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Genetic Deletion of PKR Abrogates TNF-Induced Activation of Ijba Kinase, JNK, Akt and Cell Proliferation but Potentiates P44/P42 MAPK and P38 MAPK Activation

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Genetic Deletion of PKR Abrogates TNF-Induced Activation of Ijba Kinase, JNK, Akt and Cell Proliferation but Potentiates P44/P42 MAPK and P38 MAPK Activation 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 kinase, 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 gene-deleted mice to survival; apoptosis investigate the role of PKR in TNF-induced activation of nuclear factor-jB (NF-jB), mitogen-activated protein kinases (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 interferon (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 enzyme-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 p53 (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.
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