus, chelerythrine has the potential for drug development against cancers. Since chelerythrine was described previously to be an inhibitor of PKC (32), the biological effects of chelerythrine have been presumably ascribed to the inhibition of PKC; however, a number of recent studies indicate that PKC-independent mechanisms are involved (34 -* This work was supported in part by National Institutes of Health Grants R01-CA73647 (to A.-N. T. K.) and R01-AI38649 (to T.-H. T.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Immunocomplex Kinase Assays of MAPK, SEK1, and MEKK1 Activities-After treatments, cells were washed twice with ice-cold phosphate-buffered saline (PBS) and harvested in cell lysis buffer containing 10 mM Tris-HCl (pH 7.1), 50 mM NaCl, 50 mM NaF, 30 mM Na 4 P 2 O 7 , 100 M Na 3 VO 4 , 5 M ZnCl 2 , 2 mM iodoacetic acid, 1 mM phenylmethylsulfonyl fluoride, and 0.5% Triton X-100. Cell lysates were homogenized by passing through a 23-gauge needle three times. The homogenates were left on ice for 30 min before centrifugation at 12,500 ϫ g for 15 min at 4°C. Kinase activities of JNK1, p38, ERK2, SEK1, and MEKK1 in the supernatants were determined by in vitro immunocomplex kinase assays as described previously (39). Briefly, an equal amount of protein, as measured by Bradford assays (Bio-Rad), was incubated with the indicated antibodies for 2 h at 4°C in the presence of protein A-Sepharose 4B conjugate (Zymed Laboratories Inc. Laboratories, San Francisco, CA). The immunocomplex was spun down at high speed for 1 min and washed twice with the lysis buffer and twice with kinase assay buffer (20 mM HEPES at pH 7.5, 10 mM MgCl 2 , 2 mM MnCl 2 , 0.1 mM Na 3 VO 4 , 50 mM ␤-glycerophosphate, and 10 mM -nitrophenyl phosphate). The immunoprecipitates were resuspended in a 30-l kinase assay buffer with addition of 2 Ci of [␥-32 P]ATP, 20 M ATP, and 5 g of the indicated substrates. Kinase reaction was performed at 30°C for 30 min in JNK1, p38, SEK1, and MEKK1 assays, or 15 min in ERK2 assay, and then terminated with 10 l of 4ϫ Laemmli's buffer. The reaction mixtures were heated to 95°C for 5 min and resolved by SDS-polyacrylamide gel electrophoresis (PAGE) analysis. The gel was stained with Coomassie Blue, washed overnight, and dried. The phosphorylated products were visualized by autoradiography and quantitated with a PhosphorImager (AMBIS, Inc., San Diego, CA).
PKC Preparations and Activity Assays-After washing twice with ice-cold PBS, cells were harvested in a buffer containing 20 mM Tris-HCl (pH 7.5), 1 mM EDTA, 1 mM EGTA, 150 mM NaCl, 10 mM NaF, 100 M Na 3 VO 4 , 2 mM DTT, 1 mM phenylmethylsulfonyl fluoride, 10 g/ml aprotinin, 10 g/ml leupetin, and 1% Triton® X-100. Cell lysates were homogenized by passing through a 23-gauge needle four times and left on ice for 30 min before centrifugation at 12,500 ϫ g for 20 min at 4°C. The total PKC activity in the resulting supernatants was isolated by DEAE-cellulose chromatography as described previously (40). Briefly, samples were applied to a 0.5-ml DEAE-cellulose column equilibrated with buffer A (20 mM Tris-HCl (pH 7.5), 1 mM EDTA, 1 mM EGTA, and 2 mM DTT). After washing column with 2 ml of buffer A, PKCs were eluted with 0.2 M NaCl, and protein concentration was determined by Bradford assays (Bio-Rad). For PKC activity assay, 2 g of isolated enzymes was incubated with 10 g of histone H1 in a 50-l assay buffer containing 20 mM Tris-HCl (pH 7.5), 10 mM MgCl 2 , 0.5 mM CaCl 2 , 2 Ci of [␥-32 P]ATP, 20 M ATP. The kinase reaction was performed at 30°C for 10 min and terminated with Laemmli's buffer. The phosphorylated histone H1 was separated by SDS-PAGE and visualized by autoradiography.
To assay the individual PKC isozyme activity, different isozymes were immunoprecipitated directly from cell lysates with their respective antibodies as described previously (41). The immunoprecipitates were washed four times with a buffer containing 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, and 1% Triton X-100. PKC activity was analyzed by electrophoresis as described above and quantitated with a PhosphorImager (AMBIS, Inc., San Diego, CA). The results were expressed as a percentage, relative to solvent-treated controls.
Flow Cytometric Analysis of Intracellular Peroxides-After treatment with different agents, H 2 DCFDA (5 M) was added to the medium and incubated at 37°C for 30 min as described previously (39). Cells were washed with ice-cold PBS and scraped off the plate. Following centrifugation at 1000 ϫ g for 5 min, cell pellets were resuspended in PBS containing 2% fetal bovine serum and 5 M H 2 DCFDA. The fluorescence intensities of H 2 DCFDA of more than 10,000 viable cells from each sample were analyzed by using a Becton Dickinson FACScan flow cytometer with excitation and emission settings of 488 and 525 nm, respectively. Prior to data collection, propidium iodide was added to the sample to gate out dead cells.
Nuclear Staining Assays-After treatments, floating cells were collected by centrifugation at 2,000 ϫ g for 15 min, and attached cells were first trypsinized and then harvested by centrifugation. Apoptotic cells with condensed or fragmented nuclei were visualized by DAPI staining as described previously (42). Briefly, cells were washed once with icecold PBS before fixing in a solution of methanol:acetic acid (3:1) for 30 min. Fixed cells were placed on slides. After evaporation of fixing solution, cells were stained with 1 g/ml DAPI for 15 min. The nuclear morphology of cells was examined by a fluorescence microscopy.
DNA Fragmentation Assays-Approximately 10 7 cells were lysed in a buffer containing 10 mM Tris (pH 7.4), 150 mM NaCl, 5 mM EDTA, and 0.5% Triton® X-100 for 30 min on ice. Cell lysates were vortexed and cleared by centrifugation at 12,500 ϫ g for 20 min. DNA in supernatant was extracted with an equal volume of neutral phenol:chloroform:isoamyl alcohol mixture (25:24:1) at room temperature for 15 min and precipitated with 2 volumes of 100% ethanol and one-tenth volume of 0.3 M sodium acetate (pH 5.2) overnight at Ϫ20°C. The DNA precipitates were spun down at 12,500 ϫ g for 20 min and washed once with 70% ethanol. The air-dried DNA pellets were incubated with 5 g/ml DNase-free RNase in a 40-l Tris-EDTA buffer (pH 8.0) at 37°C for 2 h. Fragmented DNA was resolved on 1.5% agarose gels in the presence of 0.5 g/ml ethidium bromide.
Transient Transfection Death Assays-HeLa cells were plated in 6-well plates (5 ϫ 10 4 cells/well) 24 h before transfection. Cells were transfected with a ␤-galactosidase-expressing reporter construct together with an empty vector or the plasmids as indicated using calcium phosphate precipitation method. Transfection mixture was removed 12 h after transfection. Cells were cultured in fresh medium for 12 h before treatment with chelerythrine. Cells were stained with 5-bromo-4-chloro-3-indoxyl-␤-D-galactopyranoside (X-gal). Result was expressed as the percentage of round blue cells among the ␤-galactosidase-positive cells.
Western Blot Analysis of JNK, p38, and PKC Isozymes-After treatment, cell lysates were prepared as described above. Twenty five g of total protein, as determined by Bradford assay, was resolved on 10% SDS-PAGE and transferred to polyvinylidene difluoride membrane using a semi-dry transfer system (Fisher). The membrane was blocked with 5% non-fat dry milk in TBS (20 mM Tris-HCl (pH 7.4), 8 g/liter NaCl, and 0.2 g/liter KCl) for 1 h at room temperature. The membrane was then incubated with 1 g/ml of the indicated primary antibodies in TBS containing 3% non-fat milk at 4°C overnight. Membrane was washed three times with TBS and blotted with horseradish peroxidaseconjugated secondary antibodies at room temperature for 1 h. After washing three times with TBS, the protein was visualized using the ECL system (Amersham Pharmacia Biotech).

RESULTS
Chelerythrine Stimulates Activities of JNK1 and p38 but Not ERK2-Chelerythrine has been reported to inhibit PKC activity with an IC 50 of 0.66 M (32). To examine the effect of chelerythrine on JNK1 activity, we treated HeLa cells with the concentrations of chelerythrine around this IC 50 . Cells were collected 2 h after treatment, and the endogenous JNK1 activity was determined by immunocomplex kinase assays. As shown in Fig. 1A, chelerythrine strongly stimulated JNK1 activity. The activation of JNK1 appeared at 1 M and was greatly enhanced with the increase of chelerythrine concentrations. Approximately a 9-fold induction of JNK1 activity was seen at a concentration of 2.5 M as compared with untreated control cells. To characterize further JNK1 activation by chelerythrine, we studied the time course. Induction of JNK1 activity became evident 30 min after treatment with 2.5 M chelerythrine and reached the maximum at 2 h (Fig. 1B). The induced JNK1 activity decreased and returned to basal levels 4 h post-treatment (Fig. 1B). Western blotting analysis revealed no change in the protein levels of JNK1 throughout dose-response and time course studies (Fig. 1, A and B), indicating that JNK1 activation by chelerythrine was not due to de novo protein synthesis.
Although different members of MAPKs are often responsive to different extracellular signals, they can also be activated by the same stimuli, e.g. proinflammatory cytokines and oxidative stresses induced JNK, p38, and ERK kinase activities concom-itantly (11,17,43). We therefore measured the activities of p38 and ERK2 following treatment of HeLa cells with various concentrations of chelerythrine. Similar to JNK1, p38 activity was strongly induced by chelerythrine in a dose-dependent manner (Fig. 1C). Western blotting showed that such induction was not due to the increase of protein levels of p38 (Fig. 1C). When ERK2 activity was measured, a strong induction was detected in PMA-treated cells but not in chelerythrine-treated cells (Fig.  1D). To exclude the possibility that ERK2 activity was measured at an unfavorable time point after treatment with chelerythrine, we also performed a time course study. But no ERK2 activation was observed (data not shown). These results revealed that chelerythrine preferentially induced JNK1 and p38 activation, but had no effect ERK2 activity.
Chelerythrine-induced JNK1 and p38 Activation Is Mediated by MEKK1 and MKK4 -Because incubation of chelerythrine with the immunoprecipitated JNK1 or p38 did not stimulate their kinase activities (data not shown), we speculate that there is the involvement of upstream kinases. Previous studies have shown that activation of JNK by various stimuli is mediated by MEKK1. We therefore examined the effects of chelerythrine on MEKK1 activity. HeLa cells were treated with chelerythrine (2.5 M) for the indicated times. Endogenous MEKK1 was immunoprecipitated with a specific antibody, and kinase activity was assayed with a kinase-inactive GST-SEK1 fusion protein as substrate. Chelerythrine induced MEKK1 activation in a time-dependent fashion ( Fig. 2A). The stimulated MEKK1 activity was detected as early as 15 min after treatment, preceding JNK1 activation shown in Fig. 1B.
Because MEKK1 is unable to phosphorylate JNK directly, we next examined the activation of MKK4 or SEK1, a kinase that has been shown to interact specifically with MEKK1 and activate JNK1 (44). As shown in Fig. 2B, chelerythrine also stimulated MKK4/SEK1 kinase activity, which appeared at 15 min and peaked 1 h after chelerythrine treatment. Thus, both MEKK1 and MKK4 are activated upon treatment with chelerythrine. Furthermore, cotransfection with a dominant-negative mutant MEKK1(KR) or MKK4(ala) impaired chelerythrine-induced JNK1 activation (Fig. 2C) as well as p38 activation (Fig. 2D). These results provide strong evidence that the upstream kinases, MEKK1 and MKK4, mediate the activation of JNK1 and p38 by chelerythrine.
Treatment with Other PKC Inhibitors or Down-regulation of PKC with PMA Does Not Affect JNK and p38 Activation by Chelerythrine-Chelerythrine has been described to be an inhibitor of PKC (32) and also strongly activated JNK and p38 as demonstrated above. We wondered whether inhibition of PKC might serve as a general signal to initiate JNK or p38 pathway. To test this, we examined the effects of two other potent PKC inhibitors, GF-109203X and Gö6983. Previous studies have shown that GF-109203X selectively inhibits the isozymes, ␣, ␤, ␥, ␦, and ⑀ (45), and Gö6983 selectively inhibits ␣, ␤, ␥, ␦, and PKC isozymes (46). Unlike chelerythrine, treatment of HeLa cells with GF-109203X or Gö6983 did not stimulate JNK1 activity (Fig. 3A). Furthermore, pretreatment with these two inhibitors had little effect on chelerythrine-induced JNK1 activation (Fig. 3B). We also examined the effect of down-regulation of PKC on JNK1 activity. HeLa cells were pretreated with PMA (100 nM) for 36 h, prior to challenge with chelerythrine for different times. The result in Fig. 3C shows that prolonged treatment with PMA had no effect on JNK1 activity and did not affect chelerythrine-induced JNK1 activation either. Similar results were obtained when p38 activity was measured (Fig.  3D). These data indicate that inhibition of PKC does not necessarily leads to JNK or p38 activation and raise the possibility that chelerythrine-induced JNK1 and p38 activation is inde- Chelerythrine Does Not Inhibit PKC Activity at Concentrations That Activate JNK1 and p38 -To define further the role of PKC in chelerythrine-induced JNK and p38 activation, we examined the effect of chelerythrine on PKC activity. GF-109203X and Gö6983 were also included in this experiment. As expected, GF-109203X and Gö6983 inhibited PKC activity when added to cell culture medium (Fig. 4A); however, at the concentrations that strongly induced JNK1 and p38 activation, chelerythrine did not show any inhibitory effect on PKC activity. To exclude the possibility that the lack of inhibitory effect of chelerythrine is due to the dissociation of the compound from PKC during the isolation procedure, we conducted the in vitro assays. Total PKC was isolated from HeLa cells and incubated with different concentrations of agents tested. PKC activity was determined by the phosphorylation of histone H1. As shown in Fig. 4B, GF-109203X and Gö6983 showed strong inhibition on PKC activity, whereas the effect of chelerythrine was dose-dependent. No significant inhibition was observed for chelerythrine at the concentrations less than 25 M. Similar results were obtained when PKC activity was assayed in the presence of activators, phosphatidylserine and diolein (data not shown).
Considering the diversity and the different natures of PKC isozymes, we further measured the effect of chelerythrine on individual isozyme activity. Western blotting showed that HeLa cells expressed six PKC isozymes, ␣, ␥, ␦, ⑀, , and (or , a human homologue of ), and prolonged treatment with PMA (100 nM, for 36 h) dramatically down-regulated conventional PKCs (␣ and ␥) and novel PKCs (␦ and ⑀) but had no effect on atypical PKCs ( and /) (Fig. 4C). Incubation of chelerythrine (5 M) with these isozymes immunoprecipitated from cell lysates did not significantly change their kinase activities as compared with solvent control (Fig. 4D). Based on these and the results described above, we conclude that chelerythrineinduced JNK1 and p38 activation does not require the inhibition of PKC. Chelerythrine-induced JNK1 and p38 Activation Is Inhibited by Antioxidants-Lack of effect of chelerythrine on PKC raises the question as to the initial signal that led to the activation of MEKK1 3 MKK4 3 JNK module. The iminium bond of chelerythrine is known to be reactive to cellular thiols including GSH and sulfhydryl group of protein (47). As a result, treatment with chelerythrine may cause depletion of intracellular GSH pool and/or protein thiols. Since GSH is a primary antioxidant responsible for the removal of extra peroxides and the maintenance of cellular redox status, depletion of GSH by chelerythrine will cause accumulation of peroxides and thereby generate oxidative stress. To test this hypothesis, we measured the peroxide levels in chelerythrine-treated cells using a peroxide-sensitive fluorescent dye, H 2 DCFDA. This chemical is freely permeable to cells. Once inside the cells, it is hydrolyzed to H 2 DCF and trapped in the cells. In the presence of peroxides, especially hydrogen peroxide (H 2 O 2 ), H 2 DCF is oxidized to fluorescent DCF. FACScan analysis revealed that cheleryth- rine treatment caused the increase of intracellular peroxides as compared with control cells (0.1% Me 2 SO-treated cells) (Fig.  5A). Addition of aminotriazole, a catalase inhibitor, slightly enhanced the chelerythrine-induced accumulation of peroxides. H 2 O 2 (1 mM) was included as a positive control (Fig. 5A).
After demonstrating the oxidative stress induced by chelerythrine, we next studied the effects of pretreatment of cells with antioxidants, NAC, DTT, and GSH prior to chelerythrine stimulation. Activation of JNK1 by chelerythrine was inhibited by NAC, DTT, and GSH pretreatment (Fig. 5B). Similarly, p38 activation was blocked in the presence of these antioxidants (Fig. 5C). Treatment with the antioxidants alone did not affect the JNK1 and p38 activity over the time period of experiments (data not shown). These results suggest that induction of oxidative stress plays a crucial role in chelerythrine-induced JNK1 and p38 activation.
Chelerythrine Induces Apoptosis in HeLa cells, Which Is Prevented by Antioxidants-Previous studies show that chelerythrine induced apoptosis in leukemia and lymphoma cells (28,33). To address the biological significance of chelerythrineinduced JNK1 and p38 activation, we examined their roles in apoptosis. As shown in Fig. 6A (bottom panel), HeLa cells after treatment with chelerythrine (5 M) for 24 h showed extensive nuclear condensation or fragmentation when stained with a fluorescent DNA-binding agent, DAPI, indicative of apoptotic cell death. In contrast, control cells (0.1% Me 2 SO-treated cells) showed normal nuclear morphology (Fig. 6A, top panel). Chelerythrine-induced apoptosis was further confirmed by DNA laddering, which showed a dose-dependent effect (Fig. 6B).
To provide primary evidence for the role of JNK1 or p38 in the induction of apoptosis by chelerythrine, we examined the effects of antioxidants, NAC, DTT, and GSH, which have been shown above to inhibit chelerythrine-induced JNK and p38 activation. Pretreatment with these antioxidants significantly inhibited chelerythrine-induced apoptosis as determined by DAPI staining (Fig. 6C) After transfection, cells were either treated with chelerythrine or with solvent (0.1% Me 2 SO) as control. To ensure that the majority of cells were attached to the plates after chelerythrine treatment, a suitable treatment time period was determined from preliminary experiments. Following treatment with chelerythrine (5 M for 12 h), the cells were stained with X-gal to examine ␤-galactosidase-expressing cells (blue in color) (Fig.  7A). Cells transfected with empty vector and treated with solvent had normal appearance (a in Fig. 7A). When treated with chelerythrine, most of cells including the transfected (blue color) and the untransfected (light color) assumed a characteristic apoptotic appearance (round shape shown in b of Fig. 7A). However, the majority of cells transfected with MEKK1(KR) (c in Fig. 7A), MKK4(ala) (d in Fig. 7A), JNK1(APF) (e in Fig. 7A), or p38(AGF) (f in Fig. 7A) showed normal shape in the presence of chelerythrine, whereas most of untransfected cells (light color) in these plates showed apoptotic morphology.
To quantitate apoptosis, the number of blue cells and their morphology were determined. The results were expressed as the percentage of round blue cells (apoptotic cells) as factor of total blue cells (22). As shown in Fig. 7B, expression of dominant-negative mutants of MEKK1 and MKK4 substantially inhibited chelerythrine-induced apoptosis. Although expression of a dominant-negative mutant of JNK or p38 alone showed less inhibition, transfection with both mutants together provided a pronounced protective effect against chelerythrine-induced cell death and even more effective than a dominant-negative mutant of MEKK1 or MKK4. This result suggests that activation of both JNK and p38 is required for the full induction of apoptosis by chelerythrine. DISCUSSION PKC plays an important role in signal transduction (23,48). In addition to its normal functions in various cellular proc-esses, activation of PKC has also been implicated in the pathogenesis of diseases, especially the role in phorbol ester-promoted tumorigenesis (49). It is therefore of considerable interest to identify or develop PKC inhibitors. By using the PKC isozymes isolated from rat brain, chelerythrine was shown to be a competitive inhibitor with respect to phosphate acceptor (histone IIIs) and a non-competitive inhibitor with respect to ATP. Furthermore, chelerythrine did not inhibit other kinases, such as tyrosine protein kinase, cAMP-dependent protein kinase, and Ca 2ϩ /calmodulin-dependent protein kinase with great efficacy (32). Accordingly, chelerythrine has been used as a specific PKC inhibitor in the studies aiming at elucidation of the roles of PKC in various cellular functions. However, by using chelerythrine, a number of studies have obtained the results that would not be anticipated by an inhibitor of PKC (34,35,38  mental conditions published previously (32). In contrast to the previous results, they found that chelerythrine did not show a potent inhibitory effect on PKC activity, regardless of the sources of PKC and the presence of different activators. In addition, chelerythrine did not affect phorbol 12,13-dibutyrate binding to the regulatory domain of PKC, and no significant alteration of PKC-␣, -␤, -␥ translocation was observed in human leukemia (HL-60) cells. These results strongly suggest that a mechanism independent of PKC should be considered as responsible for the biological activities of chelerythrine. In the present study, we demonstrated that chelerythrine activated JNK1 and p38, but not ERK2, pathways. The activation of p38 and JNK1 by chelerythrine was independent of PKC inhibition as evidenced by the following: (i) chelerythrine-induced JNK1 and p38 activation was not affected by the pretreatment with GF-109203X and Gö6983 that substantially reduced the PKC activity in HeLa cells; (ii) a prolonged treatment with PMA almost completely down-regulated cPKC and nPKC isozymes in HeLa cells but had little effect on chelerythrine-induced JNK1 and p38 activation; (iii) chelerythrine did not inhibit PKC activity present in HeLa cells at the concentrations that stimulated the activities of p38 and JNK1. Thus, activation of JNK1 and p38 pathways as shown in this study represents a novel biochemical property of chelerythrine. JNK pathway is regulated by the upstream MAPKKKs (50,51). Genetic and biochemical studies have identified a number of such kinases (52), among which MEKK1 has been shown to play a major role in JNK activation by various stimuli such as growth factors (53), microtubule disruption (54), and alkylating agents (55). The stimulated MEKK1 phosphorylates and activates MKK4, which, in turn, activates JNK (56). However, the role of MEKK1 in p38 activation appears to be controversial. Several studies demonstrated that overexpression of MEKK1 induced marked activation of JNK but not p38 (56 -58), whereas others showed that overexpressing MEKK1 can phosphorylate and activate p38 (59,60). Our results suggest that MEKK1 mediated both JNK1 and p38 activation by chelerythrine, because MEKK1 activity was stimulated in chelerythrinetreated cells and blocking this induced MEKK1 activity with its dominant-negative mutant attenuated JNK1 and p38 activation. The role of MEKK1 in chelerythrine-induced JNK1 and p38 activation is further supported by the activation of MKK4 and the inhibitory effect of its dominant-negative mutant on JNK1 and p38 activation. However, it is worthy to note that, when overexpressed at similar levels, the dominant-negative mutants of MEKK1 and MKK4 showed much stronger inhibitory effect on JNK1 activation than that on p38 activation by chelerythrine, suggesting that additional signaling pathways may exist in regulating p38 activity. Indeed, certain upstream MAPKKKs have been identified as components of p38 signaling pathways. These MAPKKKs include TAK1 (61) and ASK1 (62) that directly phosphorylate and activate MKK3 and/or MKK6, leading to the activation of p38. It should be also noted that the dominant-negative mutant of MEKK1 or MKK4 may interact with several partners and hence may block more than one of the parallel MAPK pathways. Thus, whether other related kinases are involved in chelerythrine-induced p38 and JNK activation remains to be studied.
The lack of effect of chelerythrine on PKC activity raises the questions as to the initial signals that lead to the activation of JNK1 and p38 pathways. As noted above, chelerythrine-induced JNK1 and p38 activation was suppressed by the antioxidants, NAC, GSH, and DTT, implicating a role of oxidative stress. However, the mechanisms by which chelerythrine-induced oxidative stress activates JNK1 and p38 pathways are not clear. Since iminium bond of chelerythrine is reactive to cellular thiols, including protein sulfhydryl groups, treatment with chelerythrine may cause covalent modification of one or multiple upstream components of JNK and p38 pathways, resulting in their activation. Several apical proteins in JNK and p38 pathways have been identified. These proteins include the Rac1 (63), Cdc42 (64), and/or Ras (65). Interestingly, overexpression of a dominant-negative mutant of Rac1, Cdc42, or Ras had no significant effect on chelerythrine induction of JNK and p38 (data not shown). Alternatively, chelerythrine may inhibit a specific phosphatase responsible for down-regulation of one or multiple activated upstream protein kinases. Inhibition of a constitutive dual specificity JNK phosphatase has been recently implicated in the activation of JNK and p38 by arsenite (66), an oxidant that primarily attacks protein sulfhydryls. Since chelerythrine-stimulated JNK1 and p38 activities require upstream kinases, MEKK1 and MKK4, it is possible that chelerythrine interferes with a phosphatase(s) that is able to regulate MEKK1 activity. In fact, all phosphatases have an active cysteine site that could be the target of oxidants. It is also possible that chelerythrine interacts with glutathione, resulting in its depletion and the increase of reactive oxygen species, which may, in turn, activate JNK and p38 pathways.
Apoptosis, or programmed cell death, is a highly regulated process that involves activation of a cascade of molecular events, leading to cell death that is characterized by plasma membrane blebbing, shrinkage, chromatin condensation, chromosomal DNA fragmentation, and formation of membranebound apoptotic bodies (67,68). A number of studies indicate that activation of JNK or p38 kinase pathway plays a crucial role in apoptosis induced by certain stimuli. For example, in PC-12 neuronal cells, activation of JNK and p38 pathways is required for apoptosis induced by nerve growth factor depletion (21). In U937 and Jurkat cells, interfering with JNK or p38 pathway has been shown to block apoptosis induced by ceramide and UV radiation (22,69). Activation of JNK and p38 pathways has also been implicated in anticancer drug-induced apoptosis (70,71). In the present study, we found that chelerythrine induced apoptosis at the concentrations that stimulated JNK and p38 activities. Pretreatment with antioxidants blocked JNK and p38 activation and also attenuated the induction of apoptosis by chelerythrine. Furthermore, specifically blocking JNK and p38 pathways by overexpression of a dominant-negative mutant of MEKK1, MKK4, JNK1, or p38 inhibited chelerythrine-induced apoptosis. Therefore, JNK and p38 pathways are also involved in the regulation of apoptosis induced by chelerythrine.
In summary, we demonstrate that chelerythrine induces JNK and p38 activation without inhibiting PKC activity. Activation of JNK and p38 is mediated by upstream kinases, MEKK1 and MKK4, and regulates apoptosis. Chelerythrine is known to display a variety of biological activities, such as inhibition of taxol-mediated polymerization of rat brain tubulin (47) and histamine release induced by aggregation of the IgE receptors on human basophils (34). Chelerythrine is also shown to stimulate protein phosphorylation in the mitochondrial fraction of rat retina (35). It will be interesting to study whether JNK and p38 play a role in those biological responses induced by chelerythrine.