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J. Biol. Chem., Vol. 275, Issue 28, 21416-21421, July 14, 2000

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p38 Mitogen-activated Protein Kinase Regulates a Novel, Caspase-independent Pathway for the Mitochondrial Cytochrome c Release in Ultraviolet B Radiation-induced Apoptosis^*

Zerihun Assefa, Annelies Vantieghem^§, Marjan Garmyn^¶, Wim Declercq, Peter Vandenabeele^**, Jackie R. Vandenheede, Roger Bouillon^¶, Wilfried Merlevede, and Patrizia Agostinis^**§§

From the  Division of Biochemistry and ^¶ Laboratory of Dermatology, Faculty of Medicine, Katholieke Universiteit Leuven, Herestraat 49, B-3000 Leuven, Belgium and the  Department of Molecular Biology, Flanders Interuniversity Institute for Biotechnology, University of Gent, Ledeganckstraat 35, B-9000 Gent, Belgium

Received for publication, March 23, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The mechanisms of UVB-induced apoptosis and the role of p38 mitogen-activated protein kinase (MAPK) were investigated in HaCaT cells. UVB doses that induced apoptosis also produced a sustained activation of p38 MAPK and mitochondrial cytochrome c release, leading to pro-caspase-3 activation. Late into the apoptotic process, UVB also induced a caspase-mediated cleavage of Bid. Caspase inhibitors benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone and benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethylketone substantially blocked the UVB-induced apoptosis without preventing the release of mitochondrial cytochrome c and the p38 MAPK activation. The inhibition of p38 MAPK counteracted both apoptosis and cytochrome c release as well as the DEVD-amino-4-methylcoumarin cleavage activity without affecting the processing of pro-caspase-8. These results indicate that UVB induces multiple and independent apoptotic pathways, which culminate in pro-caspase-3 activation, and that the initial cytochrome c release is independent of caspase activity. Importantly, we show that a sustained p38 MAPK activation contributes to the UVB-induced apoptosis by mediating the release of mitochondrial cytochrome c into the cytosol.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Ultraviolet (UV) radiation is the most important environmental factor involved in the etiology of non-melanoma skin cancer (1). The carcinogenic effects of the solar radiation have been mainly attributed to the UVB (280-320 nm) fraction, while the potentially dangerous UVC (100-290 nm) radiations are absorbed by the ozone layer and are therefore not of physiological importance (2). Exposure of keratinocytes to UVB leads to the expression of several genes involved in cell cycle arrest, DNA repair, and/or apoptosis, an event that is collectively known as the UV response (3). The induction of apoptosis following UV irradiation is thought to be a protective mechanism ensuring the removal of irreversibly damaged and potentially cancerous cells, and recent reports indicate that both nuclear and cytosolic events contribute critically to the overall apoptotic response (4). Therefore, the decision whether epidermal keratinocytes undergo apoptosis or not upon UV irradiation is a crucial process in photocarcinogenesis.

Recent studies have revealed that the basic mechanism of apoptosis induced by UVB irradiation involves the typical process in which caspases play a critical role (4-7). In addition, a number of reports have established that the cell surface death receptors, namely TNF¹ receptor and Fas, are clustered in a ligand-independent manner to initiate the process of UV-induced apoptosis (6, 8-10). It is well recognized that the clustering and activation of the Fas or TNF receptor leads to the recruitment and subsequent activation of pro-caspase-8 via the adapter molecule FADD (for Fas-associated death domain protein) (11). In this so-called extrinsic pathway of caspase activation, caspase-8 leads to the proteolytic activation of downstream effector caspases, cleavage of vital protein substrates and subsequent apoptotic cell death. Accordingly, UVB-induced apoptosis in keratinocytes has been shown to be significantly counteracted by inhibitors of the initiator caspase-8 such as cytokine response modifier A and zVAD-fmk, or by the expression of dominant negative FADD (6, 10).

On the other hand, the intrinsic pathway for caspase activation, induced by many apoptogenic stimuli, involves the mitochondrial release of cytochrome c into the cytosol, which then acts as a trigger for the formation of a complex including the Apaf-1 (apoptotic protease-activating factor 1) and pro-caspase-9. This event culminates in the activation of pro-caspase-9, which can directly cleave and activate the effector caspases, such as caspase-3, resulting in the orchestration of the biochemical execution of the cell (12). In the Fas- and/or TNF-mediated apoptosis, a cytochrome c-dependent mechanism of caspase activation can also amplify the effect of caspase-8 on downstream caspases. In this case, active caspase-8 cleaves Bid, a pro-apoptotic Bcl-2-interacting protein, producing an active C-terminal fragment, which binds to mitochondria and induces the release of cytochrome c (13). In many cases, however, the mitochondrial release of cytochrome c proceeds independently of caspase activity (14). An important and still open question is whether this mitochondria-mediated pathway of caspase activation is relevant to the overall UVB-induced apoptotic response.

UV irradiation has been shown to result in the activation of several members of the MAPK superfamily, comprising ERKs, JNKs, and p38 MAPK (15-19). However, depending on the UV wavelengths and the type of cells used, different regulatory effects by JNKs and ERKs on programmed cell death have been described (16-19). In human keratinocytes, the UVB-induced ERK activation was recently shown to be a protective signal against apoptosis (19), whereas the activation of the p38 MAPK cascade was reported to play a causative role in UVB-induced apoptosis (7). However, how p38 MAPK interacts with the apoptotic machinery and the caspase activation pathways during UVB-induced apoptosis is not yet established.

In this study, we show that in UVB-irradiated HaCaT cells, two independent pathways of caspase activation and apoptosis are induced: a caspase-8- and a cytochrome c-dependent pathway, both of which converge on the activation of pro-caspase-3. Importantly, we show that the p38 MAPK pathway mediates apoptosis following UVB irradiation by inducing the release of mitochondrial cytochrome c into the cytosol, which is followed by pro-caspase-3 activation. Furthermore, our results suggest that, in the late phase of the apoptotic process, a caspase-8-dependent cleavage of Bid may provide a mechanism to exacerbate cell death following UVB irradiation.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials-- Hoechst 33342 (bis-benzimide) was purchased from Sigma. [-³²P]ATP was from Amersham Pharmacia Biotech, while protein A-TSK was from Affiland (Liège, Belgium). zVAD-fmk and zDEVD-fmk were purchased from Enzyme Systems Products (Livermore, CA); DEVD-amc was from the Peptide Institute, Inc. (Osaka, Japan). The p38 MAPK inhibitor PD169316 was purchased from Calbiochem (Bierges, Belgium). Anti-p38 MAPK antibody and anti-phospho-p38 MAPK (Thr¹⁸⁰/Tyr¹⁸²) monoclonal antibody, which specifically recognizes the phosphorylated, active form of the kinase, were purchased from New England Biolabs, Inc. (Beverly, MA). Mouse anti-human PARP antibody was purchased from Biomol (Plymouth, PA), caspase-3 antibody was from Santa Cruz (Santa Cruz, CA), caspase-8 and cytochrome c antibodies were from PharMingen (San Diego, CA), and Bid antibody was from R&D Systems (Minneapolis, MN).

Cell Lines, Culture Conditions, and UVB Irradiation-- The spontaneously immortalized human keratinocyte cell line HaCaT were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 2 mM L-glutamine, and 1% penicillin/streptomycin solution (all cell culture media and supplements were purchased from Life Technologies, Inc.). Cells were incubated in a 37 °C humidified air environment containing 5% CO₂. UVB irradiation was performed exactly as described previously (15). All the drugs used in this study were added to culture medium prior to treatment for the period of time indicated in figure legends.

Evaluation of Apoptosis and Caspase Activity-- Apoptosis was evaluated either by fluorescent microscopic analysis of fragmented nuclei stained with Hoechst 33342 or by light microscopy. Caspase activity assays were performed as described in Ref. 20.

Preparation of Cell Extract and Western Blot Analysis-- Preparation of cell extracts and Western blot analysis were performed as described in Ref. 20.

Cell Proliferation Assay-- Cells (1 × 10⁵ cells/plate) were seeded onto 6-cm² plates. The caspase inhibitor zVAD-fmk and the p38 MAPK inhibitor PD169316 were added 2 and 1 h, respectively, before irradiation, and zVAD-fmk was re-added every 24 h. At each time point, cell proliferation was determined by quantification of the cellular protein content using naphthol blue black (Acros, Beerse, Belgium) as described in Ref. 21.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

UVB Irradiation Induces p38 MAPK Activation and Apoptosis in HaCaT Cells-- We initially examined the pattern of p38 MAPK activation in HaCaT cells following UVB irradiation. A dose of 60 mJ/cm² UVB irradiation resulted in a remarkable and sustained activation of p38 MAPK (Fig. 1A), which was observed with doses as low as 10 mJ/cm² (data not shown). No difference in the p38 MAPK protein level was observed in these experiments (data not shown), indicating that the kinase activity changes induced by UVB irradiation were due to post-translational modifications of pre-existing enzyme molecules. Irradiation of HaCaT cells with similar UVB doses resulted in the characteristic morphological features of cells undergoing apoptosis; cells appeared shrunk and apoptotic bodies were clearly visible (Fig. 1B, b). Fluorescent microscopic analysis with Hoechst 33342 showed that the nuclei of untreated cells were uniformly stained indicating unaltered nuclei (Fig. 1B, c), whereas exposure to UVB resulted in nuclear condensation and fragmentation (Fig. 1B, d). About 60-75% of the cells underwent apoptosis 24 h after UVB irradiation. Furthermore, under these conditions, less than 10% of the cells were stained with propidium iodide or trypan blue (data not shown). Since these two dyes label necrotic cells and, in some cases, cells at the late stages of apoptosis, we can conclude that apoptosis was the predominant, if not the only, type of cell death induced by 60 mJ/cm² UVB.

View larger version (63K): [in this window] [in a new window]   Fig. 1.   UVB induces p38 MAPK and apoptosis in HaCaT cells. A, cells irradiated with a dose of 60 mJ/cm2 were harvested at the indicated time points, and the phosphorylation of p38 MAPK was determined in total cell lysate as described under "Experimental Procedures." Fold activation was determined by densitometric scanning of the phospho-p38 MAPK bands on the shown autoradiogram. B, phase-contrast (a and b) and fluorescent (c and d) microscopic images of untreated cells (a and c) and cells treated with UVB (60 mJ/cm2) (b and d) 24 h after irradiation. C and D, time course of pro-caspase-3 and PARP cleavage in UVB-irradiated cells (60 mJ/cm2).

The induction of apoptosis in UVB-treated cells was further corroborated by the time-dependent activation of pro-caspase-3 as determined by Western blot (Fig. 1C) and DEVDase activity (see Fig. 2C). Pro-caspase-3 activation became apparent at 16 h after irradiation and remained sustained up to 24-30 h. Dose-response analysis indicated that the pro-caspase-3 activity was maximally induced at the UVB dose of 60 mJ/cm² (data not shown). Correspondingly, the cleavage of PARP (Fig. 1D), a substrate of caspase-3 and -7, followed kinetics that overlapped with those of the pro-caspase-3 processing and DEVD-amc cleavage activity.

View larger version (56K): [in this window] [in a new window]   Fig. 2.   Temporal relationship between UVB-induced apoptotic events. Lysates of cells irradiated with UVB (60 mJ/cm2) were harvested at the indicated time points and subjected to Western blot analysis (A, B, and D) using the respective antibodies as indicated. C, DEVD-amc cleavage was measured as described under "Experimental Procedures." Values indicated in A, B, and D as arbitrary units were obtained by densitometric scanning of the respective autoradiograms.

Molecular Mechanisms of UVB-induced Apoptosis: Cytochrome c Release and the Cleavage of Bid-- To evaluate the contribution of the mitochondrial pathway to the process of UVB-induced cell death, we first analyzed the kinetics of cytochrome c release into the cytosol of irradiated HaCaT cells. Fig. 2A shows that cytosolic cytochrome c was detected 8 h after irradiation and attained maximum levels in 24-30 h after UVB treatment. In addition, Fig. 2B shows that, concomitant to the translocation of cytochrome c into the cytosol, the processing of caspase-8 was also taking place, in agreement with previous studies implicating this caspase activity in the process of UV-induced apoptosis (4, 6, 8-10). These observations also indicate that the mitochondrial cytochrome c release and caspase-8 activation are among the earliest apoptotic events in the dying HaCaT cells, clearly preceding the typical cell morphologic and nuclear changes as well as the induction of DEVDase activity (Fig. 2C) and pro-caspase-3 processing (Fig. 1C).

Recent studies have shown that activated caspase-8 can mediate mitochondrial cytochrome c release through the cleavage of the pro-apoptotic protein Bid (13). Therefore, we explored the possibility that the processing of Bid could be implicated in the release of cytochrome c in our system as well. As shown in Fig. 2D, UVB irradiation of HaCaT cells caused a time-dependent proteolysis of Bid. However, Bid processing was a rather late event occurring significantly only 24 h after irradiation. This suggests that Bid cleavage cannot be the cause of the initial release of cytochrome c in UVB-treated HaCaT cells but may represent a mechanism to maintain high level of cytosolic cytochrome c in the dying cells. Furthermore, the initial release of cytochrome c following UVB irradiation was not significantly affected by caspase inhibitors (see Fig. 5E), suggesting that, in irradiated cells, caspase-8 activation and cytochrome c release are mediated by two independent pathways.

Role of the p38 MAPK and Caspase Signaling Cascades in the UVB-induced Apoptosis-- To assess the role of the sustained activation of p38 MAPK during UVB-induced apoptosis, we used its specific pharmacological cell permeant inhibitor PD169316. This is a powerful inhibitor of the p38 MAPK belonging to the class of imidazole-based compounds used both in vivo and in vitro to assess the specific involvement of the p38 MAPK pathway in many physiological processes (22). Cell treatment with this drug inhibited the myelin basic protein phosphotransferase activity of p38 MAPK in immunoprecipitation kinase assays without affecting the activities of either JNK1 or ERK2 (data not shown). In addition, the broad-spectrum caspase inhibitor zVAD-fmk and the inhibitor of caspase-3-like caspases zDEVD-fmk were used to analyze the contribution of the initiator (caspase-8) and/or effector (caspase-3) caspases, respectively, in the process of UVB-induced apoptosis. Thus, HaCaT cells were pretreated with zVAD-fmk, zDEVD-fmk, or different concentrations of PD169316 before irradiation and the level of apoptosis was analyzed 24 h after UVB treatment. Fig. 3 shows that all inhibitors could counteract, albeit to different extents, the UVB-induced apoptosis. Clearly, pretreatment with zVAD-fmk resulted in a superior protection against apoptosis in comparison to zDEVD-fmk, in agreement with its broad spectrum caspase inhibitory activity (23). The anti-apoptotic effect of the PD169316 pretreatment was dose-dependent and could be observed at concentrations as low as 2.5 µM. In addition, pretreatment of the cells with the protein synthesis inhibitor cycloheximide (100 µg/ml) prior to irradiation did not result in a significant protection against UVB-induced apoptosis (data not shown), suggesting that newly synthesized proteins are likely not required for this process.

View larger version (31K): [in this window] [in a new window]   Fig. 3.   Effect of inhibitors on UVB-induced apoptosis. Cells were left untreated or irradiated with 60 mJ/cm2 UVB in the presence or absence of the inhibitors as indicated. The caspase inhibitors (100 µM each) were added 2 h and PD169316 was added 1 h before irradiation. 20 h after irradiation, the number of cells with apoptotic morphology was counted by microscopic examination. The percentage of apoptotic cells relative to those in UVB-irradiated plate was determined. The result shown is a representative of at least three independent experiments.

In order to confirm that the observed anti-apoptotic activities of the inhibitors were not due to a mere delay in the apoptotic process, we looked into their long term effects on cell proliferation. Fig. 4 shows that, when pretreated with either zVAD-fmk or PD169316, UVB-irradiated cells started to proliferate following a brief cell cycle arrest. Fig. 4 also confirmed that zVAD-fmk was the most effective inhibitor as the pretreated cells begun to actively proliferate 24 h after irradiation and became nearly confluent in 3 days. The combined pretreatment with zVAD-fmk and PD169316 did not result in an additional protective effect when compared with cells pretreated with zVAD-fmk alone.

View larger version (37K): [in this window] [in a new window]   Fig. 4.   Effect of UVB on cell proliferation in the presence of PD169316 and zVAD-fmk. HaCaT cells were preincubated with zVAD-fmk (100 µM), PD169316 (25 µM), or their combination before exposure to 60 mJ/cm2 UVB. Cell proliferation was determined at the indicated time periods as described under "Experimental Procedures."

We next analyzed the effects of these inhibitors at the biochemical level. As shown in Fig. 5A, both zVAD-fmk and zDEVD-fmk completely prevented the cleavage of PARP, whereas pretreatment of cells with PD169316 significantly inhibited its cleavage in a dose-dependent manner. As expected, both caspase inhibitors, zVAD-fmk and zDEVD-fmk, completely obliterated the DEVD-caspase activity in UVB-irradiated HaCaT cells (Fig. 5B). In agreement with the results of the PARP cleavage, PD169316 also significantly inhibited the DEVDase activity (Fig. 5B). Western blot analysis revealed that the inhibitors suppressed the cleavage of pro-caspase-3 as well (data not shown).

View larger version (33K): [in this window] [in a new window]   Fig. 5.   Effect of inhibitors on UVB-induced responses. Lysates of untreated cells or cells irradiated in the presence or in the absence of the inhibitors were prepared 16 h after irradiation for the analysis of PARP cleavage (A), DEVD-amc cleavage activity (B), pro-caspase-8 cleavage (D, left panel), or cytochrome c release (E); 24 h after irradiation for Bid proteolysis (C) and at the indicated time points for p38 MAPK activation (D, right panel) as described under "Experimental Procedures." In A, C, D, and E, values shown as arbitrary units were obtained by densitometric scanning of the respective autoradiograms.

We were also interested in evaluating the effect of the inhibitors on the UVB-induced cleavage of Bid. As shown in Fig. 5C, the caspase inhibitor zVAD-fmk was the most efficient in counteracting Bid degradation. The inhibition of DEVD-directed caspases only partially reduced, while p38 MAPK inhibition did not affect Bid cleavage at all (Fig. 5C). This observation suggests that the extrinsic, death receptor-induced caspase-8 signaling pathway is likely to be the most relevant mediator of Bid cleavage while the p38 MAPK pathway does not contribute to this process. In addition, Fig. 5D (left panel) shows that the UVB-induced processing of pro-caspase-8 in HaCaT cells was not affected by PD169316 pretreatment and that, in turn, the caspase inhibitors did not interfere with the p38 MAPK activation (Fig. 5D, right panel). As suggested above, this result indicates that UVB initiates multiple pathways independently.

Pretreatment of the cells with either zVAD-fmk or zDEVD-fmk had no effect on the level of cytochrome c released 16 h after irradiation (Fig. 5E). This result implies that caspase activity is not required for the early release of cytochrome c following UVB treatment, in agreement with the results of the delayed Bid cleavage (Fig. 2D). Importantly, cell pretreatment with the p38 MAPK inhibitor PD169316 resulted in a dose-dependent inhibition of the mitochondrial cytochrome c efflux, when used in the concentration range of 2.5-25 µM. These concentrations of PD169316 were also shown to severely inhibit downstream signaling events such as pro-caspase-3 activation and PARP cleavage (Fig. 5, A and B). Therefore, these results indicate that the p38 MAPK pathway is an important mediator of the UVB-induced cytochrome c release from the mitochondria, the early phase of which appears to be independent of caspases.

Taken together, our overall observations strongly suggest that the p38 MAPK activation is positioned upstream of pro-caspase-3 activation and functions as an important effector mechanism for the UVB-induced apoptosis in human keratinocytes by facilitating the mitochondrial efflux of cytochrome c.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

In the present study, we show that exposure of human keratinocytes to UVB induces the activation of p38 MAPK, cytochrome c release, pro-caspase-8 and -3 activation, Bid cleavage, and apoptosis. In agreement with recent reports (7, 19), exposure of HaCaT cells to UVB led to a significant level of p38 MAPK activation in a dose- and time-dependent manner. We have shown previously that similar doses of UVB also lead to a robust activation of JNK1 (with little effect on ERK2 activity) and a dramatic increase in the mRNA expression of the immediate early genes c-jun and c-fos (15). These events are parts of the "UV response" that is mediated by phosphorylation of preexisting transcription factors c-Jun and/or c-Fos, resulting in the enhanced transcription of AP-1-responsive genes (3). Therefore, our results strongly implicate the p38 MAPK as well as the JNK pathway in the transmission of the UVB-induced stress responses in human keratinocytes.

A number of studies have reported on the role of JNK in coupling cellular stress signals and specifically in the UV responses leading to the apoptotic cell death (16-18). The role of ERK in protecting cells against these types of stress-induced apoptotic processes has also been reported recently (19). The functional role of p38 MAPK in cellular stress response is, however, less well understood and rather controversial. Some investigators have observed that p38 MAPK activation in calphostin C-induced apoptosis of glioma cells requires active caspases but is dispensable for cell death (24). Activation of p38 MAPK in Fas-treated Jurkat cells has also been shown to be dependent on the initiator caspases (25) but not on the executioner caspases (26) and, in both cases, seemingly unrelated to the process of apoptosis. Other reports have indicated that p38 MAPK activation occurs either upstream or independent of caspases and mediates apoptosis induced by various stresses (UVC, hyperosmolarity, sphingosine) in human neutrophils (27). In contrast, p38 MAPK activation has been shown to be protective against hypericin-induced apoptosis of HeLa cells (20). In order to provide a possible role for the p38 MAPK activation in UVB-irradiated HaCaT cells, we investigated its function in the process of UVB-induced apoptosis.

In line with other reports (4-6), we present here strong evidence that apoptosis induced by UVB is a typical caspase-dependent process and we implicate the extrinsic pathway of caspase activation as an important component of the UVB-induced apoptotic process. Pretreatment of cells with the caspase inhibitors zVAD-fmk or zDEVD-fmk drastically inhibited or reduced, respectively, the UVB-induced apoptosis. In addition, the selective p38 MAPK inhibitor, PD169316, substantially counteracted the induction of apoptosis in irradiated HaCaT cells (Fig. 3). Both the caspase- and p38 MAPK inhibitors considerably prevented the UVB-induced pro-caspase-3 processing and activation (Fig. 5B) as well as PARP cleavage (Fig. 5A). On the other hand, neither one of the caspase inhibitors had any effect on the activation of p38 MAPK (Fig. 5D, right panel), indicating that divergent pathways are elicited by UVB irradiation and that the p38 MAPK functions upstream of pro-caspase-3 activation.

Our results show that cytochrome c release is one of the earliest cellular responses to UVB irradiation (Fig. 2), evidently preceding pro-caspase-3 activation and the onset of apoptotic morphological changes. Interesting and novel observations were made by analyzing the effect of the different inhibitors on the release of mitochondrial cytochrome c. The PD169316 pretreatment strongly counteracted the UVB-induced cytochrome c release, whereas neither one of the caspase inhibitors zVAD-fmk or zDEVD-fmk had any substantial effect on this process. This suggests that the UVB-induced release of cytochrome c from the mitochondria occurs independently of caspase activation and requires p38 MAPK activity. Previous reports have also shown that cytochrome c release induced by several apoptogenic stimuli, including UV irradiation, staurosporine, and overexpression of Bax, is independent of caspase activity (reviewed in Ref. 14) and precedes a reduction in mitochondrial transmembrane potential (28). The observation that zVAD-fmk, while inhibiting pro-caspase-3 activation and apoptosis, does not prevent cytochrome c release, implies that it targets key components of the intrinsic (downstream of cytochrome c release) as well as the extrinsic pathway of caspase activation. Bearing in mind the inhibitory effects of PD169316 on caspase-3 activation and PARP cleavage, our results strongly suggest that this inhibitor acts by counteracting the mitochondrial cytochrome c release. Hence, p38 MAPK mediates pro-caspase-3 activation in UVB-irradiated cells by inducing the mitochondrial efflux of cytochrome c through as yet an unknown mechanism.

Recent reports have shown that the p38 MAPK inhibitor SB203580, which is structurally related to the PD169316, can trigger a significant, Ras-independent activation of c-Raf in certain cell lines in the concentration range of 8-25 µM (29, 30). Activated Raf may phosphorylate and inactivate Bax, a pro-apoptotic member of the Bcl-2 family of proteins, thereby preventing its cytochrome c releasing activity. In our system, however, treatment of HaCaT cells with PD169316 concentrations up to 25 µM, either alone or in combination with UVB irradiation, did not induce any notable activation of c-Raf (data not shown). Moreover, as shown in Fig. 5, mitochondrial cytochrome c efflux and the PARP cleavage (data not shown) were considerably inhibited by PD169316 concentrations as low as 2.5 µM. Therefore, it is unlikely that this indirect effect of the p38 MAPK inhibitor can account for the prevention of cytochrome c release and protection against apoptosis in UVB-treated HaCaT cells.

Recent reports have shown that, following UVC irradiation, p38 MAPK phosphorylates the tumor suppressor protein p53 at different Ser residues (31, 32) thereby increasing its transcriptional activity (31). Given the evidence that the mutated p53 in HaCaT cells may still have a role in apoptosis (33) and that it contains the p38 phosphorylation sites (34), an interesting hypothesis could be that p38 MAPK mediates mitochondrial cytochrome c release and apoptosis in a p53-dependent fashion. The mechanisms by which p53 mediates apoptosis are still unclear, and both transcriptional-dependent and -independent pathways seem to be involved (35, 36). In particular, a p53-mediated up-regulation of the pro-apoptotic protein Bax (35) could explain how p38 MAPK induces the release of cytochrome c from mitochondria of UVB-irradiated cells. However, in our system, UVB did not lead to any detectable transcriptional activation of Bax under apoptosis-inducing conditions² and the protein synthesis inhibitor cycloheximide did not protect HaCaT cells from apoptosis in agreement with previous findings (33). Thus, it seems that the mechanism by which p38 MAPK induces mitochondrial cytochrome c release and apoptosis in UVB-irradiated cells does not require the de novo protein synthesis, whether or not it involves p53 phosphorylation.

In some cellular systems, the cleavage of Bid, a proapoptotic Bcl-2 family member, results in the translocation of cytochrome c from mitochondria to the cytoplasm (13). In UVB-treated cells, Bid cleavage took place after 20-24 h of irradiation (Fig. 2) indicating that it is not critical for the initial process of cell death. Whereas the pretreatment of cells with zVAD-fmk, and to a lesser extent with zDEVD-fmk, inhibited the UVB-induced Bid cleavage, PD169316 was without any effect (Fig. 5C). Because PD169316 strongly inhibits pro-caspase-3 activation but does not affect pro-caspase-8 cleavage, this result suggests that caspase-8 is likely the main protease responsible for Bid cleavage in UVB-treated cells. Why Bid should be processed only at such a delayed time point is not clear. At most, Bid cleavage at such late stage of apoptosis may provide a means of maintaining high levels of cytosolic cytochrome c levels that could augment the process of cell death.

Overall, our results provide new insights into the mechanisms involved in the process of apoptosis induced by UVB in human keratinocytes and identify a novel pathway mediated by the activation of p38 MAPK that leads to the release of mitochondrial cytochrome c into the cytosol, which significantly contributes to the induction of the apoptotic process.

	ACKNOWLEDGEMENTS

We thank G. Nijs for expert technical assistance. We also thank Dr. N. E. Fusenig (German Cancer Research Center, Division of Carcinogenesis and Differentiation, Heidelberg, Germany) for kindly providing us with HaCaT cells.

	FOOTNOTES

^* This work was supported in part by Interuniversitaire Attractiepolen Grant p4/26, Grant 0211.99 from the Fonds voor Wetenschappelijk Onderzoek (FWO)-Vlaanderen, and European Biomed Program Grant BMH4-CT96-0300.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^§ Recipient of a fellowship from the Vlaams Instituut voor de Bevordering van het Wetenschappelijk-Technologisch Onderzoek in de Industrie.

^** Research leader with the FWO-Vlaanderen.

Research director with the FWO-Vlaanderen.

^§§ To whom correspondence should be addressed. Tel.: 32-16-345-715; Fax: 32-16-345-995; E-mail: patricia.agostinis@med.kuleuven.ac.be.

Published, JBC Papers in Press, March 29, 2000, DOI 10.1074/jbc.M002634200

² Z. Assefa, unpublished results.

	ABBREVIATIONS

The abbreviations used are: TNF, tumor necrosis-factor; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated protein kinase; FADD, Fas-associated death domain; JNK, c-Jun N-terminal kinase; PD169316, 4-(4-fluorophenyl)-2-(4-nitrophenyl)-5-(4-pyridyl)-1H-imidazole; zDEVD-fmk, benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethylketone; zVAD-fmk, benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone; PARP, poly(ADP-ribose)polymerase; DEVD-amc, DEVD-amino-4-methylcoumarin; DEVDase, DEVD-directed protease.

	REFERENCES

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				Z. Assefa, G. Bultynck, K. Szlufcik, N. Nadif Kasri, E. Vermassen, J. Goris, L. Missiaen, G. Callewaert, J. B. Parys, and H. De Smedt Caspase-3-induced Truncation of Type 1 Inositol Trisphosphate Receptor Accelerates Apoptotic Cell Death and Induces Inositol Trisphosphate-independent Calcium Release during Apoptosis J. Biol. Chem., October 8, 2004; 279(41): 43227 - 43236. [Abstract] [Full Text] [PDF]

				G. Mallikarjuna, S. Dhanalakshmi, R. P. Singh, C. Agarwal, and R. Agarwal Silibinin Protects against Photocarcinogenesis via Modulation of Cell Cycle Regulators, Mitogen-Activated Protein Kinases, and Akt Signaling Cancer Res., September 1, 2004; 64(17): 6349 - 6356. [Abstract] [Full Text] [PDF]

				E. J. Horn, A. Albor, Y. Liu, S. El-Hizawi, G. E. Vanderbeek, M. Babcock, G. T. Bowden, H. Hennings, G. Lozano, W. C. Weinberg, et al. RING protein Trim32 associated with skin carcinogenesis has anti-apoptotic and E3-ubiquitin ligase properties Carcinogenesis, February 1, 2004; 25(2): 157 - 167. [Abstract] [Full Text] [PDF]

				M. T. Sturniolo, S. R. Dashti, A. Deucher, E. A. Rorke, A.-M. Broome, R. A. S. Chandraratna, T. Keepers, and R. L. Eckert A Novel Tumor Suppressor Protein Promotes Keratinocyte Terminal Differentiation via Activation of Type I Transglutaminase J. Biol. Chem., November 28, 2003; 278(48): 48066 - 48073. [Abstract] [Full Text] [PDF]

				Y. Zhang, M. I. Dawson, Y. Ning, L. Polin, R. E. Parchment, T. Corbett, A. N. Mohamed, K.-C. Feng, L. Farhana, A. K. Rishi, et al. Induction of apoptosis in retinoid-refractory acute myelogenous leukemia by a novel AHPN analog Blood, November 15, 2003; 102(10): 3743 - 3752. [Abstract] [Full Text] [PDF]

				Y. Liao and M.-C. Hung Regulation of the Activity of p38 Mitogen-Activated Protein Kinase by Akt in Cancer and Adenoviral Protein E1A-Mediated Sensitization to Apoptosis Mol. Cell. Biol., October 1, 2003; 23(19): 6836 - 6848. [Abstract] [Full Text] [PDF]

				J. G. Pastorino, N. Shulga, and J. B. Hoek TNF-{alpha}-induced cell death in ethanol-exposed cells depends on p38 MAPK signaling but is independent of Bid and caspase-8 Am J Physiol Gastrointest Liver Physiol, August 8, 2003; 285(3): G503 - G516. [Abstract] [Full Text] [PDF]

				R. Weller, A. Schwentker, T. R. Billiar, and Y. Vodovotz Autologous nitric oxide protects mouse and human keratinocytes from ultraviolet B radiation-induced apoptosis Am J Physiol Cell Physiol, May 1, 2003; 284(5): C1140 - C1148. [Abstract] [Full Text] [PDF]

				Y.-Y. He, J.-L. Huang, D. C. Ramirez, and C. F. Chignell Role of Reduced Glutathione Efflux in Apoptosis of Immortalized Human Keratinocytes Induced by UVA J. Biol. Chem., February 28, 2003; 278(10): 8058 - 8064. [Abstract] [Full Text] [PDF]

				V. Poulaki, C. S. Mitsiades, A. M. Joussen, A. Lappas, B. Kirchhof, and N. Mitsiades Constitutive Nuclear Factor-{kappa}B Activity Is Crucial for Human Retinoblastoma Cell Viability Am. J. Pathol., December 1, 2002; 161(6): 2229 - 2240. [Abstract] [Full Text] [PDF]

				W. Li and J. R. Bertino Fas-mediated Signaling Enhances Sensitivity of Human Soft Tissue Sarcoma Cells to Anticancer Drugs by Activation of p38 Kinase Mol. Cancer Ther., December 1, 2002; 1(14): 1343 - 1348. [Abstract] [Full Text] [PDF]

				C. Bowen, M. Birrer, and E. P. Gelmann Retinoblastoma Protein-mediated Apoptosis After gamma -Irradiation J. Biol. Chem., November 15, 2002; 277(47): 44969 - 44979. [Abstract] [Full Text] [PDF]

				W. F. Holmes, D. R. Soprano, and K. J. Soprano Elucidation of Molecular Events Mediating Induction of Apoptosis by Synthetic Retinoids Using a CD437-resistant Ovarian Carcinoma Cell Line J. Biol. Chem., November 15, 2002; 277(47): 45408 - 45419. [Abstract] [Full Text] [PDF]

				I. Kuwabara, Y. Kuwabara, R.-Y. Yang, M. Schuler, D. R. Green, B. L. Zuraw, D. K. Hsu, and F.-T. Liu Galectin-7 (PIG1) Exhibits Pro-apoptotic Function through JNK Activation and Mitochondrial Cytochrome c Release J. Biol. Chem., January 25, 2002; 277(5): 3487 - 3497. [Abstract] [Full Text] [PDF]