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Activation of p38 MAPK is required for Bax translocation to mitochondria, cytochrome c release and apoptosis induced by UVB irradiation in human keratinocytes

AN VAN LAETHEM^*,, SOFIE VAN KELST^*,, SASKIA LIPPENS, WIM DECLERCQ, PETER VANDENABEELE, STEFAN JANSSENS, JACKIE R. VANDENHEEDE^*, MARIA GARMYN and PATRIZIA AGOSTINIS^*,1

^* Division of Biochemistry,
Laboratory of Dermatology, and
Department of Cardiology, Faculty of Medicine, Catholic University of Leuven, Leuven, Belgium; and
Department for Molecular Biomedical Research, Flanders Interuniversity Institute for Biotechnology, University of Ghent, Ghent, Belgium

¹Correspondence: Division of Biochemistry, Herestraat 49, Leuven B-3000, Belgium. E-mail: patricia.agostinis{at}med.kuleuven.ac.be

SPECIFIC AIMS

The mitochondrial pathway of apoptosis has been shown to be crucial for the elimination of UVB-damaged human keratinocytes. In spite of this, the mechanisms that lead to permeabilization of the outer mitochondrial membrane resulting in release of proapoptotic factors into the cytosol remain poorly understood. This study examines the role of p38 MAPK in the regulation of premitochondrial events leading to cytochrome c release and keratinocyte cell death in response to UVB irradiation.

PRINCIPAL FINDINGS

1. UVB induces redistribution of Bax to mitochondria and effector caspase activation in skin equivalents
Immunostaining using a conformation-specific Bax antibody revealed that in skin equivalents exposed to UVB, the pattern of Bax fluorescence was rapidly changed from a diffuse cytosolic staining to a predominantly perinuclear punctuated pattern. Co-localization with the signal obtained by the mitochondrion-selective dye MitoTrackerRed, suggested a translocation of Bax from the cytosol to the mitochondria. UVB-induced translocation of Bax started to be detectable as early as 3 h after irradiation, matching the onset of cytochrome c release into the cytosol, and clearly preceding maturation of procaspase-3 and DNA fragmentation. Increased phosphorylation/activation of p38 MAPK was concomitantly detected in UVB-treated skin equivalents.

2. p38 and p38ß MAPK are major mediators of the intrinsic pathway of apoptosis in UVB-irradiated keratinocytes
To provide more evidence for a specific role of p38 MAPK in UVB-induced apoptosis, we produced a HaCaT cell line stably overexpressing a drug resistant p38 (DRp38) MAPK mutant, which fully retains the ability to be activated by UVB but is insensitive to chemical p38 MAPK inhibitors, PD169316 and SB203580. Exposure of DRp38 MAPK HaCaT cells to UVB enhanced release of cytochrome c and overall cell death as compared with empty vector-expressing control cells (Fig. 1 A, C). DRp38 MAPK cells were substantially less sensitive to the inhibitory effects of PD169316 and SB203580 on cytochrome c release, procaspase-3 processing, and PARP cleavage (Fig. 1C ). In contrast, the cytoprotective effect of chemical p38 MAPK inhibitors was mimicked by the expression of a dominant negative p38 MAPK (Ad-DNp38) mutant in both HaCaT and NHK cells, suggesting a general function of this signaling pathway in mediating the mitochondrial pathway of apoptosis in UVB-irradiated human keratinocytes.

View larger version (42K): [in this window] [in a new window]   Figure 1. Overexpression of drug resistant p38 MAPK (DRp38) mutant enhances the kinetics of UVB induced apoptosis and reverts the protective effect of PD169316. A) Phase contrast microscopic analysis of UVB-treated HaCaT cells stably overexpressing empty vector (HaCaT-EV), upper panels, vs. DRp38 MAPK, lower panels. Pictures of irradiated untreated cells (left) vs. cells pretreated with PD169316 (5 µM; right), were taken 16 h after UVB (20mJ/cm2). B) p38 MAPK expression and activation in HaCaT-EV cells and in HaCaT-DRp38 MAPK by Western blot. C) Western blot analysis of the enhanced apoptotic kinetics in the DRp38 MAPK overexpressing cells as compared with EV transfected cells. Comparison of cytochrome c, caspase-3 cleavage, and PARP cleavage in HaCaT-EV and HaCaT-DRp38 MAPK cells in untreated vs. cells pretreated with 5 µM of PD169316.

3. p38 MAPK is required for Bax translocation to the mitochondria following UVB
We next evaluated the possibility that p38 MAPK could mediate its apoptogenic effect by mobilizing Bax to the mitochondria. HaCaT cells in which p38 MAPK was inhibited by SB203580 or PD169316, showed a marked decrease in mitochondrial Bax translocation, cytochrome c release, and _m drop in response to UVB exposure. Conversely, in UVB-irradiated DRp38 MAPK cells this death signal was not only amplified, as judged by the increased percentage of cells showing intracellular Bax relocation, but was also insensitive to inhibitory effects of SB203580, clearly indicating the selective importance of p38 MAPK in this process.

4. p38 MAPK-mediated Bax translocation is blocked by Bcl-2 and occurs in a caspase-independent fashion
Bcl-2 overexpression in HaCaT cells abolishes mitochondrial cytochrome c release, caspase activation, and efficiently protects keratinocytes from UVB. To position the antiapoptotic role of Bcl-2 in the p38 MAPK-mediated cascade leading to cytochrome c release, we investigated at what stage Bcl-2 would reverse the UVB-activated p38 MAPK-Bax cascade. Overexpression of Bcl-2 drastically abrogated conformational change of Bax and its intracellular redistribution in response to UVB irradiation as judged by immunocytochemistry. Activation of p38 MAPK by UVB was unaffected in Bcl-2 overexpressing cells, thus ordering this event upstream of the inhibition exerted by Bcl-2 on the mitochondrial translocation of Bax. Bax translocation occurred equally well in cells pretreated with pan-caspase inhibitor zVAD-fmk, although caspase activity and UVB-induced apoptosis were clearly blocked. zVAD-fmk did not alter UVB induced p38 MAPK activation, nor did it significantly inhibit onset of cytochrome c release although it blocked progression of this process with time. In addition, zVAD-fmk protected UVB-treated cells from major loss in mitochondrial transmembrane potential, which occurred at the time of maximal caspase activation 16–24 h post-irradiation.

5. Inhibition of the p38 MAPK cascade in normal epidermis reduces UVB-induced Bax relocation and apoptosis
To recapitulate UVB-mediated cellular stress responses leading to keratinocyte death in normal human epidermis, a short-term human skin organ culture was used. As shown in Fig. 2 , exposure of human skin to UVB irradiation induced a time-dependent accumulation of sunburn/apoptotic cells, scattered in basal and suprabasal layers, as revealed by DAPI staining. Immunostaining with anti-active Bax antibody revealed that apoptotic keratinocytes stained positive for Bax. Pretreatment of the whole skin organ culture with p38 MAPK inhibitor PD169316 did reduce the number of apoptotic cells in skin and it did block intracellular Bax redistribution (Fig. 2) .

View larger version (47K): [in this window] [in a new window]   Figure 2. UVB-induced Bax translocation and apoptosis in normal epidermis is reduced when p38 MAPK is blocked by PD169316. Skin organ cultures (SOC) were pretreated with 20 µM PD169316 2 h prior to UVB irradiation. At 16 h after UVB (300mJ/cm2), the SOC were fixed and processed for immunostaining with anti-active Bax antibody. Images taken with a fluorescence microscope show a clear reduction of Bax-translocated cells (green panels) and cells with nuclear condensation (DAPI staining, blue panels) in UVB irradiated SOC incubated with PD169316 prior to irradiation as compared with untreated irradiated SOC.

This result shows that inhibition of p38 MAPK can significantly protect human skin from UVB-induced killing by preventing Bax translocation to mitochondria, which represents an early commitment step in the initiation of apoptosis of UVB-damaged keratinocytes.

CONCLUSIONS AND SIGNIFICANCE

In this study we show for the first time that exposure of human skin to UVB irradiation leading to formation of sunburn/apoptotic cells is associated with a rapid p38 MAPK activation and intracellular redistribution of proapoptotic Bax protein to the mitochondria. The latter event occurs with kinetics closely paralleling those of cytochrome c release and preceding activation of procaspase-3, as well as other endpoints of the apoptotic process, such as chromatin fragmentation and loss of mitochondrial transmembrane potential. By using HaCaT cells stably expressing a DRp38 MAPK mutant, which is insensitive to the effect of pharmacological p38 MAPK inhibitors, and by expressing a DN-p38 MAPK, we provide conclusive evidence for the requirement of p38 MAPK in UVB-induced apoptosis. Most importantly, we show that UVB-mediated activation of p38 MAPK is required to induce intracellular redistribution of Bax from the cytosol to the mitochondria. This finding is supported by different experimental evidences. The conformational change of Bax leading to its mitochondrial redistribution is not observed in UVB-irradiated keratinocytes in which p38 MAPK has been inhibited. p38 MAPK inhibition is associated with the inhibition of mitochondrial cytochrome c release and effector caspase activation and with a protection from loss in mitochondrial transmembrane potential. All these effects are specifically reversed in DRp38 MAPK overexpressing cells. Inhibition of p38 MAPK in skin organ cultures leads to protection against UVB-induced cell death and concomitantly abolishes the redistribution of Bax in the basal layer of the epidermis where sunburn cells are also found (Fig. 2) . Together, these data establish that p38 MAPK plays a key physiological role in the activation of the mitochondrial cell death pathway in human keratinocytes in response to UVB-irradiation. To our knowledge, this is the first report identifying Bax as a downstream target of the proapoptotic p38 MAPK cascade in UVB irradiated human skin.

Results presented here indicate that the UVB-induced p38 MAPK-Bax death cascade is independent of caspases but can be overruled by Bcl-2 (Fig. 3 ). Bcl-2 blocks the Bax conformational change required for its mitochondrial translocation, mitochondrial membrane permeabilization, cytochrome c release, and apoptosis, without interfering with p38 MAPK activation.

View larger version (16K): [in this window] [in a new window]   Figure 3. Activation of p38 MAPK is an important initiating signal of the mitochondrial death pathway induced by UVB. Activation of the p38 MAPK-signal by UVB mediates the conformational change of proapoptotic Bax and causes its translocation to the mitochondrial membrane and subsequent release of cytochrome c. This signaling pathway is inhibited by Bcl-2. Cytosolic cytochrome c will activate the caspase cascade that feeds back to mitochondria (e.g., loss of transmembrane potential) and results in full cell death.

Our data are consistent with a model of UVB-induced apoptosis in human skin in which activated p38 MAPK signals to Bax to engage the mitochondrial pathway of apoptosis. The present study identifies new elements in the signaling cascade that leads to mitochondrial membrane permeabilization in UVB-irradiated keratinocytes and suggests that p38 MAPK by committing keratinocytes to apoptosis may play a key role in preventing photocarcinogenesis.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-2285fje;

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