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Previous Article | Next Article 
Molecular and Cellular Biology, September 2004, p. 8167-8183, Vol. 24, No. 18
0270-7306/04/$08.00+0 DOI: 10.1128/MCB.24.18.8167-8183.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.
Protein Kinase C
Regulates Keratinocyte Death and Survival by Regulating Activity and Subcellular Localization of a p38-Extracellular Signal-Regulated Kinase 1/2 Complex
Tatiana Efimova,1 Ann-Marie Broome,1 and Richard L. Eckert1,2,3,4,5*
Departments of Physiology and Biophysics,1
Dermatology,2
Oncology,3
Biochemistry,4
Reproductive Biology, Case Western Reserve University School of Medicine, Cleveland, Ohio5
Received 16 April 2004/
Returned for modification 13 May 2004/
Accepted 9 June 2004
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ABSTRACT
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Protein kinase C
(PKC) is an important regulator of apoptosis in epidermal keratinocytes. However, little information is available regarding the downstream kinases that mediate PKC-dependent keratinocyte death. This study implicates p38 mitogen-activated protein kinase (MAPK) as a downstream carrier of the PKC-dependent death signal. We show that coexpression of PKC with p38 produces profound apoptosis-like morphological changes. These morphological changes are associated with increased sub-G1 cell population, cytochrome c release, loss of mitochondrial membrane potential, caspase activation, and PARP cleavage. This death response is specific for the combination of PKC and p38 and is not produced by replacing PKC with PKC
or p38 with p38. A constitutively active form of MEK6, an upstream activator of p38, can also produce cell death when coupled with p38. In addition, concurrent p38 activation and extracellular signal-regulated kinase 1/2 (ERK1/2) inactivation are required for apoptosis. Regarding this inverse regulation, we describe a p38-ERK1/2 complex that may coordinate these changes in activity. We further show that this p38-ERK1/2 complex relocates into the nucleus in response to PKC expression. This regulation appears to be physiological, since H2O2, a known inducer of keratinocyte apoptosis, promotes identical PKC and p38-ERK1/2 activity changes, leading to similar morphological changes.
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INTRODUCTION
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Identifying the intracellular signal transduction pathways that regulate keratinocyte death and differentiation is an important goal. The protein kinase C (PKC) family of lipid-activated serine/threonine kinases appears to play a key role in this process (2, 19). The PKC family includes three subtypes: classical, novel, and atypical (49-52). The classical isoforms (PKC, -ßI, -ßII, and -
) are calcium, diacylglycerol, and phospholipid dependent, the novel isoforms (PKC, -
, -
, -
, and -µ) are calcium independent, and the atypical isoforms (PKC
and -
) are calcium and diacylglycerol independent (49-52). Each PKC isozyme displays a unique tissue distribution, subcellular localization, and substrate specificity. Epidermal keratinocytes express PKC, -, -, -, and - (18, 27, 30, 44, 55), and PKC is implicated as a regulator of epidermal apoptosis (8, 15, 16, 43). For example, Denning et al. showed that PKC is proteolytically cleaved in UVB-treated keratinocytes, and the released catalytic fragment then acts to destabilize the mitochondria and cause apoptosis (15). However, other studies, with pharmacologic agents, suggest that PKC-dependent activation of downstream signaling cascades is required for death, although the identity of relevant signaling cascade(s) has not been reported (43).
Mitogen-activated protein kinases (MAPKs) are important intracellular regulators of keratinocyte differentiation that are activated by PKC-dependent pathways (17, 23, 25). Several MAPK subtypes have been identified, including extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 MAPK (7, 9, 78). The p38 MAPK family includes p38, -ß, -, and - (54). Only p38, -ß, and - are expressed in keratinocytes (14, 20). Among these isoforms, p38 has been implicated as a regulator of keratinocyte function and is required for cell response to a variety of differentiating agents, including phorbol ester, calcium, okadaic acid, and green tea polyphenol (1, 22, 23). In addition, p38 MAPK is a known downstream target of PKC via a cascade that includes novel PKC, Ras, MEKK1, and MEK3/6 (23). Therefore, it is possible that p38 may mediate PKC-associated keratinocyte cell death. The present experiments were designed to assess this possibility. Our studies show that PKC acts to regulate the function of a novel p38-ERK1/2 complex that is constitutively present in the cell. PKC stimulation results in relocation of p38 and ERK1/2 to the nucleus, along with a coordinate increase in p38 activity and decrease in ERK1/2 activity. We hypothesize that the complex mediates the coordinate inverse regulation of p38 and ERK1/2. Both the increase in p38 activity and the decrease in ERK1/2 activity are required for subsequent procaspase and PARP activation leading to cell death. We show here that hydrogen peroxide (H2O2), a known inducer of keratinocyte apoptosis (6, 12, 29, 57), produces similar changes in keratinocyte morphology and similar enzyme activities, suggesting that the results are meaningful in normal keratinocyte physiology.
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MATERIALS AND METHODS
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Antibodies and reagents.
Keratinocyte serum-free medium, gentamicin, trypsin, and Hanks balanced salt solution were obtained from Life Technologies, Inc. Dispase was obtained from Boehringer Mannheim. Z-VAD-FMK was purchased from BD Pharmingen. Polybrene (hexadimethrine bromide) was obtained from Sigma. Anti-PKC (sc-937), anti-PKC (sc-208), anti-p38/SAPK4 (sc-7585), anti-MEK6 (sc-6073), and anti-phospho-ERK1/2 (sc-7383) antibodies were obtained from Santa Cruz Biotechnology. Anti-caspase 3 (AHZ0052) and anti-phospho-Tyr311-PKC (44-950) antibodies were from Biosource International. Anti-PARP antibody 556494 was from BD Pharmingen. Anti-ERK1/2 (M5670), anti-FLAG (F3165), and anti-ß-actin antibodies were obtained from Sigma. Anti-phospho-ATF2 antibody 9221 was from Cell Signaling Technology. Hydrogen peroxide (H2O2) was obtained from Calbiochem.
Cell culture and infection with recombinant adenovirus vectors.
The methods for culturing normal human foreskin keratinocytes were described previously (13). Adenoviruses encoding wild-type PKC and PKC and dominant-negative PKC were provided by T. Kuroki (53). Adenovirus encoding constitutively active PKC was provided by A. Samarel (69). Adenoviruses encoding constitutively active Raf-1 (Raf-BXB), constitutively active MEK6 (caMEK6), and wild-type FLAG-tagged p38 MAPK isoforms and were obtained from Y. Wang (76). An empty control adenovirus was generated by recombining pCA3 plasmid with the pJM17 adenovirus backbone in 293 cells. Recombinant adenoviruses were propagated in 293 cells and purified by cesium chloride centrifugation. The optimal multiplicity of adenoviral infection was determined by using the green fluorescent protein-encoding adenovirus (13). The adenoviruses were administered at the indicated MOIs in the presence of 2.5 µg of Polybrene/ml.
Immunoblot analysis.
Total cell extracts were prepared from cultured human epidermal keratinocytes. Equivalent amounts of protein were electrophoresed on reducing and denaturing polyacrylamide gels and transferred to nitrocellulose. The membranes were blocked, incubated with an indicated primary antibody, washed, and exposed to an appropriate horseradish peroxidase-conjugated secondary antibody. Secondary antibody binding was visualized by using chemiluminescence detection methods.
MAPK assays.
The adenovirus-delivered FLAG-tagged p38 MAPK isoforms were precipitated with anti-FLAG monoclonal antibody. Expression of individual FLAG-p38 isoforms was confirmed by immunoblotting with anti-FLAG antibody. The activities of precipitated FLAG-tagged kinases were measured by using a nonisotopic p38 MAPK assay method (Cell Signaling Tech). For p38 MAPK assays, FLAG-tagged p38 and isoforms are delivered to keratinocytes by using adenovirus. After 48 h, individual FLAG-tagged isoforms are precipitated with anti-FLAG monoclonal antibody. The activity of the precipitated FLAG-tagged kinase was measured as follows. Briefly, keratinocyte total cell lysates are prepared and equal amounts of total protein (100 µg) are precipitated for kinase assay. Precipitated kinases were then allowed to phosphorylate the p38 substrate, ATF2, in the presence of ATP. Phosphorylation of ATF2 was then analyzed by immunoblotting with an antibody specific for phospho-ATF2. ERK1/2 activity was assessed by immunoblotting with anti-phospho-ERK1/2 (Cell Signaling Tech). ERK1/2 activity was also measured by kinase assay (data not shown).
Mitochondrial membrane potential and cytochrome c release.
To monitor changes in mitochondrial membrane potential, keratinocytes growing on coverslips were stained with the MitoSensor Reagent (BD Biosciences) for 20 min at 37°C and then examined by fluorescence microscopy by using a band-pass filter that detects fluorescein and rhodamine. To assay for cytochrome c release, mitochondrial and cytosolic fractions were separated by using an ApoAlert cell fractionation kit (BD Biosciences/Clontech) in accordance with the manufacturer's instructions, and the fractions were analyzed for the presence of cytochrome c by immunoblotting with anti-cytochrome c antibody. The purity of each cytosol and mitochondrial fraction was controlled by monitoring the level of cytochrome c oxidase subunit IV (COX4, a mitochondrial marker) and ß-actin (cytosolic marker).
Flow cytometry analysis of DNA content.
To measure DNA content, keratinocytes were treated with trypsin, fixed in methanol, washed with phosphate-buffered saline (PBS), and treated with DNase-free RNase (40 µg/ml final concentration) at 37°C for 30 min. The cells were then stained with propidium iodide (50 µg/ml final concentration) and DNA content was analyzed by flow cytometry.
Confocal microscopy.
Human keratinocytes were plated onto 22-by-22-mm coverslips. After 24 h, cells were infected with the appropriate virus. After 24 to 48 h, the cells were fixed with 2% paraformaldehyde for 1 h, permeabilized with methanol for 10 min, and incubated with a primary antibody cocktail containing rabbit anti-ERK1/2 (M5670, 1:1,000; Sigma-Aldrich) and mouse anti-FLAG M2 (F3165, 1:1,000; Sigma-Aldrich). The secondary antibody cocktail contained the appropriate combination of Alexa Fluor 488-conjugated goat anti-rabbit immunoglobulin G (IgG; A11034, 1:1,000; Molecular Probes) and Cy3-conjugated sheep anti-mouse IgG (C2181, 1:1,000; Sigma-Aldrich). The coverslips were then sealed onto microscope slides by using DABCO antifade reagent (Molecular Probes) and examined by laser scanning confocal microscopy (LSM510; Zeiss, Thornwood, N.Y.) with a x63 N.A. 1.4 oil immersion plan-Apochromat objective. Green fluorescent ERK1/2 images were collected by using a 488-nm excitation light from an argon/krypton laser, a 488-nm dichroic mirror and a 500- to 550-nm band-pass barrier filter. Images of red FLAG-p38 fluorescence were collected by using a 543-nm excitation light from the HeNeI laser, a 543-nm dichroic mirror, and a 560-nm pass-filter. The images were analyzed, combined, and processed by using Adobe Photoshop (version 7.0) and are representative of at least three separate experiments in which five fields were examined.
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RESULTS
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PKC, MEK6, and p38 regulate cell morphology.
To determine whether PKC and p38 cooperate to promote keratinocyte cell death, we expressed PKC in the presence or absence of p38 and monitored the effects on cell survival. As shown in Fig. 1A, expression of PKC or p38 alone produces minimal change, and the phenotypes are similar to those observed after infection with the empty expression vector (EV). In contrast, expression of PKC in combination with p38 results in nuclear shrinkage, plasma membrane blebbing, cell rounding, detachment, and formation of spherical structures (Fig. 1A). We have previously shown that a PKC/Ras/MEKK1/MEK6 cascade regulates p38 function in keratinocytes (13, 54). Consistent with it being a member of this signaling cascade, constitutively active MEK6 (caMEK6) also cooperates with p38 to produce a remarkably similar morphological change. Figure 1B confirms that the adenovirus-delivered kinases are expressed. These morphological changes are reminiscent of those associated with programmed cell death, and so we next examined whether the cells were undergoing apoptosis. Figure 2 shows that treatment with a combination of PKC and p38 (PKC+p38) or caMEK6+p38 results in a 10-fold increase in the number of sub-G1 cells present in the population compared to untreated cultures. Individual expression of PKC, p38, or caMEK6 produces no change (not shown). It is important to note that the fraction of subG1 cells in the PKC+p38- and caMEK6+p38-treated groups represents a minimal estimate of the extent of cell damage, since a substantial fraction of dead cells is released from the dish or disintegrates during harvest. These results suggest that caMEK6+p38- and PKC+p38-expressing cells undergo cell death. However, the cell cycle evidence alone is not adequate to support a definitive diagnosis of apoptosis, and so we endeavored to measure additional death indicators.
PKC+p38 treatment stimulates cytochrome c release, mitochondrial membrane depolarization and killer caspase activation.
In many cell types, including keratinocytes (15, 16, 43), apoptosis involves the loss of the mitochondrial membrane potential with accompanied release of apoptogenic factors, including cytochrome c, and activation of cysteine-dependent aspartate-directed proteases known as caspases (65, 77, 83). We therefore measured the extent of cytochrome c release and mitochondrial membrane status. As shown in Fig. 3A, the altered morphology observed in Fig. 1A is associated with enhanced release of cytochrome c into the cytosol in PKC+p38- or caMEK6+p38-treated cells. Expression of PKC, caMEK6, or p38 alone produced no cytochrome c release (results not shown). We also measured the COX4 level. COX4 is a mitochondrial protein that is not released from damaged mitochondria and serves as a marker of the mitochondrial fraction. The ß-actin level was measured as a cytosolic marker. The distribution of these markers confirms that the compartments were successfully separated. We next assessed the effects of PKC+p38 or caMEK6+p38 on mitochondrial membrane potential. Mitochondria of healthy cells accumulate MitoSensor dye, forming intramitochondrial aggregates that fluoresce red. In apoptotic cells, dye monomers reside in the cytoplasm and fluoresce green. As anticipated, red fluorescence, which is indicative of intact mitochondria, was observed in empty vector-infected cells. In addition, cells expressing only PKC, caMEK6 or p38 also fluoresced red (Fig. 3B). In contrast, cells treated with PKC+p38 or caMEK6+p38, fluoresce green, indicating a loss of mitochondrial membrane potential.
The release of cytochrome c into the cytoplasm ultimately results in caspase cleavage and activation (28, 60, 83). Figure 4A shows that the expression of PKC+p38 or caMEK6+p38 results in increased cleavage of procaspase 3, a caspase that is activated via mitochondrion-dependent mechanisms (28, 60, 83). Figure 4B shows that the expression of PKC+p38 or caMEK6+p38 also results in enhanced cleavage of PARP, a caspase 3 target. These findings are consistent with a model wherein mitochondrion destruction permits the release of cytochrome c that, in turn, activates the caspase-dependent cell death cascade. Activation of each apoptotic marker requires the simultaneous presence of p38 plus the upstream activator kinase (i.e., MEK6 and PKC).
If the process is really caspase dependent, it should be possible to attenuate the response by inhibiting caspase activity. We therefore treated cells with empty vector, PKC+p38, or caMEK6+p38 in the presence or absence of Z-VAD-FMK, a pan-caspase inhibitor. Indeed, as shown in Fig. 4C, treatment with this agent nearly completely inhibited the appearance of the apoptotic morphology. In addition, Z-VAD-FMK blocks procaspase 3 and PARP degradation induced by the coexpression of p38+PKC or p38+caMEK6 (not shown).
PKC, MEK6, and regulation of p38 activity.
The findings presented above suggested that PKC- or MEK6-dependent activation of p38 may be required for keratinocyte cell death. To test this hypothesis, PKC or caMEK6 was expressed with p38 and, after 48 h, FLAG-p38 was precipitated with a FLAG-specific antibody. The activity of the precipitated enzyme was measured based on its ability to phosphorylate the p38 substrate, ATF2 (23). When expressed alone, FLAG-p38 is active at a low level that is only detected when the blot in Fig. 5 is overexposed (not shown). In contrast, expression of PKC or caMEK6 with p38 results in marked p38 activation (Fig. 5).
Required reduction in ERK1/2 activity.
It has been suggested, in a variety of cell models, that the balance between activity of JNK and p38 versus ERK1/2 activity regulates cell fate (22, 46, 79). We therefore considered whether ERK1/2 activity is altered in cells displaying increased p38 activity and undergoing apoptosis. Cells were infected with PKC, caMEK6, or p38 encoding adenovirus, and the endogenous ERK1/2 activity was monitored after 48 h. Figure 6 shows that when p38 activity is increased (i.e., cells expressing PKC or caMEK6 with p38), the P-ERK1/2 level is markedly reduced. This represents an authentic reduction in activity, since the absolute level of ERK1/2 protein is not altered (ERK1/2).
To determine whether reduced ERK1/2 activity is necessary for apoptosis, we forced maintenance of ERK1/2 activity by expressing constitutively active Raf-1 (caRaf-1), a specific upstream activator of ERK1/2, in the presence of various combinations of PKC, caMEK6 and p38. Figure 7A confirms that PKC+p38 expression suppresses ERK1/2 activity and that this suppression is partially reversed when caRaf-1 is present. We were also concerned to demonstrate that shifting the p38/ERK1/2 activity balance in favor of ERK1/2 inhibits the apoptotic response. We therefore determined whether forced ERK1/2 activation inhibits the PKC+p38-dependent activation of PARP cleavage. As shown in Fig. 7B, expressing PKC+p38 in keratinocytes results in PARP cleavage. Figure 7B also shows that expression of caRaf-1 inhibits the PKC+p38-dependent PARP cleavage.
Evidence for PKC-dependent intranuclear relocation of p38-ERK1/2.
Our results (Fig. 5 and 6) clearly show that p38 activity and ERK1/2 activity are coordinately and inversely regulated by PKC and MEK6. One possible explanation for the p38-dependent reduction in ERK1/2 activity is an interaction of p38 with ERK1/2. A p38-ERK1/2 complex has been identified in HeLa cells (80), and we have previously shown that an endogenous p38-ERK1/2 complex is constitutively present in keratinocytes (22). Regulatory stimuli, including treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA) and okadaic acid, alter activity in this complex to increase p38 and decrease ERK1/2 activity (22). We therefore assessed whether such a p38-ERK1/2 complex, which could function to integrate the reciprocal regulation, is observed during PKC/MEK6/p38-dependent keratinocyte apoptosis. Cells were infected for 48 h with FLAG-p38-encoding virus in the presence or absence of PKC- or caMEK6-encoding virus. Extracts were prepared and FLAG-p38 was immunoprecipitated. The p38 immunoblot in Fig. 8A (lower blot) confirms that FLAG-p38 is precipitated. The ERK1/2 immunoblot (Fig. 8A, upper panel) demonstrates that ERK1 is precipitated with p38. We also performed the inverse experiment: precipitation with anti-ERK1/2. The anti-ERK1/2 immunoblot shown in Fig. 8B (lower panel) confirms that ERK1 and ERK2 are precipitated. The anti-FLAG blot (Fig. 8B, upper panel) shows that FLAG-p38 coprecipitates with ERK1/2.
These findings confirm that a FLAG-p38-ERK1/2 complex exists in keratinocytes (Fig. 8) and that PKC or caMEK6 cause p38 activation with coordinate ERK1/2 inactivation (Fig. 5 and 6). A recent study indicates that mitogen stimulation of cells results in rapid ERK1/2 accumulation in the nucleus and that prolonged stimulation results in intranuclear ERK1/2 inactivation (58, 75). We therefore explored the possibility that some p38 and ERK1/2 may relocate to the nucleus after PKC stimulation. As shown in Fig. 9A, p38 and ERK1/2 colocalize in the cytoplasm of resting keratinocytes (empty vector). PKC expression, in contrast, results in the appearance of some p38 and ERK1/2 in the nucleus. To provide additional evidence of FLAG-p38 and ERK1/2 movement into the nucleus and to assess the activation state of these kinases, we isolated nuclear extracts from keratinocytes expressing PKC in the presence or absence of p38. Figure 9B shows that a substantial quantity of FLAG-p38 is nucleus associated in the absence of PKC and that this amount does not change in the presence of PKC. However, only when PKC is present does activated, P-FLAG-p38, accumulate inside the nucleus. A similar distribution is observed for endogenous p38 (not shown). The lower panels in Fig. 9B show that intranuclear ERK1/2 levels increase in the presence of PKC; however, this ERK1/2 is dephosphorylated (inactive). The association of FLAG-p38 with the nuclear fraction in Fig. 9B, in the absence of PKC, is surprising considering that no nuclear FLAG-p38 is present as measured by immunohistology (Fig. 9A). However, additional images (not shown) suggest that some FLAG-38 is constitutively associated with the outer nuclear surface in the absence of PKC and only moves into the nucleus in the presence of PKC.
Active PKC is required for regulation.
The above results suggest a key role for PKC in driving apoptosis. However, it was not clear whether PKC catalytic activity is required for a response. We therefore monitored changes in cell morphology and p38 activity after expression of intact or kinase-inactive (dominant-negative) PKC. As shown in Fig. 10A, expression of intact PKC increases p38 activity (Fig. 10A) and stimulates morphological change (Fig. 10B), but inactive, dominant-negative PKC (dnPKC) does not activate p38 or cause altered morphology. These finding indicate that PKC kinase activity is required to promote apoptosis. We also anticipated that dominant-negative PKC should inhibit the ability of native PKC to induce apoptosis. Indeed, Fig. 10C shows that expression of dnPKC inhibits the PKC+p38-associated apoptosis.
A second important issue is whether activation of apoptosis is a property shared by all PKC and p38 isoforms. We first sought to determine whether the classical PKC isoform, PKC, can promote cell death when coupled with p38. As shown in Fig. 11A, this combination does not cause altered morphology. We also show that the combination of PKC+p38 produces no response. Thus, PKC does not promote cell death in keratinocytes when coupled to or isoforms of p38 MAPK. We next determined whether p38 plays a role as a downstream mediator of PKC-induced death signal. As shown in Fig. 11A, coupling of PKC with p38 does not stimulate morphological change. In addition, coupling of caMEK6 with p38 does not produce an apoptotic response (not shown). Moreover, p38 activity can be inhibited without altering p38-dependent cell death. Treatment of cells with SB203580, an inhibitor of p38 and -ß, but not p38 (11, 40), does not suppress PKC+p38 (Fig. 11B)- or caMEK6+p38 (not shown)-associated cell death, further supporting the notion that p38 and p38ß do not have a role in this process.
PKC is a member of the novel PKC subfamily (49, 50). Thus, it is possible that novel PKC isoforms may share an ability to function with p38 to promote keratinocyte apoptosis, since multiple novel PKC isoforms can promote keratinocyte apoptosis (8, 15, 16). To address this possibility, we tested the combination of PKC+p38. As shown in Fig. 11C, PKC+p38 expression produces a morphological change that is very similar to that produced by PKC+p38. Moreover, PKC induces increased p38 activity and decreased ERK1/2 activity similar to that observed when PKC or caMEK6 are expressed with p38 (not shown). In addition, the combination of PKC+p38 promotes increased procaspase 3 cleavage (not shown). The immunoblot in Fig. 11D confirms that the viral expression vectors actually delivered the p38, PKC, and PKC proteins.
These results suggest that the PKC+p38 combination does not influence morphology. We would anticipate that this combination should also not regulate the biochemical responses associated with the morphological change. Figure 12A tests the ability of PKC to increase p38 and reduce ERK1/2 activity. These results confirm that PKC cannot substitute for PKC as a regulator of p38 or ERK1/2 activity. As a measure of a downstream biochemical response, we measured PKC+p38-dependent regulation of procaspase 3 cleavage. The results shown in Fig. 12B confirm that PKC cannot substitute for PKC and function, in conjunction with p38, to promote procaspase 3 cleavage.
PKC tyrosine phosphorylation.
Covalent modification can alter PKC stability and activity (3, 37, 50, 68). Phosphorylation of PKC at Y311 is thought to increase activity (29, 39, 63). We therefore examined whether PKC Y311 phosphorylation is altered in cells undergoing PKC+p38-dependent apoptosis. Keratinocytes were treated with empty vector, PKC, or PKC+p38. Cell extracts were then assayed by immunoblotting for the presence of PKC and PKC-P-Y311. Figure 13A shows that vector-delivered PKC is expressed, and Fig. 13B shows that it is phosphorylated at Y311. In contrast, endogenous PKC is not phosphorylated at Y311 in unstimulated cells. This finding will be considered further in the context of H2O2-dependent regulation of endogenous PKC phosphorylation (below).
H2O2 promotes keratinocyte apoptosis via a PKC/p38-dependent signaling pathway.
We next determined whether known inducers of keratinocyte apoptosis operate via a PKC- and p38-dependent mechanism. H2O2 is a known keratinocyte stress agent, produced in response to UV light treatment, that promotes keratinocyte apoptosis (6, 12, 29, 57). We determined whether H2O2-dependent cell death is mediated via the PKC and p38-ERK1/2 pathway. As shown in Fig. 14A and B, H 2O2 treatment of keratinocytes produces marked morphological changes, including shrinkage of the nuclei, cell rounding, formation of balloon-like blebs (spheres) (Fig. 14B, arrow), and cell detachment. The phenotype is virtually indistinguishable from that observed following overexpression of PKC+p38 (Fig. 1). The H2O2-induced apoptotic morphology is completely blocked by pretreatment of the cells with rottlerin, a PKC-selective inhibitor (31). A recent report indicates that rottlerin also uncouples respiration and thus is not an ideal PKC inhibitor (67). However, we believe it can still have value, when used in conjunction with other evidence, since it inhibits the PKC-dependent keratinocyte morphological change.
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FIG. 14. H2O2 treatment causes apoptosis-associated morphological changes. (A) Keratinocytes were pretreated for 1 h with 10 µM rottlerin and then treated for 2 h with 10 mM H2O2 in the presence or absence of 10 µM rottlerin. The cells were then photographed. (B) Photographic enlargement of the H2O2-treated morphology. The arrow indicates an apoptotic-associated sphere. (C) H2O2 treatment enhances loss of mitochondrial membrane potential. Human keratinocytes, grown on coverslips, were treated in the absence or presence of H2O2 for 4 h and then stained with MitoSensor reagent. Fluorescence was monitored by epifluorescence microscopy. The arrows show the location of a representative apoptotic sphere.
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H2O2 enhances keratinocyte cell death.
We next determined whether treatment with H2O2 causes keratinocyte apoptosis as measured by loss of mitochondrial membrane potential. Keratinocytes were treated in the absence or presence of H2O2 for 4 h and stained with MitoSensor dye prior to the detection of fluorescence. The dye fluoresces red in untreated cells, a finding indicative of intact mitochondria, but green in H2O2-treated cells, a result indicative of a loss of mitochondrial membrane potential (Fig. 14C).
H2O2 causes PKC phosphorylation and inversely regulates p38-ERK1/2 activity.
We next examined the role of PKC and p38-ERK1/2 signaling in mediating the response to H2O2. We monitored the effects of H2O2 treatment on PKC phosphorylation and on ERK1/2 and p38 activity. As shown in Fig. 15A, H2O2 treatment increases phosphorylation of PKC at Y311, a modification that is known to be associated with PKC activation (29, 39, 63). This response is similar to that observed after expression of PKC in keratinocytes (compare with Fig. 13B). We would anticipate that if H2O2 works to promote apoptosis via a mechanism similar to that observed after PKC+p38 expression, then treatment would increase p38 and decrease ERK1/2 activity. Indeed, as shown in Fig. 15B and C, H2O2 treatment increased p38 and inhibited ERK1/2 activity. It should be noted that, although keratinocytes are relatively tolerant of H2O2 (74, 81), all of the H2O2-dependent responses described in Fig. 14 and 15 were observed at concentrations ranging from 1 to 10 mM. The only difference was the rate of response: treatment with 1 mM H2O2 for 24 h matched the response after 10 mM H2O2 treatment for 2 h.
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DISCUSSION
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The PKC family includes 11 serine/threonine kinases classified into three groups. Conventional/classical PKCs (cPKC, -ßI, -ßII, and -) are calcium, phospholipid-, and diacylglycerol-dependent, novel PKCs (nPKC, -, -, -, and -µ) are calcium independent, and atypical PKCs (aPKC and -) are calcium- and diacylglycerol independent (49, 50, 52). Differences in subcellular localization, cofactor requirements, substrate specificity, and tissue distribution suggest distinct biological functions for each isozyme (47, 52). Each of the five PKC isozymes (PKC, -, -, -, and -), expressed in keratinocytes (18, 27, 30, 44, 55), differentially regulate keratinocyte proliferation and differentiation. For example, PKC and - regulate keratinocyte cell cycle protein function (34-36, 53), and expression of differentiation-dependent genes (41) such as type I transglutaminase (73), involucrin (22-25), and transcriptional regulators (23, 25). Taken together, these findings suggest that novel PKCs are prodifferentiation and antisurvival (21). In contrast, classical PKC isoforms (e.g., PKC) can oppose these responses (17, 21).
PKC isoforms have also been implicated in cell death responses. In keratinocytes, cell death is a distinct, but related process, to cell differentiation. Apoptotic keratinocyte cell death involves killer caspase activation and is frequently associated with exposure to a toxic stimuli (e.g., UVB irradiation, etc.) (15, 16, 46, 66, 71). UVB-dependent apoptosis is associated with release of the PKC catalytic subunit (15). In addition, apoptotic stimuli may induce translocation of PKC from the cytoplasm to the plasma membrane, mitochondria, Golgi, endoplasmic reticulum, or nucleus (3). Translocation of PKC to the cell membrane in response to UVB irradiation has been reported in mouse epidermal cell lines (8). Yuspa and coworkers have shown that treatment of PKC overexpressing keratinocytes with phorbol ester results in activation of PKC and its subsequent movement to mitochondria, followed by loss of mitochondrial membrane potential and apoptosis (43).
PKC and p38 act to stimulate mitochondrion-dependent apoptosis.
Given the central role of PKC isoforms in regulating keratinocyte death and differentiation, understanding the signaling pathways that mediate PKC action is an important goal. Our previous studies have implicated a signaling cascade that includes novel PKCs, Ras, MEKK1, and MEK6 that targets p38 and ERK1/2 to regulate keratinocyte differentiation (25). In the present study, we identify this pathway as having a central role in regulating keratinocyte cell death. Our studies show that PKC, when coupled with p38, causes keratinocyte apoptosis. This response requires functional PKC kinase activity and is associated with potent and sustained PKC-stimulated activation of p38. This pathway produces a classical apoptotic response that includes accumulation of cells in sub-G1 growth phase, mitochondrial cytochrome c release, loss of mitochondrial membrane potential, caspase activation, and PARP degradation. The absence of procaspase 8 cleavage (not shown) suggests that apoptosis does not involve nonmitochondrial, death receptor-dependent pathways (28, 72). This is interesting, since receptor-dependent caspase 8 activation and apoptosis is observed in keratinocytes treated with UVB (38), undergoing anoikis (59) or treated with DNA-damaging agents (61). Thus, although UVB, for example, can trigger both receptor-dependent and mitochondrion-associated caspase activity, PKC/MEK6/p38 appears to specifically activate only mitochondrion-associated apoptosis.
Which PKC isoforms activate apoptosis?
Our results indicate that PKC, when coupled with p38, promotes apoptosis. Substitution of PKC for PKC results in a similar pattern of regulation; however, if PKC, a classical isoform, replaces PKC, no apoptosis is observed. These observations are consistent with previous reports suggesting that classical PKC isoforms (e.g., PKC) promote cell survival and inhibit differentiation (21, 32). Moreover, the observation that two novel PKC isoforms (PKC and PKC) promote a similar apoptotic process, suggests that the novel PKC isoforms are major mediators of this response. These findings are consistent with other studies of human keratinocytes (8, 15, 21, 34-36, 53) and of other cell types, suggesting that the novel PKCs are prodifferentiation/antisurvival kinases. However, it is clear that the regulation is not always straightforward (32). In our cell culture model, coupling PKC with p38 fails to cause apoptosis (T. Efimova and R. L. Eckert, unpublished data). This is despite the fact that PKC is a potent p38 activator (23) and is consistent with the observed opposite roles of PKC and PKC in UVB-induced apoptosis. In this context, PKC is a negative regulator of the apoptotic response (45). In addition, both classical PKC and novel PKC mediate apoptosis in prostate cancer cells (70), and PKC is prosurvival in cardiac myocytes (33). Moreover, in PKC-overexpressing transgenic mouse skin, treatment with TPA causes apoptosis (5). Thus, it is clear that additional studies will be required to understand the subtle differences that account for this diverse regulation.
Phosphorylation of expressed PKC is associated with PKC/MEK6/p38-dependent keratinocyte apoptosis.
PKC can be modified by enzymatic cleavage and by phosphorylation (3, 37). We find that expressed PKC is phosphorylated constitutively at Y311. Phosphorylation at Y311 renders PKC resistant to caspase cleavage and appears to promote the ability of the intact PKC to mediate death (37). It is possible that the tyrosine phosphorylation activates PKC - thereby facilitating its apoptotic role (37). As noted below, PKC Y311 phosphorylation is also observed when keratinocytes are treated with physiological stress agents.
Moreover, PKC proteolysis during apoptosis is a caspase 3-dependent event that results in release of the active PKC catalytic domain (3, 26). Catalytic fragment release is observed after keratinocyte treatment with UV radiation (15). In contrast, in the present study, we detected no increase in PKC catalytic fragment production during PKC+p38-dependent apoptosis. This finding agrees with previous reports indicating that caspase-dependent PKC cleavage is not necessarily required for apoptosis (3, 37).
Role for p38.
The p38 MAPK cascade includes four isozymes (i.e., p38, -ß, -, and -), and each can produce distinct cellular responses (54). Activation of p38 isoforms can be either pro- or antiapoptotic (48, 54). p38 isoform is by far the most thoroughly studied member of the p38 family (48, 54). Numerous reports document proapoptotic and prosurvival effects of p38 (10, 76, 82). Keratinocytes express p38, -ß, and -, but not p38 (14). Several lines of evidence presented here suggest that p38 activity is required for keratinocyte apoptosis. First, PKC and MEK6 can couple with p38 to drive apoptosis. Second, p38 cannot couple with PKC or MEK6 to induce apoptosis. Third, treatment with an inhibitor, SB203580, that inhibits p38 and -ß, but not p38 (11, 40), does not inhibit apoptosis. It is interesting that although a role for p38 has been confirmed in only a limited number of systems, a clear role for p38 as a keratinocyte prodifferentiation regulator has been confirmed in several contexts and in response to several stimuli (1, 20, 22, 23). The present study is the first to document a role for p38 in regulating keratinocyte apoptosis.
Physiological role of PKC- and p38-ERK1/2-associated cell death.
An important issue is whether similar changes in morphology and signaling kinase activity occur in the presence of physiologic regulators of keratinocyte apoptosis. In this regard, okadaic acid, a known inducer of keratinocyte apoptosis, promotes keratinocyte death via a mitochondrial/caspase/PARP pathway, and this response requires increased endogenous p38 activity and decreased ERK1/2 activity (Efimova and Eckert, unpublished). PKC expression has been reported to sensitize keratinocytes to phorbol ester-dependent apoptosis (43). If p38 acts downstream of PKC to promote apoptosis, we would expect that p38 expression would also facilitate cell death in response to phorbol ester treatment. Indeed, we observed that p38 expression renders keratinocytes susceptible to phorbol ester-induced apoptosis with changes identical to that observed in PKC/p38-expressing cells (results not shown).
Moreover, in the present study, we show that hydrogen peroxide, an important mediator of oxidative stress-induced cell death and a known activator of keratinocyte apoptosis, promotes PKC Y311 phosphorylation, increases p38 activity, and decreases ERK1/2 activity. Hydrogen peroxide is produced in response to keratinocyte exposure to UV light (6, 12). These changes are coupled with the same morphological alterations (nuclear shrinkage, cell death, membrane blebbing, and sphere formation) that accompany PKC+p38 overexpression-dependent apoptosis. Taken together, these findings suggest that the PKC- and p38-ERK1/2-dependent pathway mediates cell death in response to keratinocyte apoptotic agents. These findings are in agreement with data from PKC knockout mice which show that PKC-deficient smooth muscle cells are resistant to apoptosis induced by various stimuli, including H2O2 (42). These cells exhibit diminished cytochrome c release, caspase activation, and PARP cleavage in response to H2O2 treatment, suggesting that PKC function is required for this response. Moreover, stress-induced p38 activity is significantly reduced in these cells, a finding consistent with our suggestion that PKC is an upstream activator of p38.
p38 activation and ERK1/2 suppression: coordination mediated via a p38-ERK1/2 complex?
As noted above, our studies suggest that p38 activity is required for PKC-induced keratinocyte apoptosis. However, a remarkable feature of this regulation is the coupling of p38 activation with suppression of ERK1/2 activity. This finding is consistent with the suggestion that cell fate is decided by a balance between prosurvival ERK1/2 signaling and prodeath stress-activated protein kinase signaling (46, 79). Our studies reveal two significant aspects of this regulation. First, as first described in our recent report (22) and as confirmed here, p38 and ERK1/2 are part of a complex that is constitutively present in stimulated and nonstimulated keratinocytes. The presence of this complex suggests that p38 activation is, either directly or indirectly, coupled to suppression of ERK1/2 activity. Second, the present study indicates that PKC stimulation of keratinocytes results in coaccumulation of p38 and ERK1/2 into the cell nucleus. Comovement of the p38/ERK partners to the nucleus in response to PKC is an important finding. In resting fibroblasts, ERK1/2 is anchored to the upstream ERK1/2 activator, MEK1, in the cytoplasm (58). In response to MAPK stimulation, ERK1/2 is released from MEK1 and ERK1/2 accumulates in the nucleus (4, 56). Nuclear accumulation of ERK1/2 is ultimately associated with activation of phosphatases that inactivate ERK1/2 (75). Our studies suggest that p38 and ERK1/2 are distributed in the cytoplasm and on the outer nuclear surface in resting keratinocytes. In the presence of PKC, some p38 and ERK1/2 kinases are distributed to the nucleus interior. Both kinases remain nuclear even 48 h after initiation of treatment, and this localization is associated with maintenance of p38 activity and inactivation of ERK1/2. Although ERK1/2 dephosphorylation may also occur in the cytoplasm, our findings are consistent with the idea that that the nucleus is a site of ERK1/2 inactivation by resident phosphatases (75). Interestingly, a recent study describes a direct association of a p38 splice isoform, Mxi2, with ERK1/2 that has a profound effect on the function of ERK1/2 in the nucleus (64). Specifically, Mxi2 binding to ERK1/2 appears to hinder ERK1/2 dephosphorylation, which results in prolonged ERK1/2 activation in the nucleus (
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Abstract
Introduction
