Inhibition of p38 mitogen-activated protein kinase reduces TNF-induced activation of NF-κB, elicits caspase activity, and enhances cytotoxicity

Experimental Cell Research 293 (2004) 196 – 206 www.elsevier.com/locate/yexcr

Inhibition of p38 mitogen-activated protein kinase reduces TNF-induced activation of NF-nB, elicits caspase activity, and enhances cytotoxicity Silke Lu¨schen, a Gudrun Scherer, a Sandra Ussat, a Hendrik Ungefroren, b and Sabine Adam-Klages a,* b

a Institute of Immunology, Christian-Albrechts-University, D-24105 Kiel, Germany Research Unit Molecular Oncology of the Clinic for General Surgery and Thoracic Surgery, Christian-Albrechts-University, D-24105 Kiel, Germany

Received 28 March 2003, revised version received 9 October 2003

Abstract Among other cellular responses, tumor necrosis factor (TNF) induces different forms of cell death and the activation of the p38 mitogenactivated protein kinase (MAPK). The influence of p38 MAPK activation on TNF-induced apoptosis or necrosis is controversially discussed. Here, we demonstrate that pharmacological inhibition of p38 MAPK enhances TNF-induced cell death in murine fibroblast cell lines L929 and NIH3T3. Furthermore, overexpression of dominant-negative versions of p38 MAPK or its upstream kinase MKK6 led to increased cell death in L929 cells. While overexpression of the p38 isoforms alpha and beta did not protect L929 cells from TNF-induced toxicity, overexpression of constitutively active MKK6 decreased TNF-induced cell death. Although the used inhibitors of p38 MAPK decreased the phosphorylation of the survival kinase PKB/Akt, this effect could be ruled out as cause of the observed sensitization to TNF-induced cytotoxicity. Finally, we demonstrate that the nuclear factor nB (NF-nB)-dependent gene expression, shown as an example for the antiapoptotic gene cellular inhibitor of apoptosis (c-IAP2), was reduced by p38 MAPK inhibition. In consequence, we found that inhibition of p38 MAPK led to the activation of the executioner caspase-3. D 2003 Elsevier Inc. All rights reserved. Keywords: Apoptosis; Necrosis; Mitogen-activated protein kinase; Nuclear factor-nB; L929 cells

Introduction Tumor necrosis factor (TNF) is a proinflammatory cytokine that exerts a variety of effects, which depend on cell type, and additional signals that influence the physiological Abbreviations: c-IAP, cellular inhibitor of apoptosis; CHX, cycloheximide; cPLA2, cytosolic phospholipase A2; ERK, extracellular signalregulated kinase; FADD, Fas-associated death domain protein; FLICE, Fasinduced ICE-like protease; Flip, FLICE-inhibitor protein; NF-nB, nuclear factor nB; MAPK, mitogen-activated protein kinase; MAPKAPK, MAPKactivated protein kinase; MEK1, MAPK ERK kinase 1; MKK, MAPK kinase; PARP, Poly(ADP-ribose) polymerase; PI3K, phosphatidyl-inositol3-phosphate kinase; RIP, receptor-interacting kinase; SAPK/JNK, stressactivated protein kinase/Jun N-terminal kinase; TNF, tumor necrosis factor; TR55, p55-TNF receptor; TR75, p75-TNF receptor; TRADD, TNF receptor-associated death domain protein; TRAF2, TNF receptor-associated factor 2; zDEVD-AFC, benzyloxycarbonyl-Asp-Glu-Val-Asp-amino-4trifluoromethylcoumarine. * Corresponding author. Institute of Immunology, Christian-AlbrechtsUniversity Kiel, Michaelisstr. 5, D-24105 Kiel, Germany. Fax: +49-431597-3335. E-mail address: [email protected] (S. Adam-Klages). 0014-4827/$ - see front matter D 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.yexcr.2003.10.009

status of cells. Stimulation with TNF can induce gene expression, leading, for example, to the release of other proinflammatory cytokines, or activates signaling cascades, which mediate differentiation, proliferation, or cell death [1]. The signal is transduced by two TNF receptors, TR55 (TNFR1) and TR75 (TNFR2), from which the first one is mostly responsible for TNF-induced cell death [2,3]. After binding of the noncovalent TNF trimer, the receptor is also trimerized and, in consequence, an intracellular platform is formed, where different adapter proteins bind to compose a signaling complex [4]. Thereby, the signal is transmitted to a variety of effector cascades leading to different biological responses. The adapter molecules TNF receptor-associated death domain protein (TRADD) and Fas-associated death domain protein (FADD) bind to the death domain of TR55 [4], which is also present in other death receptors of the TNF receptor superfamily like Fas/APO-1/CD95, TRAIL-R1 (DR4) and TRAIL-R2 (DR5), DR3, and DR6 [5]. Thereby, the initiator caspase-8 is recruited to the receptor, activated by autoproteolysis [2], and, in consequence, a cascade of proteolytic processes is started leading to the execution of

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apoptotic cell death [6]. TNF receptor-associated factor 2 (TRAF2) and receptor-interacting protein kinase (RIP) also bind to TRADD and thereby transmit the signal for the activation of transcription factor nuclear factor nB (NF-nB) [7]. Consequently, the expression of proinflammatory cytokines like IL-6 or TNF itself on one hand, and of antiapoptotic genes like inhibitor of apoptosis (c-IAP1), c-IAP2, TRAF1, TRAF2 [8,9], and c-FLIP (FLICE-inhibitor protein) [10] on the other hand, is induced. The expression of these anti-apoptotic genes is most likely the reason why in most cell types TNF induces cell death only in the presence of inhibitors of macromolecular synthesis (actinomycin D or cycloheximide (CHX)). One of few exceptions is the murine fibrosarcoma cell line L929, in which TNF alone induces cell death by necrosis even though NF-nB is activated [11]. In addition to the signaling pathways described above, stimulation with TNF also activates protein kinase cascades like the mitogen-activated protein kinase (MAPK) pathways, which include extracellular signal-regulated kinase (ERK) 1/ 2, stress-activated protein kinase/Jun N-terminal kinase (SAPK/JNK), and p38 MAPK [2,12], or the phosphatidylinositol-3-phosphate kinase (PI3K)/Akt pathway [13]. These protein kinases are phosphorylated and thereby activated after stimulation with TNF. The exact molecular mechanisms for the activation of these kinases after stimulation with TNF are not entirely clear. In addition, the functional role of the activation of these kinases is often controversially discussed. The activation of ERK1/2 is thought to transmit mainly proliferative and survival signals [14], while SAPK/JNK or p38 MAPK mediates stress signals [15]. SAPK/JNK has been reported to contribute to cell death by induction of AP1-dependent gene expression [16]. P38 MAPK is necessary for stress-induced expression of proinflammatory cytokines and growth factors like IL-6, TNF itself, or GM-CSF [17]. This is regulated by p38 MAPK not only on the transcriptional level, but also by stabilization of cytokine mRNA using AU-rich elements in the 3V untranslated region [18]. Recently, it was shown that the p38 MAPK pathway is additionally involved in cell death processes, cell cycle regulation, osmoregulation, and differentiation of distinct cell types [19 – 22]. In this study, we examined the contribution of p38 MAPK to TNF-induced cell death in murine fibroblast cell lines L929 and NIH3T3. We provide evidence that the TNF-induced p38 MAPK activation contributes to NF-nB activation, thereby generating a survival rather than a deathpromoting signal in these cells.

Materials and methods Reagents Highly purified human TNF (3  107 units/mg) was provided by G. Adolf (Bender, Vienna, Austria). The protein synthesis inhibitor cycloheximide (CHX) and

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G418 were purchased from Sigma (Deisenhofen, Germany), the p38 MAPK inhibitors SB202190, SB203580, and the PI3K inhibitor Wortmannin were purchased from Calbiochem, and were solubilized in DMSO as a 10-mM stock solution. The polyclonal antibody against p38a- and hisoforms (H-147) was obtained from Santa Cruz Biotechnology (Santa Cruz, USA). The monoclonal antibody against poly-(ADP)-ribose-polymerase (PARP, #65196E) was purchased from BD Biosciences (Heidelberg, Germany), the monoclonal anti-FLAG antibody (M2) from Sigma, the polyclonal antibody against the proform and cleavage products of caspase-3, the phosphospecific antip38 antibody, and the phosphospecific anti-PKB/Akt (Ser 473) antibody from Cell Signaling Technology (MA, USA). The vector containing the neo gene (BMGNeo) has been published [23]. Expression cloning RNA was isolated from L929 cells using the High Pure RNA Isolation Kit according to the instructions of the manufacturer (Roche Molecular Biochemicals, Penzberg, Germany). The cDNA was prepared with the First Strand c-DNA Synthesis Kit for RT-PCR (AMV) (Roche Molecular Biochemicals) using oligo-p(dT)-Primer. For cloning of murine p38a, the cDNA of L929 cells was amplified using the primers 5V-TAT ggA TCC ATg TCg CAg gAg Agg C3V and 5V-TTg CAA gCT TCC CAT gAg AAg gTC TTC C-3V and the product was subcloned into the expression vector pRK5 as a BamHI/HindIII fragment. The murine p38h isoform was amplified in a nested PCR using the first primers 5V-TCC AgC TgC TTC TgT gg-3V and 5V-CAg gTT AAg TgT CAg gg-3V, followed by a second PCR using the primers 5V-TAT gAA TTC ATg TCg ggT CCg CgC-3V and 5V-TTg CAA gCT TgA ggA AAg CCg gAA Ag-3V. The PCR product was also subcloned into pRK5 as an EcoRI/HindIII fragment. Retroviral expression vectors containing the coding sequences for dominant-negative human p38a MAPK (p38a-DN, containing Thr to Ala and Tyr to Phe mutations at codons 180 and 182), dominant-negative human MKK6 (MKK6-DN, containing an Ala mutation at the conserved lysine residue in the ATP binding site), and constitutive active MKK6 (MKK6-EE, containing Ser to Glu and Thr to Glu mutations at codons 207 and 211) have been described previously [24]. Cell culture and transfection L929 and NIH3T3 cells were grown in DMEM without HEPES supplemented with 10% fetal calf serum, 3.5xhmercaptoethanol, and 50 Ag/ml each of streptomycin and penicillin. The p38 MAPK inhibitors, Wortmannin, or DMSO used as control were preincubated for 30 min or 2 h before the cells were stimulated as indicated. For transient expression of p38 constructs, 5  104 NIH3T3 were transfected with 1 Ag plasmid DNA using

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the calcium phosphate precipitation method [25]. To generate L929 cells stably expressing p38 constructs, 2  105 cells were cotransfected with 3 Ag p38 expression construct and 0.3 Ag BMGNeo plasmid using the Lipofectamine reagent (Gibco-BRL) according to the instructions of the manufacturer. After 48 h, cells were incubated with 1 mg/ml G418 to select for stable transfectants. Medium containing G418 was changed every 2 days and the cells were further incubated for 6 days. After trypsination and reseeding, L929 cells were further cultured with G418 for another 4 days and the expression of p38 constructs was confirmed using Western blot analysis. Retroviral transduction with the p38a-DN and the MKK6 constructs, and the generation of transiently or stably expressing L929 pools were performed as previously described [24]. Cell cycle analysis Cells were seeded in 6-well plates at 2  105 cells/well. After stimulation, cells were detached using trypsin, washed twice with PBS/5 mM EDTA, and resuspended in 1 ml of PBS/5 mM EDTA. Cells were fixed by adding 1 ml of ethanol and incubation for 30 min at room temperature. Fixed cells were harvested and resuspended in 0.2 ml PBS/5 mM EDTA. RNA was digested by adding 40 Ag/ml RNaseA for 30 min at room temperature. After an additional 1h incubation with 0.2 ml staining solution (125 Ag/ml propidium iodide in PBS/5 mM EDTA), cell cycle analysis was performed by flow cytometry using a FACSCalibur Analyzer (BD Biosciences). TUNEL assay Cells were seeded at a density of 1.5  106 cells per 10cm dish. After stimulation, cells were harvested by trypsination, washed with PBS, and analyzed for DNA strand brakes using the In situ Cell Death Detection Kit, Fluorescein, from Roche Molecular Biochemicals according to the instructions provided by the supplier. Quantification of TUNEL-positive cells was performed using the FACSCalibur Analyzer. Cytotoxicity assay TNF-induced cytotoxicity was determined in L929 cells using a crystal violet staining solution as described previously [26]. The amount of surviving cells was quantified as absorption at 570 nm.

142 mM KCl, 5 mM MgCl2, 1 mM EDTA, 0.2% NP40 with freshly added 1 mM DTT, and 1 mM Pefabloc SC, Roche Molecular Biochemicals) to yield a high protein concentration. The cells were lysed for 20 min on ice followed by vigorous mixing. After centrifugation at 4jC for 15 min at 14000 rpm, the protein concentration in the supernatant was determined using Coomassie Reagent (Pierce). For the determination of caspase activity, 50 Ag of protein was incubated with 100 Al assay buffer (20 mM PIPES, pH 7.2, 100 mM NaCl, 1 mM EDTA, 0.1% CHAPS, 10% sucrose with freshly added 1 mM DTT) containing 100 AM of the fluorogenic substrate z-DEVDAFC (Calbiochem) to measure caspase-3 activity in a black 96-well multiplate at 37jC. During incubation, the fluorescence was measured in intervals of 2 min for 2 h at an excitation wavelength of 405 nm and an emission wavelength of 510 nm in a fluorometer (Fluoroscan, Abion). For positive control, caspase-3 was activated in lysates of unstimulated cells by incubation with 10 AM cytochrome c and 1 mM dATP at 30jC for 1 h. The same lysates were used to perform Western blots with anti-caspase-3 and antiPARP antibodies. Western blot analysis Cells were seeded in 6-well plates at a density of 2  105 NIH3T3 cells and 2.5  105 L929 cells/well. After stimulation, the plates were immediately placed on ice, the medium was removed, the monolayers were washed twice with cold PBS, scraped with cold TNE lysis buffer (40 mM Tris, pH 8.0, 4 mM EDTA, 2% NP40, 20 mM NaF, 2 mM Na3VO4), supplemented with protease inhibitor mix Completek (Roche Diagnostics), and incubated on ice for 10 min. After precipitating cell debris at 4jC for 10 min at 14000 rpm, the protein concentration of the supernatants was determined using Coomassie Reagent. From each lysate, 20 Ag of total protein was separated on 10% SDS-PAGE and transferred to Porablot NCL membranes (0.45 Am, Macherey-Nagel). Blots were blocked for 30 min at room temperature in PBST (PBS containing 0.1% Tween 20) supplemented with 5% milk powder and incubated at 4jC overnight with the different antibodies diluted in PBST. After three washes, the membranes were incubated with a 1:10,000 dilution of the appropriate peroxidase-conjugated secondary antibody (Dianova) for 1 h at room temperature. After additional washing steps, they were developed with ECL detection reagent (Amersham). NF-nB dependent luciferase expression

Determination of caspase activity L929 cells were seeded at a density of 1.5  106 cells per 10-cm dish. After stimulation, cells were harvested by trypsination, washed with PBS, and resuspended in a small volume of caspase lysis buffer (10 mM HEPES, pH 7.4,

To analyze NF-nB-dependent luciferase expression, NIH3T3 cells were seeded in 12-well plates at a density of 7.5  104/well and transfected with 0.01 Ag reporter construct coding for NF-nB-dependent firefly luciferase gene using the calcium phosphate precipitation method.

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After 15 h, transfected cells were stimulated and luciferase expression was measured with the Luciferase Assay System according to the instructions of the manufacturer (Promega, Mannheim, Germany). After stimulation, the medium was removed, the cells were washed with PBS, and scraped in 200 Al CCLR lysis buffer. After removing cell debris by centrifugation at 14000 rpm for 2 min, the luciferase expression was determined incubating 20 Al of supernatant with 100 Al of Luciferase Assay Reagent for 5 s and measuring luminescence using a FB12 Luminometer (Berthold Detection Systems, Pforzheim, Germany). Real-time PCR The cells were seeded in 6-well plates at a density of 2.5  105/well. After stimulation, cells were detached using trypsin and cells from three in parallel stimulated wells were pooled. Total RNA was isolated and reverse transcription was performed as described above. CDNA was frozen in aliquots and freshly thawed for each PCR run. To determine the c-IAP2 expression, the cDNA of L929 cells was left undiluted and the cDNA of NIH3T3 cells was diluted 1:10. The cDNAs were subjected in duplicates to real-time PCR using the LightCycler and the FastStart DNA Master SYBR Green I kit (Roche Molecular Biochemicals). For quantification of c-IAP2 mRNA, the cDNA was amplified using the primers 5V-ggg CTg Agg TgC Tgg gAA TC-3V and 5V-ATC TgC CgC TgA ACC gTC Tg-3V. Standard curves were generated for each cDNA separately by RT-PCR of different dilutions with primers for the house keeping gene glyceraldehyde-3-phosphate dehydrogenase (5V-ACC ACA gTC CAT gCC ATC AC-3V and 5V-TCC ACC ACC CTg TTg CTg TA-3V). Exact quantification was achieved by comparison of each standard curve with the respective PCR result of c-IAP2 using the LightCycler software (Roche Molecular Biochemicals). The amount of specific product of c-IAP2 amplification in the cDNA of

Fig. 1. The p38 MAPK inhibitors SB202190 and SB203580 increase the toxicity of TNF. (A) L929 cells were preincubated with the indicated inhibitors for 30 min at various concentrations or with DMSO as control, stimulated with TNF (50 ng/ml) for an additional 10 h, and analyzed for DNA fragmentation using cell cycle analysis. The assay was run in duplicates. (B) NIH3T3 cells were preincubated with SB202190 for 30 min at the indicated concentrations and stimulated with TNF (50 ng/ml) alone for a further 24 h or in combination with CHX (10 Ag/ml) for a further 12 h. The cells were harvested and DNA fragmentation was analyzed using cell cycle analysis. (C) L929 cells were stimulated as described for A with the exemption that TNF stimulation was carried out for 16 h. Cell death was quantified using TUNEL assay. Shown are the average values of three independent experiments with the corresponding standard deviations. (D) NIH3T3 cells were stimulated as described for B and cell death was quantified using TUNEL assay. Shown are the average values of two independent experiments. (E) L929 and NIH3T3 cells were stimulated with TNF (50 ng/ml) for the indicated times, lysed, and analyzed for p38 MAPK phosphorylation on Western blots using a phosphospecific anti-p38 antibody.

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L929 cells was analyzed by stopping the amplification in the exponential phase of the PCR and separating the products on agarose gels.

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Results Inhibition of the p38 MAPK pathway increases TNFinduced cytotoxicity in L929 and NIH3T3 cells Stimulation with TNF in many cell types leads to the activation of a variety of protein kinase cascades, for example, activation of the PI3K/Akt pathway and of different MAP kinase pathways like ERK, JNK/SAPK, and p38 MAPK. The contribution of these pathways to TNF-induced toxicity or survival is controversially discussed. Here, we provide evidence that inhibition of p38 MAPK contributes to TNF-induced cytotoxicity. Figs. 1A, C show using two different apoptosis assays that in L929 cells, preincubation with the structurally similar pharmacological p38 MAPK inhibitors SB202190 or SB203580 led to a pronounced increase in TNF-induced toxicity. The incubation with these inhibitors alone was not toxic. In another murine fibroblast cell line, NIH3T3, a moderate sensitization effect to TNF alone was observed, while classical apoptosis induced by costimulation with TNF and CHX was clearly augmented (Figs. 1B, D). The ability of SB202190 to inhibit immunoprecipitated active p38 MAPK, but not SAPK/JNK1, was shown elsewhere ([27] and data not shown). In both cell lines, TNF stimulation led to a time-dependent phosphorylation of p38 MAPK indicative of its activation (Fig. 1E). To more specifically demonstrate that inhibition of the p38 MAPK pathway leads to enhanced cytotoxicity in response to TNF, we chose to overexpress dominantnegative mutants of p38 MAPK (p38a-DN) and of the p38-phosphorylating upstream kinase MKK6 (MKK6-DN) in L929 cells. We used human proteins, which exhibit 99% and 97% sequence homology, respectively, to their murine counterparts and, therefore, were very likely to function properly. Both dominant-negative mutants were retrovirally expressed in L929 cells and cytotoxicity was quantified using the crystal-violet staining assay. As shown in Fig. 2A, TNF-induced cytotoxicity was enhanced in cell pools transfected with p38a-DN as well as with MKK6-DN. In addition, a slight increase in cytotoxicity was still seen in cells overexpressing MKK6-DN after cotreatment with the p38 MAPK inhibitors (Fig. 2A). A reduction of p38 MAPK phosphorylation by overexpressed MKK6-DN was confirmed in L929 cells stimulated with TNF for 15 min in Western-blots using the phosphospecific anti-p38 antibody (data not shown). Successful overexpression was verified in Western blots using anti-p38 or anti-FLAG antibody (Fig. 2B). Costimulation with SB202190 and TNF induces caspase activity In L929 cells, stimulation with TNF alone induces cell death. We and others have found that this death is independent of caspase activity resembling features of both apoptosis and necrosis [26,28]. In Fig. 3A, we demonstrate

Fig. 2. Overexpression of dominant-negative mutants of p38 MAPK and MKK6 leads to increased cytotoxicity in TNF-treated L929 cells. (A) Cell pools of L929 cells stably transfected with the empty retroviral vector, dominant-negative p38a-MAPK (p38a-DN), or MKK6 (MKK6-DN) were stimulated as indicated for 16 h. Cytotoxicity was quantified using a crystal-violet staining assay. Cell viability is expressed relative to untreated controls. The values shown represent the means of eight parallel determinations with the corresponding standard deviations. (B) The same cell pools used for A were lysed and analyzed for expression of dominant negative p38 MAPK using the p38 MAPK-specific antibody. The FLAGtagged dominant-negative MKK6 was visualized using an anti-FLAG antibody.

that treatment with TNF alone, as expected, did not activate caspase-3. Caspase-3 activity, which was measured as cleavage activity against the fluorogenic substrate zDEVD-AFC, however, was strongly induced by incubation with the classical apoptotic stimulus TNF in combination with CHX. Interestingly, we show that pretreatment of L929 cells with SB202190 resulted in the induction of caspase-3 activity (Fig. 3A). As positive control, caspase-3 activity was measured in lysates of unstimulated cells, in which the activation of caspases was induced by incubating the lysate with cytochrome c and dATP ([29]; data not shown). Caspases are activated by sequential cleavage of the proform at defined cleavage sites, releasing a large and a small subunit forming an active caspase tetramer [30]. In Fig. 3B, activation of caspase-3 in L929 cells is demonstrated by the detection of the p19 and p17 subunits in a Western blot. After 3 and 6 h, stimulation with TNF + CHX and also with TNF + SB202190 induced the appear-

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[26], after stimulation with TNF alone, the immunoreactivity of full-length PARP also disappeared without generation of the specific cleavage product (Fig. 3C). The same lysates were used to analyze the cleavage of cPLA2, which could also be detected after treatment with TNF + CHX and TNF + SB202190 (data not shown). Overexpression of constitutively active MKK6, but not of wild-type p38 MAPK, protects against TNF-induced cell death The inhibitors SB202190 and SB203580 were described to specifically block the activity of p38 isoforms alpha and beta [33], while they do not inhibit the p38 isoforms gamma and delta [34,35]. Because inhibition of p38 MAPK activity increased the TNF-induced toxicity, we postulated that enhanced p38 MAPK activity should protect cells against TNF-induced cell death. To differentiate between the protective potential of the p38 alpha and beta isoforms, we generated cells overexpressing both p38 isoforms. NIH3T3 cells were transiently transfected with expression vectors coding for p38 alpha or beta. The

Fig. 3. Costimulation with p38 MAPK inhibitors and TNF induces caspase activation in L929 cells. (A) L929 cells were preincubated with SB202190 (10 AM) for 30 min or with DMSO for control, and then stimulated with TNF (50 ng/ml) alone or in combination with CHX (6 Ag/ml). After 6 h, the cells were harvested and caspase activity in the lysates was determined by adding the fluorogenic caspase-3-substrate z-DEVD-AFC (100 AM). (B) L929 cells were stimulated as described above and lysed. From each lysate, 140 Ag of protein was analyzed by immunoblotting to detect the proform and the p19 and p17 cleavage products of caspase-3. (C) After 10 h of stimulation, the cells were harvested and lysed in caspase lysis buffer. The appearance of PARP and its 85-kDa cleavage product was analyzed by immunoblotting. For positive control, a lysate from unstimulated cells was prepared using caspase lysis buffer and incubated with cytochrome c and dATP to induce caspase activity.

ance of the p19 and p17 subunits, while stimulation with TNF or SB202190 alone did not. In addition, the caspase-3 cleavage products could be detected in lysates of unstimulated cells incubated with cytochrome c and dATP (Fig. 3B). To further confirm that pretreatment with p38 MAPK inhibitors leads to caspase activation, we analyzed the specific cleavage of the caspase substrates PARP [31] and cytosolic phospholipase A2 (cPLA2) [32]. Fig. 3C demonstrates the appearance of the specific 85-kDa PARP cleavage product and the loss of full-length PARP after stimulation with TNF + CHX or TNF + SB202190 and in cytochrome c/dATP-activated lysates. As previously shown

Fig. 4. Overexpression of p38 MAPK isoforms alpha or beta does not protect cells from TNF-induced toxicity. NIH3T3 cells were transiently transfected with expression plasmids coding for p38a or p38h or empty vector for control. (A) After 24 h, the cells were scraped in TNE lysis buffer and the expression of the p38 constructs was detected by immunoblotting. (B) After 24 h, the cells were preincubated with SB202190 (10 AM) for 30 min and then stimulated with TNF (50 ng/ml) alone or in combination with CHX (10 Ag/ml). After additional 15 or 24 h, the cells were harvested and DNA fragmentation was analyzed using cell cycle analysis. (C) Stable pools of L929 cells transfected with expression vector for p38h isoform or empty vector pRK5 in combination with G418 resistance plasmid BMGNeo were scraped in TNE lysis buffer and analyzed in Western blots for p38h expression. (D) Stable pools of L929 cells were stimulated as described above for 15 or 24 h and analyzed for DNA fragmentation using cell cycle analysis.

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Because the levels of p38 MAPK are already relatively high in L929 cells (Fig. 4A), the overexpression of wildtype p38 MAPK might just not have led to increased activity and, therefore, not to protection. Thus, we chose to overexpress constitutively active MKK6 (MKK6-EE) in L929 cells. Because we were not able to obtain stable transfectants, we generated transient transfectants. As shown in Fig. 5A, overexpression of MKK6-EE increased survival in response to TNF treatment compared to vectortransfected cells. However, the observed protection was only partial. Treatment with TNF together with the p38 MAPK inhibitors led to about the same amount of cell death as that seen in vector-transfected controls (Fig. 5A). Overexpression of MKK6-EE led to enhanced p38 MAPK phosphorylation in untreated L929 cells, which was not further increased after TNF stimulation (data not shown). Successful overexpression of MKK6-EE was verified in Western blots using the anti-FLAG antibody (Fig. 5B). Taken together, our data show that constitutive activation of the p38 MAPK pathway has a protective effect on TNFinduced cytotoxicity in L929 cells, while inhibition of p38 MAPK enhances cell death. Treatment with p38 MAPK inhibitors results in dephosphorylation of PKB/Akt

Fig. 5. Overexpression of constitutively active MKK6 leads to partial protection from TNF-induced cytotoxicity in L929 cells. (A) Cell pools of L929 cells stably transfected with the empty retroviral vector or constitutive active MKK6 (MKK6-EE) were stimulated as indicated for 24 h. Cytotoxicity was quantified using a crystal-violet staining assay. Cell viability is expressed relative to untreated controls. The values shown represent the mean of eight parallel determinations with the corresponding standard deviations. (B) The same cell pools used for A were lysed and analyzed for expression of MKK6-EE in a Western blot using the antiFLAG antibody.

expression of the constructs was confirmed by Western blot analysis (Fig. 4A). Transfected cells were stimulated with TNF or TNF + CHX and cell death was analyzed using cell cycle analysis. Unexpectedly, overexpression of neither p38 construct protected NIH3T3 cells from TNFinduced death (Fig. 4B). In parallel, we performed experiments where the pRK5-GFP plasmid was cotransfected in a 10-fold lower amount. Subsequently, transfected green fluorescent cells were gated during FACS analysis. After stimulation, however, we again could not demonstrate any protective influence of the overexpressed p38 constructs by measurement of propidium iodide uptake (data not shown). In addition, we generated pools of L929 cells stably overexpressing the p38 alpha or beta isoforms as shown by Western blotting (Fig. 4C and data not shown). Again, these cells were not protected from TNF-induced cytotoxicity (Fig. 4D and data not shown).

The PI3K/Akt pathway has been shown to mediate proliferative and survival signals [36]. Therefore, we investigated for an influence of SB202190 and SB203580 on the phosphorylation of protein kinase PKB/Akt in L929 cells by performing Western blot analysis with a phosphospecific anti-Akt antibody. In unstimulated growing L929 cells, Akt was already constitutively phosphorylated. After stimulation with TNF for 15 min, the phosphorylation was slightly enhanced. Pretreatment with both inhibitors alone

Fig. 6. Treatment of L929 cells with p38 MAPK inhibitors leads to a decreased phosphorylation of protein kinase PKB/Akt. L929 cells were preincubated for 30 min with SB202190 (10 AM), SB203580 (10 AM), PD98059 (20 AM), or DMSO for control (A), and Wortmannin at the indicated concentrations (B). After stimulation with TNF (50 ng/ml) for 15 min, cells were harvested and the phosphorylation of PKB/Akt was analyzed in Western blots.

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Fig. 7. Pretreatment with the PI3K inhibitor Wortmannin does not enhance TNF-induced cell death in L929 cells. L929 cells were preincubated with Wortmannin at the indicated concentrations or with DMSO for control and then stimulated with TNF (50 ng/ml) for further 24 h. Cells were harvested and DNA fragmentation was analyzed by cell cycle analysis.

clearly reduced Akt phosphorylation (Fig. 6A). Also, the following stimulation with TNF after preincubation with both p38 MAPK inhibitors failed to enhance phosphorylation of Akt. As control, we used PD98059, which is an inhibitor of the ERK1/2 upstream kinase MEK1. In contrast to p38 MAPK inhibitors, pretreatment of L929 cells with PD98059 had no effect on the phosphorylation of Akt (Fig. 6A). To investigate whether the increased toxicity of TNF after pretreatment with p38 MAPK inhibitors is caused by the reduced phosphorylation of Akt, we tested an indirect inhibitor of Akt phosphorylation, the PI3K inhibitor Wortmannin, for its influence on TNF-induced cytotoxicity. Preincubation of L929 cells with up to 10 nM Wortmannin resulted in a clear reduction of Akt phosphorylation after 15-min stimulation with TNF (Fig. 6B). However, in contrast to pretreatment with the p38 MAPK inhibitors, pretreatment with Wortmannin did not sensitize but rather protect L929 cells from TNF-induced death (Fig. 7). Thus, the inhibition of Akt phosphorylation could be excluded as a mechanism through which p38 MAPK inhibitors augment cell death.

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expression after stimulation with TNF alone (Fig. 8A). The suppression of NF-nB-dependent gene expression was observed after treatment with p38 MAPK inhibitors over a concentration range of 0.1– 10 AM (Fig. 8A). To analyze the influence of pharmacological p38 MAPK inhibition on TNF-induced expression of an endogenous NFnB-dependent anti-apoptotic gene, we extracted RNA from NIH3T3 and L929 cells and prepared cDNA. Using realtime PCR, we measured the TNF-induced transcription of murine c-IAP2 gene. In both cells lines, the induction of the c-IAP2 gene after pretreatment with SB202190 was suppressed, while treatment with inhibitor alone had no effect (Fig. 8B). These data are in concordance with the above data concerning the NF-nB-dependent luciferase expression. In summary, we conclude that the inhibition of p38 MAPK during TNF stimulation results in the reduced induction of NF-nB-dependent protective genes like cIAP2. This reduction then leads to the appearance of caspase activity and to enhanced TNF-induced cell death.

Pretreatment with p38 MAPK inhibitors reduces NF-nBdependent gene expression Stimulation with TNF leads to activation of the transcription factor NF-nB and, in consequence, of NF-nB-dependent gene expression. Some of these genes, like c-IAP1, c-IAP2, or TRAF-2, have a protective role against apoptosis [8]. To examine whether the p38 MAPK inhibitors influence the NF-nB activation signal, we transiently transfected NIH3T3 cells with a reporter gene construct conferring NF-nBdependent firefly luciferase gene expression. After stimulation, the luciferase expression was measured using luminescence detection. While treatment with SB202190 or SB203580 alone had no effect on NF-nB-dependent gene expression, pretreatment with these inhibitors and subsequent stimulation with TNF resulted in reduced induction of luciferase expression compared to induction of luciferase

Fig. 8. Pretreatment with p38 MAPK inhibitors reduces TNF-induced NFnB activation. (A) NIH3T3 cells were transiently transfected with a reporter construct containing a NF-nB-dependent luciferase gene. After 15 h, the cells were preincubated for 2 h with SB202190 (10 AM), SB203580 (10 AM), or DMSO for control, and stimulated with TNF (50 ng/ml) for additional 4.5 h. Cells were scraped in lysis buffer and the expression of the NF-nB-dependent luciferase gene was analyzed. Shown are triplicate determinations with the respective standard deviations. (B) NIH3T3 and L929 cells were preincubated for 2 h with SB202190 (10 AM) or DMSO for control and stimulated with TNF (50 ng/ml) for further 4.5 h. Total RNA was extracted, converted into cDNA, and c-IAP2 mRNA was amplified by real-time PCR. The signal was quantified in comparison to the expression of glyceraldehyde-3-phosphate dehydrogenase mRNA. For L929 cells, the amplification was terminated in the exponential phase and the PCR products were separated on an agarose gel.

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Discussion Besides the regulation of inflammatory processes by induction of cytokine expression, TNF induces cell death. The mechanisms leading to death are not fully understood and differ depending on cell type and additional signals, which affect the cells. Even the mode of death and its characteristic features can vary between classical apoptosis involving caspase activation, and necrosis, which is characterized by a lack of caspase activity [11]. Between these two extremely different types of death, many intermediate states are described [37]. In the past, the role of p38 MAPK in context with cell death was mostly considered to be sensitizing to or enhancing apoptosis. For example, it was shown that apoptosis induced in human FS-4 fibroblasts by the nonsteroidal anti-inflammatory drug sodium salicylate was suppressed by a p38 MAPK inhibitor [38], and that the p38 MAPK inhibitor reversed the sodium salicylate-induced reduction in InBa phosphorylation after stimulation with TNF [39]. Similarly, overexpression of a constitutive active form of an upstream kinase of p38 MAPK reduced InBa phosphorylation and NF-nB-dependent reporter gene activity after stimulation with TNF [40]. Furthermore, in RAT-1 fibroblasts, cell death induction by serum starvation was inhibited by blocking the p38 MAPK activity. Finally, p38 MAPK inhibition protected PC12 cells from apoptosis after NGF withdrawal [41]. In L929 cells, stimulation with TNF induces a type of cell death, which shows features of necrosis and is executed independently of caspase activity [26,28]. Stimulation with TNF in the presence of the protein synthesis inhibitor CHX, however, converts necrotic cell death to apoptosis including the activation of caspase-3 (Fig. 3). Previously, Beyaert et al. [17] have demonstrated that pretreatment with SB203580 had no effect on TNF-induced cytotoxicity of a subclone of L929 cells (L929sA), which undergo necrosis even in the absence of gene expression. In contrast, we show here that inhibition of p38 MAPK activity clearly enhanced TNFinduced cell death of wild-type L929 cells, most likely by activation of caspases. Similarly, p38 MAPK inhibition increased TNF-induced cell death in NIH3T3 cells as well as the degree of death induced by stimulation with classical apoptosis inducers TNF and CHX. From these data, we concluded that the p38 MAPK pathway has a protective function against TNF-induced cell death in L929 and NIH3T3 cells. This conclusion was supported by the fact that overexpression of constitutively active MKK6 protected L929 cells from TNF-induced cytotoxicity. Similar results have been described by Guo et al. [42], who reported that rat mesangial cells were sensitized to TNF by pretreatment with SB203580. Furthermore, in the human myelomonocytic cell line U937 and in primary murine splenic macrophages, pharmacological inhibition of p38 MAPK increased cell death after stimulation with TNF [43]. This report describes an increase in caspase-9 and caspase-8 activity after pretreatment with p38 MAPK inhibitor PD169316. In contrast,

TNF-induced toxicity was not affected by inhibition of p38 MAPK in the human Jurkat T cell line [43]. Roulston et al. [44] have shown that overexpression of dominant-negative MKK4 and MKK6, which are activating kinases of p38 MAPK and SAPK/JNK, increased the TNF-induced cell death in NIH3T3 cells and, vice versa, the overexpression of MKK4 and MKK6 protected cells from death. They also found an increase in caspase activation after pretreatment with p38 MAPK inhibitor in L929 cells. However, these authors have used an L929 subclone that activates caspases already after stimulation with TNF alone. Finally, Franco et al. [45] have published that HeLa cells were sensitized to TNF-induced toxicity by hyperosmotic stress and that the pharmacological inhibition of p38 MAPK further increases the TNF-induced toxicity under these conditions. In contrast, we could not detect any influence of SB202190 on cell death induced by TNF in HeLa cells (S. L., unpublished results). The p38 MAPK inhibitors used in this study only affect isoforms alpha and beta. To confirm our conclusion concerning the protective function of p38 MAPK and to discriminate between the protective potential of the two isoforms, we analyzed the effect of overexpressing both wild-type p38 isoforms separately using various transfection and expression systems. However, neither the overexpression of p38a nor p38h resulted in protection against TNF-induced cell death. In contrast, Guo et al. [42] described that overexpressed p38h, but not p38a, protected rat mesangial cells from TNF-induced toxicity. Similarly, Nemoto et al. [46] published that overexpression of p38a induced cell death in HeLa cells, which was increased after treatment with SB202190. Overexpression of p38h, however, did not induce cell death, but diminished the death induced by treatment of SB202190. In addition, they demonstrated that overexpressing p38a increased cell death induced by stimulation with UV or anti-Fas, while cells overexpressing p38h were protected. The discrepancy to our results is unclear, but might be explained with dissimilar levels of basal expression of the two p38 MAPK isoforms in the different experimental settings. Following those lines, constitutive activation of the p38 MAPK pathway finally led to protection from TNF-induced cell death in our system. The pyridinyl imidazoles had been developed for their potential to inhibit the expression of proinflammatory cytokines. It has been shown that they bind to p38 MAPK in a reversible manner and block their ATPase activity [47]. The binding depends mostly on a single threonine residue and is highly specific [33]. However, there are descriptions that pyridinyl imidazoles can influence TGF beta receptor type 1 [48], c-Raf [49], and distinct isoforms of SAPK/JNK, which are not immunoprecipitated by an anti-JNK1-antibody [50]. Nevertheless, pyridinyl imidazoles are frequently used to examine the functions of p38 MAPK. While searching for the reason of the sensitizing effect of p38 MAPK inhibition, we detected a reduction in the phosphorylation of Akt/PKB

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after treatment with the p38 MAPK inhibitors. This might be an additional nonspecific effect or a direct inhibitory function of the p38 MAPK inhibitors. Because Akt/PKB activates a well-described survival pathway [36], it seemed possible that inhibition of PI3K/Akt mediated the sensitizing effect. However, pretreatment of L929 cells with Wortmannin in concentrations that reduced Akt phosphorylation to a comparable level than pretreatment with p38 MAPK inhibitors did not increase TNF-induced cell death. Therefore, the reduced phosphorylation of Akt/PKB after pretreatment with p38 MAPK inhibitors is unlikely to be the reason why the cells are sensitized against TNF-induced toxicity by these inhibitors. As a likely explanation for the observed sensitizing effect, we demonstrate here that pretreatment of L929 as well as NIH3T3 cells with p38 MAPK inhibitors led to a diminished induction of TNF-induced NF-nB-dependent gene expression. As an example, we show that the induction of an important anti-apoptotic gene, c-IAP2, is reduced in both cell lines. Because c-IAP2 inhibits the active forms of caspases-3 and -7 [51] and possibly even the initiator caspase-8 [8], this might explain the induction of caspase activity and thus the increase in TNF-induced apoptosis after p38 MAPK inhibition. Similar results have been described by Varghese et al. [43]. In accordance with our results, it has been shown in other systems that blocking p38 MAPK led to a decreased induction of NF-nB-dependent reporter gene expression, although the translocation of NFnB into the nucleus, the DNA binding, and the general phosphorylation of the NF-nB subunits p65/RelA and p50 were not affected [17,43,45,52]. Vanden Berghe et al. [52] have demonstrated that the transcriptional activating potential of the p65/RelA subunit depending on two transactivation domains TA1 and TA2 was reduced after pretreatment with a p38 MAPK inhibitor. For full activation of p65/RelA, it has to interact with the coactivator CBP/p300, which is also a target of phosphorylation [53]. Further analysis is needed to elucidate, whether inhibition of p38 MAPK in murine fibroblasts leads to a reduced phosphorylation of the p65/RelA subunit of NF-nB or of coactivator proteins. In addition, it has to be explored whether this phosphorylation is performed either by p38 MAPK itself or by one of its substrate kinases MAPKAPK2 or MAPKAPK3.

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This work was supported by the Deutsche Krebshilfe. We thank Stefan Ludwig for p38 MAPK and MKK6 constructs.

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