RelA and MAPK Pathways in Keratinocytes in Response to Sulfur Mustard

ORIGINAL ARTICLE

Role of NF-jB/RelA and MAPK Pathways in Keratinocytes in Response to Sulfur Mustard Bernd Rebholz1,3, Kai Kehe2,3, Thomas Ruzicka1 and Rudolf A. Rupec1 Sulfur mustard (SM) is a strong vesicant that has been used as a chemical warfare agent. To understand the molecular mechanisms that underlie the inflammatory skin reaction in response to SM, we analyzed the activation pattern of the NF-kB and mitogen-activated protein kinase (MAPK) pathways. Keratinocytes responded with an induction of the canonical NF-kB pathway, including activation of IkB kinase 2, followed by phosphorylation and degradation of IkBa and of the transactivating subunit RelA at Ser536. The biphasic NF-kB response was strictly dependent on the transactivating subunit RelA, as demonstrated by keratinocytes lacking RelA. Parallel to NF-kB activation, we observed an induction of the Raf-1/MEK1/2/ERK1/2/MSK1 and MKK3/ 6/p38/MSK1 pathways. Although mitogen and stress-activated kinase 1 has been described as a RelA kinase with Ser276 as its target, this site remained unphosphorylated in response to SM. A further MAPK pathway induced by SM was the MKK4/7/JNK1/2 pathway, which resulted in phosphorylation of the transcription factor activating transcription factor-2, but not c-Jun. Our results indicate that SM induces a complex cellular response in keratinocytes, with the activation of three MAPK pathways and the NF-kB pathway. Journal of Investigative Dermatology (2008) 128, 1626–1632; doi:10.1038/sj.jid.5701234; published online 17 January 2008

INTRODUCTION Sulfur mustard (SM) [bis-(2-chloroethyl)-sulfide], also termed mustard gas, is a potent vesicant that has been used as a chemical warfare agent in various conflicts during the twentieth century and recently in 1988 during the Iraq–Iran conflict. Several delayed complications can be observed in individuals exposed to SM (Hefazi et al., 2006). To date, SM remains a significant military and civilian threat. SM penetrates the skin and depending on concentration, skin moisture, body site, and temperature, induces inflammation, blistering, ulceration, and necrosis. At high doses, SM has been associated with bone marrow and immune suppression (Hassan and Ebtekar, 2001; Ghanei, 2004). Besides its inflammation-inducing properties, SM is mutagenic and carcinogenic. The clinical and histopathological changes of the skin following SM exposure have been described in detail (Requena et al., 1988; Smith et al., 1995; Balali-Mood and Hefazi, 2005; Kehe and Szinicz, 2005; Naraghi et al., 2005). 1

Department of Dermatology and Allergology, Ludwig-Maximilian-University Munich, Munich, Germany and 2Bundeswehr Institute of Pharmacology and Toxicology, Munich, Germany

3

These two authors contributed equally to this work.

Correspondence: Dr Rudolf A. Rupec, Department of Dermatology, LudwigMaximilian-University Munich, Frauenlobstrasse 9-11, Munich D-80337, Germany. E-mail: [email protected] Abbreviations: AP-1, activator protein 1; ATF, activating transcription factor; ERK, extracellular signal-regulated kinase; IKK, IkB kinase; JNK, Jun-Nterminal kinase; MAPK, mitogen-activated protein kinase; MKK, mitogenactivated protein kinase kinase; MSK, mitogen- and stress-activated kinase; SM, sulfur mustard Received 1 February 2007; revised 25 October 2007; accepted 12 November 2007; published online 17 January 2008

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The inflammatory response is mediated by cytokines that are mainly released by keratinocytes, fibroblasts, and skininfiltrating inflammatory cells (Arroyo et al., 2000; Ricketts et al., 2000; Rogers et al., 2004; Sabourin et al., 2004). The transcription factor NF-kB is a complex formed by homo- and heterodimerization of the NF-kB/Rel family members p50 (NF-kB1), p52 (NF-kB2), RelA (p65), RelB, and c-Rel. NF-kB, most often composed of p50 and p65/ RelA, is a crucial mediator of inflammatory processes (Karin and Greten, 2005). Inflammatory stimuli that are capable of activating the catalytic IkB kinase 2 (IKK2) and the regulatory subunit IKKg result in serine phosphorylation, ubiquitination, and subsequent degradation of IkBa, which allows translocation of NF-kB to the nucleus (Karin and Ben-Neriah, 2000; Li and Verma, 2002; Hayden and Ghosh, 2004). In addition to degradation of IkBa, phosphorylation of the transactivating subunit RelA is an essential regulatory step for its transcriptional activity (Viatour et al., 2005). The three typical elements of a classic mitogen-activated protein kinase (MAPK) cascade are a MAPK, a kinase that activates the MAPK (mitogen-activated protein kinase kinase (MKK)), and the kinase that activates the MKK (MAP3K) (Chang and Karin, 2001). The extracellular signal-regulated kinase (ERK), the Jun-N-terminal kinase (JNK), and p38 MAPK are the most common cascades. Up to the level of the MAPKs, the cascades resemble a funnel, as each MAPK can be activated by two MAP2K, which in turn are activated by multitudinous MAP3Ks. We used mouse keratinocytes to analyze the effect of SM on NF-kB and MAPK signal transduction pathways. The canonical NF-kB pathway and MAPKs are substantially involved in the regulation of genes coding for inflammatory & 2008 The Society for Investigative Dermatology

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cytokines. The NF-kB signal transduction pathway is induced through activation of IKK2, degradation of IkBa, and phosphorylation of RelA at Ser536. The relevance of RelA was demonstrated in RelA-deficient keratinocytes that lacked NF-kB activation following SM exposure. In addition, ERK, JNK, and p38 MAPK were induced by SM and phosphorylated activating transcription factor-2 (ATF-2) as a downstream target. ATF-2-binding sites are located in the promoter region of tumor necrosis factor-a and other inflammatory cytokines (Tsai et al., 1996). ATF-2 deficiency results in a reduced induction of inflammatory genes (Reimold et al., 2001). The findings presented here provide an insight into the inflammation-regulating signal transduction pathways induced by SM.

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RESULTS SM activates RelA through phosphorylation at Ser536

The transcription factor NF-kB is a potential candidate for mediating SM-induced cellular response. To study NF-kB activation by SM, keratinocytes isolated from wild-type mice were exposed to 100 mM SM and subsequently analyzed with specific antibodies that indicate the activation status of the canonical NF-kB activation pathway. Three hours after exposure to SM there was no reduction in the percentage of surviving cells compared to unexposed cells. 24 hours after exposure 92% of the cells survived. Both components of the IKK signalosome, IKK1 and 2, are present in keratinocytes. However, only IKK2 became phosphorylated as early as 2 minutes after exposure, and for at least 10 minutes following SM (Figure 1, lane 3). IKK1 phosphorylation could not be detected with a phosphospecific antibody (data not shown). The activation of IKK2 resulted in a phosphorylation of IkBa at Ser32 and Ser36, followed by IkBa degradation (Figure 1, lanes 4–6). In addition to IkBa, Ser536 located in the transactivation domain of RelA is yet another target of IKK2 (Sakurai et al., 1999). Phosphorylation of Ser536 favors transcription mediated by TATA-binding, protein-associated factor II31, a component of transcription factor II D (TFIID) (Buss et al., 2004). In the absence of phosphorylation, the hydrogen bond favors binding of the corepressor N-terminal enhancer of split to the RelA terminal transactivation domain. Treatment of keratinocytes with SM resulted in phosphorylation of Ser536 within less than 10 minutes and remained constant for at least 30 minutes (Figure 1, lanes 3–6). Thus, NF-kB/RelA is activated by SM via IKK2 phosphorylation, with subsequent IkBa phosphorylation and degradation. Next, we studied the effect of SM on the DNA-binding activity of the nuclear transcription factor NF-kB in keratinocytes. SM treatment induced a biphasic and strong activation of NF-kB-binding activity. A first induction was observed between 1 and 3 hours (Figure 2a, lanes 2–4). Binding activity dropped after 4 hours. A second peak of NF-kB activity appeared after 6 hours (Figure 2a, lane 7). To confirm the relevance of the transactivating subunit RelA, keratinocytes that were deficient for RelA (RelAK5D/K5D) were stimulated with SM. In contrast to wild-type keratinocytes, no induction of NF-kB was observed over the entire time period (Figure 2a and b). The activated NF-kB complex is composed of the

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P -RelA (Ser 536) β-Actin Figure 1. Activation of the canonical NF-jB pathway by SM. Keratinocytes were stimulated with SM for the indicated times. Cell lysates were fractionated by SDS-PAGE and then analyzed by Western blotting using antibodies that detected IKK1, IKK2, IkBa, and RelA in their unphosphorylated as well as phosphorylated condition.

subunits p50 and RelA (Figure 2b). These results demonstrate that the canonical NF-kB pathway via RelA is activated by SM. NF-kB activation by SM depends on RelA, which cannot be bypassed by other NF-kB family members. SM activates MAPK pathways

Besides the NF-kB pathway, the MAPK pathways are also important regulators of the cellular response to exogenous inflammatory stimuli. We were interested in the extent to which the ERK, JNK, and p38 MAPK cascades are involved in the cellular response to SM. The Raf/MEK/ERK pathway is evolutionarily conserved. Raf-1 is activated by SM, as demonstrated by phosphorylation at Ser338 (Figure 3, lanes 2–6), and activates MEK1/2 by phosphorylation at Ser217 and Ser221 in the MEK activation loop (Figure 3, lanes 3–6). A downstream target of ERK1/2 (Figure 3, lanes 2–6) is the mitogen- and stress-activated kinase 1 (MSK1), which is phosphorylated within 10 minutes, with maximal values within 60 minutes (Figure 3, lanes 4–6; Deak et al., 1998; Ryder et al., 2000). MSK1 is also a RelA kinase that phosphorylates RelA at Ser276, enhancing transcriptional activation of NF-kB (Vermeulen et al., 2003). We could not detect any phosphorylation of RelA at this residue (Figure 3), indicating that phosphorylation of RelA at Ser276 does not seem to be involved in the cellular response to SM. www.jidonline.org 1627

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Figure 2. The effect of SM on the activity of NF-jB in keratinocytes, and the relevance of RelA. Keratinocytes from wild-type (a) and RelA-deficient (b) mice were challenged for the indicated times (lanes 2–7) with SM. The first lane shows unstimulated cells that were used as control (Co). PMA (50 ng ml1) was used as positive control. Nuclear extracts were incubated with a 32P-labeled NF-kB probe and analyzed by electrophoretic mobility-shift assay and ELISA, and specified for NF-kB subunits by ELISA. The arrow indicates the position of specific complexes.

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β-Actin Figure 4. Phosphorylation of p38 MAPK by SM-triggered keratinocytes. Keratinocytes were treated for the indicated times with SM. The first lane shows unstimulated cells that were used as control (Co). Protein extracts were subjected to western blotting using antibodies against the unphosphorylated and phosphorylated p38 MAPK, as well as the phosphorylated form of MKK3/6.

β-Actin Figure 3. Analysis of the effect of SM on the activation of the Raf/MEK/ERK pathway. Keratinocytes were treated for the indicated times with SM. The first lane shows unstimulated cells that were used as control (Co). Cell lysates were analyzed with antibodies against the unphosphorylated and phosphorylated forms of Raf-1, MEK1/2, ERK1/2, MSK1, and RelA after fractionation by SDS-PAGE.

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As numerous cellular stress stimuli can activate p38 MAPK, we investigated whether p38 MAPK is also involved in the keratinocyte response to SM. p38 MAPK is activated by phosphorylation of a conserved Thr180–Gly–Tyr182 motif. Phosphorylation of Thr180 and Tyr182 was detected within 10 minutes after exposure to SM (Figure 4, lanes 3–6).

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β-Actin Figure 6. SM induces an upregulation of Cxcl1 and Cxcl2 mRNA levels. Induction of Cxcl1 and Cxcl2 upon treatment with SM. Semi-quantitative reverse transcriptase-PCR for Cxcl1 and Cxcl2 was performed and b-actin was used for standardization. Inhibition of Cxcl1 and Cxcl2 induction after 1-hour preincubation with PTD-p65–P6 (lane 3), a specific inhibitor of RelA phosphorylation at Ser536. No inhibition was detected after preincubation with control peptide (PTD) (lane 4).

P -ATF-2 β-Actin Figure 5. The effect of SM JNK cascade. Total cellular extracts were prepared and analyzed by western blotting for the activation status of JNK1/2. The first lane shows unstimulated cells that were used as control (Co). The activation of JNK1/2 was paralleled by the activation of two JNK kinases, MKK4 and MKK7. Activation of JNK1/2 was followed by phosphorylation of ATF-2 but not c-Jun.

Upstream kinases are mitogen-activated protein kinase kinase (MKK)3 and MKK6. Their kinetics of phosphorylation at Ser198 and Ser207 after stimulation with SM was similar that of p38 phosphorylation and activation (Figure 4, lanes 3–6). As MSK1 is a target of p38 MAPK (Deak et al., 1998), it can be activated by the Raf/MEK/ERK pathway as well as by the MKK/p38 pathway. Phosphorylation of JNK1/2 at Thr183 and Tyr185 was induced at slower kinetics compared with that for ERK1/2 and p38 MAPK (Figure 5, lanes 3–6). JNK1/2 induction was paralleled by phosphorylation of mixed-lineage kinase 3 at Thr277 and Ser281, which in turn phosphorylates MKK4 at Ser257 and Thr261 (Figure 5, lanes 3–6) and MKK7 at Ser271 and Thr275 (Figure 5, lanes 4–6). MKK4 phosphorylates specifically JNK and p38 MAPK (Derijard et al., 1995), whereas MKK7 is a MAP kinase that serves as a specific activator of the JNK/SAPK pathway (Moriguchi et al., 1997). c-Jun and ATF-2, which belong to the activator protein 1 (AP-1) and CRE-binding protein family of transcription factors, respectively, are downstream targets of JNKs. Members of the Jun, ATF, Fos, and Maf transcription factor families that form different combinations of hetero- and homodimers are summed up as AP-1 (Angel and Karin, 1991; Jochum et al., 2001). ATF-2 can be activated through the JNK as well as the p38 MAPK pathway (Gupta et al., 1995; van Dam et al., 1995; Wang et al., 1997). ATF-2 was phosphorylated at Thr69 and Thr71 in response to treatment of keratinocytes with SM

(Figure 5, lanes 3–6). In contrast, no phosphorylation of c-Jun at Ser63/73, another target of JNK1/2 and a potential dimerization partner of ATF-2, could be detected (Figure 5). To examine the effect of SM on gene expression and the potential relevance of the identified signal transduction pathways in keratinocytes, we analyzed the kinetics of the induction of Cxcl1 and Cxcl2, two AP-1 and NF-kB target genes (De Plaen et al., 2006; Verhaeghe et al., 2007). Induction of both genes was observed and could be inhibited by preincubation of keratinocytes with a specific RelA inhibitor peptide before SM exposure (Figure 6). DISCUSSION When skin is exposed to SM, marked inflammation is induced along with the mutagenic and carcinogenic effects. Keratinocytes are of particular importance for the initiation of cutaneous inflammation. Transcription factors that are activated by the respective signal transduction pathways in keratinocytes coordinate the activation of genes that code for cell-adhesion molecules such as CD54 (ICAM-1) or chemotactic factors such as CXCL1 and CXCL2. The mechanisms implicated in this response are not well defined. We studied the effect of SM on two major pathways, MAPK and NF-kB, which are known to regulate numerous genes coding for inflammatory mediators. We show that treatment of keratinocytes with SM induces NF-kB in a time-dependent and biphasic manner, as demonstrated by electrophoretic mobility shift assay. The activation was preceded by activation of IKK2, as we could observe by use of phosphospecific antibodies that detect the activated protein, and subsequent IkBa phosphorylation and degradation. Besides phosphorylation and subsequent degradation of inhibitory molecules, protein kinases are also required for optimal NF-kB activation by targeting functional domains of NF-kB proteins themselves. RelA is phosphorylated at Ser536 by a variety of kinases via various signaling pathways. In most cases, these www.jidonline.org 1629

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phosphorylations enhance RelA transactivation potential. In addition to IkBa phosphorylation at Ser32 and Ser36, IKK2 can phosphorylate RelA at Ser536 (Sakurai et al., 1999). These results support the idea that phosphorylation at Ser536 is relevant for gene induction of SM-treated keratinocytes, as IKK2 activation preceded phosphorylation of RelA at Ser536. Such a regulation has been demonstrated for myocytes as well (Hall et al., 2005). The overall importance of the transactivating NF-kB subunit RelA for SM-induced NF-kB activation is demonstrated by exposure of RelA-deficient keratinocytes to SM. In these mutant keratinocytes, no NF-kB-binding activity can be detected. We conclude that NF-kB-binding activity in response to SM is strictly dependent on RelA. Ser276 can be phosphorylated in response to tumor necrosis factor-a through MSK1 (Vermeulen et al., 2003) and to lipopolysaccharide through PKAc (Zhong et al., 1997). A simultaneous phosphorylation of both sites, Ser536 and Ser276 of RelA, has been demonstrated for such stimuli as Haemophilus influenzae, Streptococcus pneumoniae, and Herpes simplex virus 1 (Hargett et al., 2006; Kweon et al., 2006). Despite activation of MSK1 in response to SM, we could not detect any phosphorylation of RelA at Ser276. This might be due to the differences in the cellular stimulus or cellular system. Whereas Vermeulen and co-workers stimulated fibroblasts with tumor necrosis factor-a, we stimulated keratinocytes with SM. Our results imply that activation of MSK1 does not inevitably result in RelA phosphorylation at Ser276, and phosphorylation of this site is not essential for RelA activity in keratinocytes. The function of RelA in epidermal homeostasis is controversial. Zhang et al. (2004) reported that RelA expression suppresses epidermal proliferation. We did not observe any spontaneous skin phenotype in mice lacking epidermis-specific RelA, and found a hyperproliferative, inflammatory skin in mice expressing RelA constitutively in keratinocytes (Rebholz et al., 2007). In the context of keratinocyte exposure to SM, it has been shown in endothelial cells that SM activates NF-kB by a redox-dependent mechanism (Atkins et al., 2000). In a recent publication, NF-kB activation by SM was described in human epidermal keratinocytes (Minsavage and Dillman, 2007). This observation is contrary to our findings, as NF-kB activation did not correlate with a fast degradation of IkBa. The reason for this difference is difficult to interpret, as the phosphorylation status of Ser32/36 and Tyr42 of IkBa in the human epidermal keratinocytes was not analyzed by Minsavage and Dillman. An alternative mechanism to the canonical NF-kB signal transduction pathway is the phosphorylation of IkBa at Tyr42, where NF-kB is activated without IkBa degradation (Imbert et al., 1996). Furthermore, our experiments were performed with freshly isolated keratinocytes that had been cultured for a short time period. In the other study, HaCaT cells or long-term cultured keratinocytes were used, with significant differences in the degradation profile of IkBa, which points to a relevant influence of long-term culture and immortalization on the cellular response. The high conservation of the pathways between mouse and human makes the 1630 Journal of Investigative Dermatology (2008), Volume 128

mouse system with its targeted deletion of genes attractive and valuable for understanding mechanistic principles in the human system. Both transcription factors NF-kB and AP-1 are known to regulate various genes encoding inflammatory mediators (Manning and Mercurio, 1997; Karin and Delhase, 2000). Several studies have demonstrated an interaction between these two pathways. A negative regulation of the JNK pathway by NF-kB (De Smaele et al., 2001; Tang et al., 2001) as well as an inhibition of NF-kB-activated genes by AP-1, due to recruitment of the histone deacetylase dHDAC1 to the promoter have been demonstrated (Kim et al., 2005). Another possible inhibiting mechanism that has been shown for the human tumor necrosis factor promoter, is a direct interaction of an AP-1 complex with NF-kB-binding sites (Udalova and Kwiatkowski, 2001). On the other side, a synergistic effect has been demonstrated for GM-CSF (Thomas et al., 1997). The activation of MAPK pathways by SM, as well as of the NF-kB pathway, most probably results in a complex time-dependent gene regulation pattern. Analysis of the ERK, JNK, and p38 MAPK pathway activation in response to SM in RelA-deficient keratinocytes revealed no difference from wild-type keratinocytes (data not shown). Therefore, a direct influence of NF-kB/RelA on the studied MAPK signal cascades can be excluded in response to SM. To what extent the gene expression is affected by this mutation, is subject of subsequent study. The question remains at which level and how SM induces the MAPK and NF-kB pathways. Raf-1/MEK/ERK signaling, activated by SM, indicates that SM acts at or above the level of Raf-1 and the MLK3/MKK/JNK pathway, above the level of mixed lineage kinase3. Similarly, it remains to be investigated how the IKK complex is activated by SM. Our analyses demonstrate that SM induces the canonical NF-kB pathway as well as the three major MAPK pathways that among other proteins activate components of the transcription factor AP-1. This expands our understanding of how SM induces an inflammatory process. Further investigation using keratinocytes deficient for components of these signal transduction pathways, and the difference in the genomic response to SM, will show how these pathways interact during the inflammatory response. MATERIALS AND METHODS All treatments and sample acquisitions, including skin biopsies, were approved by the institutional review committees on investigations involving animals at the Faculty of Medicine, University of Munich, Munich, Germany.

Mice and genotyping Mice carrying a floxed RelA allele (RelAf/f) (Algu¨l et al., 2007) were crossed with transgenic mice expressing Cre recombinase under the control of the keratinocyte-specific keratin5 promoter (Tarutani et al., 1997) (IkbaK5D/K5D). The genetic background was C57BL/6 for wild-type and RelAK5D/K5D mice. Mice were genotyped by PCR. For the RelA gene, the expected sizes were 294 bp for the floxed allele, 260 bp for the wild-type allele, and 400 bp for the deleted allele.

B Rebholz et al. Role of NF-kB in Keratinocytes Response to SM

Reverse transcriptase-PCR

CONFLICT OF INTEREST

Total RNA was isolated using Trizol reagent (Gibco, Carlsbad, CA). For all reverse transcriptase-PCR and real-time quantitative reverse transcriptase-PCR analyses, 1 mg total RNA was digested with Dnase I (Sigma, St Louis, MO) to remove DNA contamination, and reversetranscribed with SuperScript IITM according to the manufacturer’s instructions (Gibco/BRL). PCR amplifications were performed using standard conditions. Each PCR product was electrophoresed on agarose gels in 1  TAE, stained with ethidium bromide, and photographed. Each gene was analyzed using specific primer pairs. CXCL1 expression was determined with primers 50 -GGCCCCACTG CACCCAAACC-30 and 50 -CCGAGCGAGACGAGACCAGGAGA-30 , with an expected size of 661 bp, and CXCL2 expression was determined with primers 50 -CTCTCAAGGGCGGTCAAAAAGGT-30 and 50 -TCAGACAGCGAGGCACATCAGGTA-30 , with an expected size of 208 bp. b-Actin expression was determined with 50 -CACCCG CCACCAGTTCGCCA-30 and 50 -CAGGTCCCGGCCAGCCAGGT-30 , with an expected size of 554 bp.

The authors state no conflict of interest.

Exposure to SM and RelA inhibition SM was obtained from the TNO (The Hague, the Netherlands). Cellculture medium was removed from mouse keratinocytes. SM was freshly dissolved in minimum essential medium supplemented with 1% L-glutamine, and added to the cultures for 30 minutes. After the indicated incubation time, cell suspension was washed with phosphate-buffered saline to remove SM, and fresh keratinocyte growth medium (Cambrex, North Brunswick, NJ) was added. Inhibition experiments were performed with PTD–p65–P6, a p65 decoy (Imgenex, San Diego, CA), or control peptide, PTD, at a concentration of 150 mM, added 1 hour before SM exposure.

Electrophoretic mobility-shift assay and NF-jB ELISA Electrophoretic mobility-shift assays were performed as described previously (Rupec et al., 2005). NF-kB ELISA was performed according to the manufacturer’s instructions (Active Motif, Carlsbad, CA).

Isolation and culture of mouse keratinocytes Keratinocytes were isolated from the skin of newborn mice as described (Roper et al., 2001), and cultured in collagen type IVcoated plates in serum-free keratinocyte medium (Gibco) on feeder cells.

Western blot analyses Protein extracts and western blot analyses were performed according to standard procedures, using 20 mg of whole-cell extracts. Phosphorylation of IkBa, RelA, MAPKs, ATF-2, c-Jun (all from Cell Signaling Technology, Danvers, MA), and IKK2 (Santa Cruz Biotechnology, Santa Cruz, CA) was evaluated according to the protocol supplied by the manufacturer of the phosphospecific antibody. Identity of the bands was confirmed using molecular mass markers and positive controls supplied by the manufacturer. b-Actin was used as the cell-extract loading control.

Viability determination Cell viability was determined with the CASYs Cell Counter and Analyzer TTC, according to the manufacturer’s instructions.

ACKNOWLEDGMENTS This work was supported by the German Ministry of Defence under contract number M-SAB1-5-A004 (to R.A.R.).

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