Oncogene (2007) 26, 1201–1212 & 2007 Nature Publishing Group All rights reserved 0950-9232/07 $30.00 www.nature.com/onc ORIGINAL ARTICLE Genetic deletion of PKR abrogates TNF-induced activation of IjBa , JNK, Akt and cell proliferation but potentiates p44/p42 MAPK and p38 MAPK activation

Y Takada1, H Ichikawa1, A Pataer2, S Swisher2 and BB Aggarwal1

1Cytokine Research Laboratory, Department of Experimental Therapeutics, The University of Texas MD Anderson Cancer Center, Houston, TX, USA and 2Department of Thoracic and Cardiovascular Surgery, The University of Texas MD Anderson Cancer Center, Houston, TX, USA

Double-stranded RNA-dependent protein kinase (PKR), a positively regulated, whereas p44/p42 MAPK and p38 ubiquitously expressed serine/threonine kinase, has been MAPK were negatively regulated. implicated in the regulation or modulation of cell growth Oncogene (2007) 26, 1201–1212. doi:10.1038/sj.onc.1209906; through multiple signaling pathways, but how PKR published online 21 August 2006 regulates tumor necrosis factor (TNF)-induced signaling pathways is poorly understood. In the present study, we Keywords: PKR; Akt; MAPK; JNK; NF-kB; TNF; used fibroblasts derived from PKR -deleted mice to survival; investigate the role of PKR in TNF-induced activation of nuclear factor-jB (NF-jB), mitogen-activated protein (MAPKs) and growth modulation. We found that in wild-type mouse embryonic fibroblast (MEF), TNF induced NF-jB activation as measured by DNA binding Introduction but deletion of PKR abolished this activation. This inhibition was associated with suppression of inhibitory Double-stranded RNA (dsRNA)-dependent protein subunit of NF-jB(IjB)a kinase (IKK) activation, IjBa kinase (PKR), a ubiquitously expressed serine/threonine phosphorylation and degradation, p65 phosphorylation and kinase, has been implicated as a signal integrator in nuclear translocation, and NF-jB-dependent reporter gene translational and transcriptional control pathways transcription. TNF-induced Akt activation needed for IKK (Williams, 2001). It was initially identified as an activation was also abolished by deletion of PKR. NF-jB (IFN)-induced translational inhibitor in the activation was diminished in PKR-deleted cells transfected IFN-induced antiviral pathway. However, more re- with TNF receptor (TNFR) 1, TNFR-associated death cently, PKR has been shown to mediate cell growth domain and TRAF2 plasmids; NF-jB activated by NF-jB- and stress response. It has two distinct domains, an inducing kinase, IKK or p65, however, was minimally N-terminal dsRNA-binding regulatory domain and a affected. Among the MAPKs, it was interesting that C-terminal kinase catalytic domain. Besides dsRNA and whereas TNF-induced c-Jun N-terminal kinase (JNK) IFNs, PKR has been shown to be activated by cytokines activation was abolished, activation of p44/p42 MAPK (e.g. tumor necrosis factor (TNF), interleukin (IL)-3 and and p38 MAPK was potentiated in PKR-deleted cells. platelet-derived growth factor) (Ito et al., 1994; TNF induced the expression of NF-jB-regulated gene Mundschau and Faller, 1995; Yeung et al., 1996) and products cyclin D1, c-Myc, matrix metalloproteinase-9, bacterial products (e.g. lipopolysaccharide (LPS) and survivin, X-linked inhibitor-of-apoptosis protein (IAP), lipoprotein) (Gusella et al., 1995; Jiang et al., 2003). IAP1, Bcl-xL, A1/Bfl-1 and Fas-associated death domain Once activated, PKR can phosphorylate downstream protein-like IL-1b-converting -inhibitory protein in À/À substrates, including protein phosphatase 2A (Xu and wild-type MEF but not in PKR cells. Similarly, TNF Williams, 2000) and initiation factor eIF-2 (Carroll induced the proliferation of wild-type cells, but this et al., 1993). proliferation was completely suppressed in PKR-deleted Besides protein phosphatase 2A and eIF-2, PKR has cells. Overall, our results indicate that PKR differentially alsobeen showntophysically interact with signal regulates TNF signaling; IKK, Akt and JNK were transducers and activators of transcription (STAT) 1 (Wong et al., 1997), STAT3 (Deb et al., 2001), inhibitory Correspondence: Professor BB Aggarwal, Cytokine Research Labora- subunit of NF-kB(IkB)a (Kumar et al., 1994), IkBa tory, Department of Experimental Therapeutics, The University of kinase (IKK) (Bonnet et al., 2000; Gil et al., 2000; Texas MD Anderson Cancer Center, 1515 Holcombe Boulevard, Unit Zamanian-Daryoush et al., 2000), MAPK kinase kinase 143, Houston, TX 77030, USA. E-mail: [email protected] 6 (Silva et al., 2004), cellular protein activator of PKR Received 25 April 2006; revised 11 July 2006; accepted 11 July 2006; (Patel and Sen, 1998; Peters et al., 2001), PKR- published online 21 August 2006 associated protein X (Ito et al., 1999), TNF receptor Role of PKR in TNF signaling Y Takada et al 1202 (TNFR)-associated factor (TRAF) (Gil et al., 2004), TNF induces PKR activation apoptosis signal-regulating kinase-1 (Takizawa et al., We first examined the effect of TNF on PKR activation 2002) and (Cuddihy et al., 1999). PKR has alsobeen by Western blot analysis. PKR protein was only shown to regulate the expression of cyclin D1 (Kron- detected in wild-type MEF, and TNF induced the feld-Kinar et al., 1999), c-Myc (Raveh et al., 1996), activation of PKR in a time-dependent manner matrix metalloproteinase (MMP)-9 (Gilbert et al., 2004) (Figure 1b). In PKRÀ/À fibroblasts, neither activation and E-selectin (Bandyopadhyay et al., 2000), and of PKR by TNF nor expression of PKR protein was activate NF-kB (Kumar et al., 1994; Cheshire et al., detected. 1999; Zamanian-Daryoush et al., 2000; Gil et al., 2001) by a wide variety of agents (Demarchi et al., 1999; Deb PKR is required for TNF-dependent NF-kB activation et al., 2001) possibly through the activation of IKK (Gil We examined the effect of PKR deletion on TNF- et al., 2000; Ishii et al., 2001). The role of PKR in the induced NF-kB activation. Cells were stimulated with activation of p38 mitogen-activated protein kinase TNF, nuclear extracts were prepared and analysed for (MAPK) (Goh et al., 2000; Iordanov et al., 2000) and NF-kB activation by electrophoretic mobility shift c-Jun N-terminal kinase (JNK) (Iordanov et al., 2000) assays (EMSA). As shown in Figure 1c, TNF induced has also been reported. PKR has been found to be a NF-kB activation in a time-dependent manner in mediator of apoptosis induced by various agents (Yeung control fibroblasts. However, TNF-induced NF-kB et al., 1996; Balachandran et al., 1998; Srivastava et al., activation was significantly suppressed in the PKRÀ/À 1998; Gil et al., 2000). fibroblasts. TNF is a proinflammatory cytokine produced pri- Because activation of NF-kB by TNF is more robust marily by macrophages that is known to activate at higher concentrations (Chaturvedi et al., 1994), we apoptosis, NF-kB, JNK, p44/p42 MAPK and Akt determined the effect of PKR deletion on NF-kB (Aggarwal, 2003, 2004). In some cells, TNF has been activation induced by higher concentrations of TNF shown to promote proliferation of cells. Sequential (Figure 1d). At a concentration of 1000 pM, TNF recruitment of TNFR, TNFR-associated death domain induced NF-kB activity strongly in wild-type cells but (TRADD), TRAF2, receptor-interacting protein and not in PKRÀ/À cells. These results show that PKR is IKK leads toNF- kB activation by TNF; TRAF2 is also required for TNF-induced NF-kB activation. required for the activation of various MAPKs by TNF Because various combinations of Rel/NF-kB protein (Aggarwal, 2003, 2004). Similarly, sequential interaction can constitute an active NF-kB heterodimer that binds of TNF with TNFR, TRADD, Fas-associated death toa specific DNA sequence (Ghosh and Karin, 2002), domain protein (FADD), FADD-like IL-1b-converting we next determined whether the retarded band visua- enzyme (FLICE) and caspase-3 leads to apoptosis lized by EMSA in TNF-treated cells was indeed NF-kB. (Aggarwal, 2003, 2004). The signals required for the We incubated nuclear extracts from TNF-stimulated induction of cell proliferation by TNF, however, are cells with antibodies to either the p50 (NF-kB1) or p65 less well understood. p44/p42 MAPK is most likely (RelA) subunit of NF-kB. Both shifted the band to a needed for the TNF-induced proliferation of fibroblasts higher molecular mass (Figure 1e), suggesting that the (Aggarwal, 2003, 2004), but the role PKR plays in this TNF-activated complex consisted of p50 and p65 aspect of TNF signaling is less clear. Although there are subunits. Preimmune serum had noeffect. Excess un- some reports implicating PKR in TNF-induced apopto- labeled NF-kB (100-fold) caused complete disappearance sis, its role in TNF-induced NF-kB and MAPK activa- of the band, but the mutant oligonucleotide of NF-kB tion is controversial (Maran et al., 1994; Kumar et al., did not affect NF-kB-binding activity. 1997; Cheshire et al., 1999; Zamanian-Daryoush et al., 2000). In the current report, we used genetically PKR- deleted cells tosystematically investigate the role ofPKR PKR is required for TNF-dependent IkBa degradation in TNF signaling. Our results indicate that PKR plays a Translocation of NF-kB tothe nucleus is preceded by differential role in TNF-induced signal transduction. proteolytic degradation of IkBa (Ghosh and Karin, 2002). To determine whether inhibition of TNF-induced NF-kB activation in PKRÀ/À cells was due toinhibition Results ofIkBa degradation, we exposed cells to TNF for the indicated intervals and assayed for degradation of IkBa The aim of this study was to investigate the effects of by Western blot analysis. TNF induced IkBa degrada- PKR on TNF signaling. This proinflammatory cytokine tion, which preceded NF-kB translocation by as much À/À has been shown to activate NF-kB through IKK and to as 10 min in wild-type fibroblasts. In PKR fibroblasts, activate MAPKs and proliferation in fibroblasts. To however, TNF had no effect on IkBa degradation investigate the role of PKR in TNF signaling, we used (Figure 2a, upper panel). These results suggest that PKR wild-type mouse embryonic fibroblast (MEF) and PKR is required for degradation of IkBa. gene-deleted fibroblasts (MEF/PKRÀ/À). We evaluated the expression of TNFR1 and TNFR2 in both MEF PKR is required for TNF-dependent IkBa and MEF/PKRÀ/À cells by Western blot analysis and phosphorylation found TNFR1 was expressed equally in both cell lines The proteolytic degradation of IkBa is known to require (Figure 1a). TNFR2 was not detected (data not shown). phosphorylation at serine residues 32 and 36 (Ghosh

Oncogene Role of PKR in TNF signaling Y Takada et al 1203

Figure 1 PKR is required for TNF-induced NF-kB activation. (a) Effect of PKR deletion on expression levels of TNF receptors. Whole-cell extracts were prepared, resolved by SDS–PAGE and electrotransferred to a nitrocellulose membrane, and then performed Western blot analysis using anti-TNFR1 antibody. (b) TNF induces PKR activation. One million cells were treated with 1 nM TNF for the indicated times, prepared whole-cell extracts and then performed Western blot analysis using indicated antibodies. (c) Time- dependent effects of NF-kB activation on PKRÀ/À fibroblasts by TNF. One million cells were treated with 0.1 nM TNF for the indicated times, prepared nuclear extracts and analysed NF-kB activation by EMSA. (d) Dose-dependent effect of NF-kB activation in PKRÀ/À fibroblasts by TNF. One million cells were treated with the indicated concentrations of TNF for 30 min, prepared nuclear extracts and analysed NF-kB activity by EMSA. (e) Composition of NF-kB induced by TNF. Nuclear extracts from untreated or 0.1 nM TNF- treated MEFs were incubated with different antibodies, preimmune serum or unlabeled NF-kB oligoprobe (competitor) or mutant oligoprobe (mutant oligo), and then analysed NF-kB activity by EMSA.

and Karin, 2002). Todetermine whether PKR deletion PKR deletion inhibits TNF-induced IKK activation affects TNF-induced IkBa phosphorylation, we Because IKK is required for TNF-induced phospho- performed Western blot analysis, using an antibody rylation of IkBa (Ghosh and Karin, 2002), we that recognizes the serine-phosphorylated form of next determined the effect of PKR deletion on TNF- IkBa. TNF induced IkBa phosphorylation as early induced IKK activation. As shown in Figure 2b, PKR as 5 min in wild-type fibroblasts but in PKRÀ/À deletion completely suppressed TNF-induced activation fibroblasts, IkBa phosphorylation induced by TNF of IKK without any effect on the expression of IKK-a was almost completely suppressed (Figure 2a, middle or IKK-b. These results suggest that PKR is required panel). for TNF-induced IKK activation. PKR deletion

Oncogene Role of PKR in TNF signaling Y Takada et al 1204

Figure 2 PKR is required for TNF-induced degradation and phosphorylation of IkBa, activation of IKK, and phosphorylation and translocation of p65. (a) One million cells were treated with 0.1 nM TNF for the indicated times, prepared cytoplasmic extracts, resolved by SDS–PAGE and electrotransferred to a nitrocellulose membrane, and then performed Western blot analysis using the indicated antibodies. (b) PKR is required for TNF-induced IKK activation. One million cells were stimulated with 1 nM TNF for the indicated times. Whole-cell extracts were incubated with anti-IKK-a antibody for 2 h, immunoprecipitated with protein A/G-sepharose beads, and then analysed by immunocomplex kinase assay. To examine the level of expression of IKK proteins, the same whole-cell extracts were resolved by SDS–PAGE and performed Western blot analysis using anti-IKK-a and anti-IKK-b antibodies. (c) One million cells were treated with 0.1 nM TNF for the indicated times, prepared nuclear extracts, resolved by SDS–PAGE and electrotransferred to a nitrocellulose membrane, and then performed Western blot analysis using the indicated antibodies. (d) Immunocytochemical analysis of p65 localization. Cells were treated with 1 nM TNF for 15 min, then cells were subjected to immunocytochemical analysis as described in Materials and methods.

suppressed TNF-induced activation of IKK but not PKRÀ/À fibroblasts, TNF failed to induce nuclear complete. translocation of p65. TNF induces the phosphorylation of p65, which is PKR is required for TNF-induced phosphorylation and required for its transcriptional activity (Ghosh et al., 1998). nuclear translocation of p65 Therefore, we also determined the effect of PKR deletion Degradation of IkBa leads to nuclear translocation of on TNF-induced phosphorylation of p65 by Western blot the p65 subunit of NF-kB. Therefore, we also deter- analysis. As shown in Figure 2c (lower panel), TNF mined the effect of PKR deletion on TNF-induced induced phosphorylation of p65 in a time-dependent nuclear translocation of p65 by Western blot analysis. manner; as early as 5 min after TNF stimulation, p65 As shown in Figure 2c (upper panel), TNF induced was phosphorylated in wild-type fibroblasts. In PKRÀ/À nuclear translocation of p65 in a time-dependent fibroblasts, TNF failed to induce phosphorylation of p65. manner; as early as 5 min after TNF stimulation, Tofurther confirmthe effect of PKR deletion on nuclear p65 was noted in wild-type MEF cells. In the suppression of nuclear translocation of p65, we

Oncogene Role of PKR in TNF signaling Y Takada et al 1205 performed an immunocytochemical assay. The PKR is required for TNF-induced activation of JNK results showed that p65 was localized in the cytoplasm, We investigated the effect of PKR on activation of JNK, TNF induced nuclear translocation of p65 in one of the earliest events induced by TNF (Aggarwal, wild-type fibroblasts and TNF failed to induce p65 2003). We treated cells with TNF for the indicated translocation to the nucleus in PKRÀ/À fibroblasts intervals, prepared whole-cell extracts and analysed (Figure 2d). them for JNK activity by immunocomplex kinase assay. TNF induced time-dependent activation of JNK in wild-type fibroblasts but not in PKRÀ/À fibroblasts PKR is required for TNF-induced activation of Akt (Figure 3b). These results suggest that PKR is required Activation of Akt is the earliest event induced by TNF for TNF-induced JNK activation. (Ozes et al., 1999). To explore the specific role of PKR in TNF-induced Akt activation, we treated cells with TNF PKR potentiates TNF-induced activation of p44/p42 for the indicated intervals, prepared whole-cell extracts, MAPK resolved them by sodium dodecyl sulfate–polyacryla- TNF has been shown to activate p44/p42 MAPK mide gel electrophoresis (SDS–PAGE) and performed through the Ras/Raf/MAPK kinase cascade (Aggarwal, Western blot analysis using phosphospecific anti-Akt 2003). To explore the specific role of PKR in TNF- antibody. Time-dependent activation of Akt occurred in induced p44/p42 MAPK activation, we treated the wild-type fibroblasts but not in PKRÀ/À fibroblasts fibroblasts with TNF for the indicated intervals, (Figure 3a). Thus, PKR is needed for TNF-induced prepared whole-cell extracts, resolved them by SDS– Akt activation. PAGE and performed Western blot analysis using

Figure 3 PKR modifies TNF-induced Akt, JNK, p44/p42 MAPK andp38 MAPK activation. One million cells were treated with 1 nM TNF for the indicated times, prepared whole-cell extracts and subjected to Western blot analysis for Akt, JNK, p44/p42 MAPK and p38 MAPK assay. (a) Whole-cell extracts were subjected to Western blot analysis using indicated phosphospecific antibodies. The same membranes were reblotted with relevant antibodies for Akt. (b) Whole-cell extracts were incubated with anti-JNK1 antibody for 2 h, and then immunoprecipitated with protein A/G-sepharose beads. The beads were washed and subjected to immunocomplex kinase assay. The same protein extracts were resolved by SDS–PAGE and electrotransferred to a nitrocellulose membrane, and then performed Western blot analysis using anti-JNK1 antibody. Whole-cell extracts were subjected to Western blot analysis using indicated phosphospecific antibodies. The same membranes were reblotted with relevant antibodies for p44/p42 MAPK (c) and p38 MAPK (d).

Oncogene Role of PKR in TNF signaling Y Takada et al 1206 phosphospecific anti-p44/p42 MAPK antibody. We co-transfected the cells with the NF-kB reporter found that TNF-induced p44/p42 MAPK phosphoryla- construct along with the PKR plasmid and then tion, unlike the other kinases, was potentiated in PKRÀ/À monitored TNF-induced NF-kB-regulated reporter fibroblasts as compared to the control cells (Figure 3c). . We found that reconstitution of These results suggest that PKR negatively regulates PKRÀ/À fibroblasts cells reverses the TNF-induced TNF-induced p44/p42 MAPK activation and this NF-kB activation (Figure 4c), indicating the critical regulation is different from that of IKK, Akt or JNK. role of PKR.

PKR potentiates TNF-induced activation of p38 MAPK PKR is required for TNF-induced NF-kB-dependent TNF has been shown to activate p38 MAPK through cyclin D1, c-Myc and MMP-9 proteins expression the MAPK kinase kinase 3 cascade (Aggarwal, 2003). TNF-induced NF-kB has been shown to induce cyclin To explore the specific role of PKR in TNF-induced p38 D1, c-Myc and MMP-9 (Guttridge et al., 1999; Esteve MAPK activation, we treated the fibroblasts with TNF et al., 2002; Takada et al., 2004b); thus, we determined for the indicated intervals, prepared whole-cell extracts, whether PKR is required for this induction. Cells were resolved them by SDS–PAGE and performed Western treated with TNF for different intervals, prepared blot analysis using phosphospecific anti-p38 MAPK whole-cell extracts and Western blot analysis was antibody. Time-dependent phosphorylation of p38 performed to determine the expression of cyclin D1, MAPK occurred in PKRÀ/À fibroblasts. Like p44/p42 c-Myc and MMP-9 (Figure 5a). Cyclin D1, c-Myc and MAPK, TNF-induced p38 MAPK activation was MMP-9 expression was induced by TNF in a time- negatively regulated in control fibroblasts by PKR dependent manner in wild-type MEF but not in PKRÀ/À (Figure 3d). MEF.

PKR is required for TNF-induced NF-kB-dependent PKR is required for TNF-induced NF-kB-dependent reporter gene expression antiapoptotic protein expression DNA binding does not always correlate with NF-kB- Because NF-kB also regulates the expression of several dependent gene transcription (Hsu et al., 1996; antiapoptotic proteins, including survivin (Zhu et al., Nasuhara et al., 1999; Simeonidis et al., 1999). To 2001), X-linked inhibitor-of-apoptosis protein (XIAP) determine the role of PKR in TNF-induced NF-kB- (Stehlik et al., 1998), inhibitor-of-apoptosis protein dependent reporter gene expression, we transiently (IAP) 1 (You et al., 1997), Bcl-xL (Tamatani et al., transfected cells with the NF-kB-regulated secretory 1999), A1/Bfl-1 (Grumont et al., 1999; Zong et al., 1999) alkaline phosphatase (SEAP) reporter construct and and FLICE-inhibitory protein (FLIP) (Kreuz et al., then stimulated the cells with different concentrations of 2001), we tested the effect of PKR on the TNF-induced TNF. NF-kB-regulated reporter gene expression was expression of these antiapoptotic gene products. As activated by TNF in a dose-dependent manner in wild- shown in Figure 5b, TNF induced antiapoptosis protein type MEF, but minimal activation was seen in PKR- expression in a time-dependent manner in wild-type À/À deleted cells (Figure 4a). These results suggest that PKR fibroblasts but not in PKR fibroblasts. These results is required not only for the activation of IKK, nuclear further strengthen our theory that PKR is required for translocation of p65 and p65 binding to DNA but also TNF induction of NF-kB-regulated gene products. for NF-kB-regulated reporter gene expression. TNF-induced NF-kB activation is mediated through PKR is required for TNF-induced cell proliferation the sequential interaction of the TNFR with TRADD, TNF has been shown to induce cell proliferation TRAF2, NF-kB inducing kinase (NIK) and IKK, (Aggarwal, 2003). Our results suggest that deletion of resulting in phosphorylation of IkBa (Hsu et al., 1996; PKR might inhibit cell proliferation induced by TNF Simeonidis et al., 1999). Todetermine the effect ofPKR through suppression of NF-kB activation. 3-(4,5- deletion on NF-kB-dependent reporter gene expression dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide activated by TNFR1, TRADD, TRAF2, NIK, IKK and (MTT) assay showed that TNF induced cell prolifera- p65, we transiently co-transfected the cells with the tion and PKR deletion suppressed it (Figure 6a). NF-kB reporter construct with TNFR1, TRADD, Furthermore, 3H-thymidine incorporation assay also TRAF2, NIK, IKK or p65-expressing plasmids and showed that TNF induced DNA synthesis and PKR then monitored them for SEAP expression. We found deletion suppressed it (Figure 6b). that cells transfected with TNFR1, TRADD, TRAF2, Whether TNF-induced proliferation of MEF is NIK, IKK and p65 plasmids showed NF-kB-regulated through PKR-induced NF-kB activation, was examined. reporter gene expression in wild-type MEF. However, in A specific NF-kB inhibitory peptide (Takada et al., PKR-deficient MEF, TNFR1, TRADD and TRAF2 2004c) was used to evaluate the role of NF-kBin failed toinduce NF- kB reporter gene expression PKR-mediated cell proliferation by TNF. The results (Figure 4b). NF-kB activated by NIK and IKK was shown in Figure 6c indicate that suppression of NF-kB minimally affected. by specific peptide inhibited the TNF-induced prolifera- To investigate whether suppression of PKR-mediated tion of MEF cells. These results suggest that PKR NF-kB activation in PKRÀ/À fibroblasts can be reversed mediates its proliferative effects through TNF-induced by transfection with the PKR plasmid, we transiently NF-kB activation.

Oncogene Role of PKR in TNF signaling Y Takada et al 1207

Figure 4 PKR is required for TNF-induced NF-kB-dependent gene expression. (a) PKR is required for TNF-induced NF-kB- dependent gene expression. Cells were transiently transfected with a NF-kB-containing plasmid for 24 h. After transfection, cells were treated with the indicated concentrations of TNF for a further 24 h. The supernatants of the culture medium were assayed for SEAP activity as described in Materials and methods. (b) PKR is required for NF-kB-dependent reporter gene expression induced by TNF, TNFR1, TRADD and TRAF2. Cells were transiently transfected with a NF-kB-containing plasmid alone or with the indicated plasmids. After transfection, media were changed, and the cells were incubated for an additional 24 h. For TNF-treated cells, media were changed after transfection and treated with 1 nM TNF for an additional 24 h. The supernatants of the culture medium were assayed for SEAP activity. (c) Cells were transiently transfected with a NF-kB-containing plasmid or with PKR plasmid. After transfection, media were changed, and the cells were incubated for an additional 24 h. For TNF-treated cells, media were changed after transfection and treated with 1 nM TNF for an additional 24 h. The supernatants of the culture medium were assayed for SEAP activity. Control was indicated as ‘C’.

Deletion of PKR potentiates TNF-induced apoptosis Discussion Activation of NF-kB has been shown to inhibit TNF- induced apoptosis (Verma and Stevenson, 1997). The goal of the study presented here was to determine Whether suppression of NF-kBbyPKR deletion affects the role of PKR in TNF signaling. Our findings show TNF-induced apoptosis was therefore investigated. Our that TNF-induced NF-kB activation, IKK activation, results indicate that deletion of PKR enhanced TNF- IkBa phosphorylation, IkBa degradation and NF-kB induced apoptosis from 2 to 49.2% (Figure 6d). reporter gene transcription were all suppressed in PKR

Oncogene Role of PKR in TNF signaling Y Takada et al 1208 whereas Ishii et al. (2001) showed that a kinase-inactive mutant of PKR diminished TNF-induced NF-kB binding and increased the IkBa phosphorylation in unstimulated cells. Similarly, Deb et al. (2001) reported that TNF-induced NF-kB DNA binding was defective in PKR-null cells. A PKR inhibitor, 2-aminopurine, failed tosuppress TNF-induced NF- kB activation as examined by DNA binding (Cheshire et al., 1999) and in another study, deletion of PKR did not affect TNF- induced IkBa degradation (Gil et al., 2000). Why the results of these reports differ so vastly is not clear. None of these reports, however, directly addressed the role of PKR in TNF-induced NF-kB activation. In almost all instances, a single dose and time interval of exposure to TNF was evaluated in the context of dsRNA. No studies involved a detailed analysis of the NF-kB activation pathway induced by TNF. Our results also suggest that deletion of PKR affects a step upstream from TRAF2. Gil et al. (2004) previously reported that PKR–TRAF interaction is structurally feasible, suggest- ing alternative docking positions for the two structural motifs. They also showed that TRAF proteins (at least TRAF2, -5 and -6) are universal adapters linking NF-kB activation not only to membrane receptor-triggered pathways but also to dsRNA-depen- dent NF-kB activation. Figure 5 PKR is required for TNF-induced NF-kB-dependent Our results also showed that PKR is needed for TNF- protein expression. PKR is required for the expression of TNF- induced Akt activation. There have been no reports induced cyclin D1, c-Myc, MMP-9 (a) and antiapoptotic proteins about the role of PKR in Akt activation. We found that (b). Cells were treated with 1 nM TNF for the indicated times. Whole-cell extracts were prepared and subjected to Western blot TNF-induced activation of JNK was also defective in analysis using the relevant antibodies. PKR-null cells. Our results agree with those of Goh et al. (2000), who found that PKR was needed for efficient activation of JNK by TNF. By using kinase- gene-deleted fibroblasts. Deletion of PKR alsoabolished defective PKR, however, Ishii et al. (2001) found that TNF-induced JNK and Akt but not p44/p42 MAPK PKR had norolein TNF-induced JNK activation.The and p38 MAPK activation. TNF-induced the expression role of PKR in dsRNA-induced JNK activation has of NF-kB-regulated gene products, cyclin D1, c-Myc, been described previously (Iordanov et al., 2000). MMP-9, survivin, IAP1, Bcl-xL, A1/Bfl-1, and FLIP Our results alsoindicate that TNF-induced p38 were all downmodulated in the PKRÀ/À fibroblasts and MAPK is potentiated on deletion of PKR. These results this correlated with the suppression of TNF-induced cell differ from those of reports that indicate that PKR is proliferation. needed for p38 MAPK activation induced by dsRNA Our results clearly show that deletion of the PKR gene (Goh et al., 2000; Iordanov et al., 2000). These results abolishes TNF-induced NF-kB activation. This is the suggest that there are multiple pathways for p38 MAPK first report to systematically evaluate the role of PKR in activation depending on the stimulus. We found that TNF-induced NF-kB DNA binding, IkBa phosphoryla- TNF-induced activation of p44/p42 MAPK was also tion and degradation, Akt and IKK activation, p65 enhanced in PKR-null cells. dsRNA has been shown to phosphorylation and nuclear translocation and NF-kB- activate p44/p42 MAPK in macrophages in a PKR- dependent reporter gene transcription. Although the independent manner (Maggi et al., 2003). role of PKR in dsRNA-induced NF-kB activation has We found that deletion of the PKR gene abrogated been well established (Yang et al., 1995; Kumar et al., expression of several NF-kB-regulated gene products, 1997), its role in TNF-induced NF-kB activation has including cyclin D1, c-Myc, MMP-9, survivin, XIAP, been controversial. By using DNA-binding assay, Der IAP1, Bcl-xL, A1/Bfl-1 and FLIP. Raveh et al. (1996) et al. (1997) showed that PKR gene deletion had no found that PKR mediated c-Myc suppression induced effect on TNF- or LPS-induced NF-kB activation. The by type I IFNs, and Kronfeld-Kinar et al. (1999) also lack of effect in their study may be because they used found that PKR overexpression downregulated the only a single dose and time interval to evaluate NF-kB expression of cyclin D1 and c-Myc in myogenesis. Our activation. In fact, it was found that PKR was required results, however, indicate that PKR regulates TNF- for sustained response to TNF (Zamanian-Daryoush induced cyclin D1 and c-Myc synthesis. We demon- et al., 2000). By using a trans dominant-negative strated that TNF-induced MMP-9 expression was mutant, Srivastava et al. (1998) found that PKR had abolished in PKRÀ/À cells. This finding is in agreement norolein TNF-induced NF- kB reporter activity, with that of Gilbert et al. (2004), whodetermined the

Oncogene Role of PKR in TNF signaling Y Takada et al 1209

Figure 6 PKR is required for TNF-induced cell proliferation. (a) Five thousand cells were seeded in a 96-well plate and incubated with indicated concentrations of TNF for 48 h. Thereafter, relative cell growth was analysed by the MTT method as described in Materials and methods. (b) Ten thousand cells were seeded in a 96-well plate and incubated with indicated concentrations of TNF for 18 h. Cells were then incubated with 3H-thymidine for a further 6 h. Thereafter, 3H-thymidine incorporation assay was performed as described in Materials and methods. (c) Ten thousand wild-type MEF were seeded in a 96-well plate and treated with indicated concentrations of p65-inhibitory peptide for 1 h, and incubated with 1 nM TNF for 18 h. Cells were then incubated with 3H-thymidine for a further 6 h. Thereafter, 3H-thymidine incorporation assay was performed as described in Materials and methods. (d) Cells (1 Â 105 cells/well) were pretreated with 1 mg/ml cycloheximide for 1 h and then incubated with 1 nM TNF for 16 h. Cells were stained with Live and Dead assay as described under ‘Materials and methods.’

role of PKR in MMP-9 induction by TNF using PKR D1, c-Myc, survivin, XIAP, IAP1, Bcl-xL, A1/Bfl-1 and inhibitor 2-aminopurine. FLIP. Overall, we found that PKR plays a critical role The downmodulation of cyclin D1 and c-Myc found not only in TNF-induced NF-kB activation but also in in our study suggests that deletion of PKR suppresses TNF-induced activation of IKK, JNK, Akt and cell cell proliferation. Indeed, we found that TNF-induced proliferation because of the critical role of TNF and cell proliferation was significantly suppressed by PKR NF-kB in cancer, inflammation, diabetes and neuro- deletion. Although PKR is known to mediate TNF- generative diseases. induced apoptosis in certain cells (Yeung and Lau, 1998), its role in TNF-induced proliferation of cells is not known. The difference in results may be owing to Materials and methods cell types used; human myeloid U-937 cells used by Yeung and Lau (1998) may have multiple genetic Reagents defects, as compared to PKR-deleted MEF used in All the reagents employed in these studies were as described our studies. Consistent with our results, Donze et al. (Takada and Aggarwal, 2004). (2004) showed that PKR activates a cell survival pathway through the expression of NF-kB-regulated Cell lines cIAP and A20 gene products. We also found that The spontaneously transformed MEF from PKR-deficient mice and its wild-type control were kindly provided by Dr deletion of PKR suppressed Akt activation, which plays Glen Barber (University of Miami, Miami, FL, USA), as a pivotal role in the cell survival pathway (Luo et al., described (Yang et al., 1995). These cells were cultured in 2003). All these results indicate that PKR has a role in Dulbecco’s modified Eagle’s medium supplemented with promoting survival. Our results demonstrate that 10% fetal bovine serum, 100 U/ml penicillin and 100 mg/ml this role is mediated through the expression of cyclin streptomycin.

Oncogene Role of PKR in TNF signaling Y Takada et al 1210 Electrophoretic mobility shift assays Live and dead assay Todetermine NF- kB activation, we performed EMSA as To measure the apoptosis induced by TNF, assay was described previously (Takada and Aggarwal, 2004). performed by a method described previously (Takada et al., 2004c). Western blot analysis To determine the levels of protein expression in the cytoplasm or nucleus, we performed Western blot analysis as described Abbreviations previously (Takada and Aggarwal, 2004). dsRNA, double-stranded RNA; PKR, dsRNA-dependent protein kinase; NF-kB, nuclear factor-kB; IkB, inhibitory IKK assay subunit of NF-kB; IKK, IkBa kinase; SEAP, secretory To determine the effect of TNF on IKK activation, the IKK alkaline phosphatase; IAP, inhibitor-of-apoptosis protein; assay was performed by a method described previously IL, interleukin; FADD, Fas-associated death domain protein; (Takada et al., 2004b). FLICE, FADD-like IL-1b-converting enzyme; FLIP, FLICE- inhibitory protein; MMP, matrix metalloproteinase; LPS, Immunocytochemical analysis for NF-kB p65 localization lipopolysaccharide; JNK, c-Jun N-terminal kinase; MAPK, The effect of TNF on the nuclear translocation of p65 was mitogen-activated protein kinase. examined by an immunocytochemical method as described previously (Takada et al., 2004a). Acknowledgements

MTT assay We thank Ann Sutton for carefully proofreading the manu- The effects of TNF on cell growth were determined by the script and providing valuable comments. Dr Takada is an MTT uptake assay as described previously (Takada and Odyssey Program Special Fellow at The University of Texas Aggarwal, 2003). MD Anderson Cancer Center. Dr Aggarwal is a Ransom Horne Jr, Professor of Cancer Research. This work was supported by a grant from the Clayton 3H-thymidine incorporation assay Foundation for Research (to BBA), Department of Defense The effect of TNF on DNA synthesis was monitored by the US Army Breast Cancer Research Program Grant BC010610 3H-thymidine incorporation assay. Ten thousand cells in 100 ml (to BBA), National Institutes of Health PO1 Grant CA91844 of medium were cultured in triplicate in a 96-well plate with the on lung chemoprevention (to BBA), National Institutes of indicated concentrations of TNF for 18 h. Then, cells were Health P50 Head and Neck SPORE Grant P50CA97007 (to pulse-treated with 0.5 mCi 3H-thymidine for 6 h, and the uptake BBA) and grants from the Odyssey Program and the Theodore of 3H-thymidine was monitored using a Matrix-9600 b-counter N Law Award for Scientific Achievement Fund from The (Packard Instruments, Downers Grove, IL, USA). University of Texas MD Anderson Cancer Center (to YT).

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