Involvement of Double-stranded RNA-activated Protein Kinase in the Synergistic Activation of Nuclear Factor-kappa B by Tumor Necrosis Factor-alpha and gamma -Interferon in Preneuronal Cells*

Jeanette L. CheshireDagger §, Bryan R. G. Williamsparallel , and Albert S. Baldwin Jr.Dagger **

From the Dagger  Lineberger Comprehensive Cancer Center, University of North Carolina School of Medicine, Chapel Hill, North Carolina 27599, and the  Department of Cancer Biology, Lerner Research Institute, Cleveland, Ohio 44195

    ABSTRACT
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Abstract
Introduction
References

Tumor necrosis factor-alpha (TNF-alpha ) and gamma -interferon (IFN-gamma ) cooperate during a variety of biological responses and ultimately synergistically enhance the expression of genes involved in immune and inflammatory responses. Recently, we demonstrated that IFN-gamma can significantly potentiate TNF-alpha -induced nuclear factor (NF)-kappa B nuclear translocation in neuronal derived and endothelial cell lines. The mechanism by which these two cytokines exert their synergistic effect on NF-kappa B involves the de novo degradation of the NF-kappa B inhibitor, Ikappa Bbeta . The double-stranded RNA-dependent kinase PKR is IFN-inducible and has been implicated in the activation of NF-kappa B; therefore, we examined the possibility that PKR may play a role in the synergistic activation of NF-kappa B during TNF-alpha /IFN-gamma cotreatment. The PKR inhibitor 2-aminopurine (2-AP) inhibited TNF-alpha /IFN-gamma -induced NF-kappa B nuclear translocation in neuronal derived cells but not in endothelial cells. The induced degradation of Ikappa Bbeta , which is normally observed upon TNF-alpha /IFN-gamma cotreatment, was blocked completely by 2-AP in neuronal derived cells. Also, 2-AP treatment or overexpression of a catalytically inactive PKR inhibited the TNF-alpha /IFN-gamma -induced synergistic activation of kappa B-dependent gene expression. Our results suggest that the signal generated by IFN-gamma during TNF-alpha /IFN-gamma cotreatment may require PKR to elicit enhanced NF-kappa B activity, and this signal may affect the stability of the Ikappa Bbeta protein.

    INTRODUCTION
Top
Abstract
Introduction
References

The transcription factor nuclear factor-kappa B (NF-kappa B)1 is activated by a variety of stimuli including cytokines, mitogens, cellular stress, and bacterial or viral products (for review, see Refs. 1-5). The family of mammalian NF-kappa B transcription factors consists of at least five distinct members: c-Rel, p50 (NF-kappa B1), p52 (NF-kappa B2), p65 (RelA), and RelB, which form a variety of active homo- and heterodimers (for review, see Refs. 1-5). Classic NF-kappa B exists as a p50-p65 heterodimer that is sequestered in the cytoplasm by inhibitor proteins collectively referred to as inhibitors of kappa B (Ikappa Bs) (for review, see Ref. 4). The two major forms of Ikappa B are Ikappa Balpha and Ikappa Bbeta . Upon stimulation, an activated Ikappa B kinase (IKK) complex (6-9) phosphorylates the Ikappa B proteins, which targets these inhibitor proteins for ubiquitination and degradation (10-13). This process allows NF-kappa B to translocate to the nucleus and regulate gene-specific transcription. Structurally, Ikappa Balpha and Ikappa Bbeta are similar, and both interact with p65- and c-Rel-containing dimers through similar binding domains (14). Additionally, both forms of Ikappa B are phosphorylated on analogous serine residues by the activated IKK complex (6). However, Ikappa Balpha is characteristically involved in the transient activation of NF-kappa B, whereas Ikappa Bbeta has been implicated in the persistent activation of NF-kappa B (14-17). There is also evidence that the stimuli that ultimately target the Ikappa Bs for degradation may differ, although this may be cell type-specific or may depend on the concentration of the inducer (14, 15, 18).

The pleiotropic cytokines tumor necrosis factor-alpha (TNF-alpha ) and interferon (IFN) can function together to coregulate gene expression synergistically in a variety of cell lines. Typically, the coregulatory effects involve the independent activation of NF-kappa B by TNF-alpha (for review, see Ref. 4) and of IFN-responsive factors by IFNs (for review, see Refs. 19 and 20), permitting these transcription factors to bind their unique sites within the promoters of target genes such as MHC class I, ICAM-1, VCAM-1, inducible iNOS, interleukin-6, and interleukin-8 (21-26). Recently, we reported that IFN-gamma , which typically does not activate NF-kappa B, synergistically enhances TNF-alpha -induced nuclear translocation of p50-p65 NF-kappa B heterodimers and synergistically activates kappa B-dependent gene expression (27). We also demonstrated that the mechanism for this synergistic activation involved the de novo degradation of the Ikappa Bbeta protein and that the TNF-alpha /IFN-gamma coactivation of NF-kappa B in PC12 cells is sensitive to the protein tyrosine kinase inhibitor genistein (27).

An important signal transduction molecule that is targeted by IFNs is the double-stranded RNA (dsRNA)-activated protein kinase (PKR). This serine/threonine kinase was first discovered as a translation inhibitor because of its ability to phosphorylate and deactivate the translation initiation factor, eIF-2 (for review, see Ref. 28). It plays a role in cellular antiviral responses and growth control and is a candidate tumor supressor gene (for review, see Refs. 29 and 30). PKR is IFN-inducible, is present at low levels in most cells, and is found in the nucleus as well as in the cytoplasm (31). Recent evidence indicates that activation of NF-kappa B by dsRNA, but not by TNF-alpha or interleukin-1beta , may involve PKR and that PKR may phosphorylate Ikappa Balpha in vitro (32-35). Therefore, we examined the possibility that PKR may be involved in TNF-alpha /IFN-gamma -induced synergistic activation of NF-kappa B.

In this report, we provide evidence supporting a role for PKR involvement in the synergistic activation of NF-kappa B by TNF-alpha /IFN-gamma cotreatment in the preneuronal derived cell line, PC12. The PKR inhibitor 2-aminopurine (2-AP) (36, 37) blocks synergistic TNF-alpha /IFN-gamma -induced NF-kappa B nuclear translocation. The requirement for PKR may be specific for cells of neuronal origin because 2-AP was able to block the synergy in another neuronal derived cell line (B12), but not in an endothelial cell line (EA.hy926). The synergistic activation of kappa B-dependent gene expression can be inhibited by 2-AP or by the overexpression of a catalytically inactive, dominant negative form of PKR. Also, 2-AP inhibits the de novo degradation of Ikappa Bbeta observed during TNF-alpha /IFN-gamma cotreatment in PC12 cells and B12 cells but does not affect the normal pattern of Ikappa Balpha degradation. Therefore, the mechanism by which the IFN-inducible kinase PKR functions in this system may involve targeted phosphorylation and degradation of Ikappa Bbeta . These data indicate a novel role for PKR in the activation of NF-kappa B.

    EXPERIMENTAL PROCEDURES

Cell Culture and Treatments-- The rat preneuronal adrenal pheochromocytoma cell line, PC12 (CRL 1721, American Type Culture Collection, Rockville, MD) (38), was maintained in Dulbecco's modified Eagle's medium F-12 supplemented with 10% fetal bovine serum and antibiotics. The central nervous system-derived rat preneuronal cell line B12 (gift of Dave Schubert, The Salk Institute, La Jolla, CA) (39) was maintained in Dulbecco's modified Eagle's medium H supplemented with 10% fetal bovine serum and antibiotics. The human vascular endothelial cell line EA.hy926 (gift of Cora-Jean S. Edgell, University of North Carolina, Chapel Hill) (40) was maintained in Dulbecco's modified Eagle's medium H supplemented with 10% fetal bovine serum, 1× hypoxanthine-aminopterin-thymidine medium supplement (Boehringer Mannheim), and antibiotics.

Cells were incubated for the times indicated under "Results" with 0.025-10 ng/ml human recombinant TNF-alpha (Boehringer Mannheim), 50-100 units/ml rat recombinant IFN-gamma (Life Technologies, Inc.), or 10 mM 2-AP (Sigma).

Nuclear and Cytoplasmic Extracts-- The day before treatment, cells were plated in 10 ml of complete media in 100-mm tissue culture plates at 1 × 107 cells/plate (PC12), 1 × 106 cells/plate (B12), or 2 × 106 cells/plate (EA.hy926). After treatment, nuclear and cytoplasmic extracts were made using a procedure described previously (27). Briefly, cells were washed with phosphate-buffered saline, scraped from plates, transferred to microcentrifuge tubes, and lysed on ice in 3 pellet volumes of cytoplasmic extraction buffer (10 mM Hepes, pH 7.6, 60 mM KCl, 1 mM EDTA, 0.1% Nonidet P-40, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, and 2.5 µg/ml each of aprotinin, leupeptin, and pepstatin). Nuclei were pelleted, and cytoplasmic supernatants were transferred to fresh tubes. Nuclei were washed with 100 µl of extraction buffer without Nonidet P-40 and then repelleted. Supernatants were discarded, and nuclear pellets were resuspended by vortexing in 2 pellet volumes of nuclear extraction buffer (20 mM Tris, pH 8.0, 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM phenylmethylsulfonyl fluoride, 25% glycerol, and 2.5 µg/ml each of aprotinin, leupeptin, and pepstatin) in which the final salt concentration was adjusted to ~400 mM NaCl. All cytoplasmic and nuclear extracts were cleared and transferred to fresh tubes. Next, glycerol was added to the cytoplasmic extracts to a final concentration of 20%, protein concentrations were determined by the Bradford assay using the Bio-Rad protein assay dye reagent (500-0006), and all extracts were stored at -70 °C until analyzed.

EMSAs-- Electrophoretic mobility shift assays (EMSAs) were performed as described previously (27). Briefly, equal amounts of nuclear extracts were incubated for 15 min at room temperature with a 32P-labeled probe containing a kappa B site from the class I MHC promoter (41, 42) in binding buffer (10 mM Tris, pH 7.7, 50 mM NaCl, 0.5 mM EDTA, 1 mM dithiothreitol, and 10% glycerol) (43) plus 2 µg of poly(dI-dC)·poly(dI-dC) (Amersham Pharmacia Biotech). Complexes were separated in 5% polyacrylamide gels in Tris-glycine-EDTA buffer (25 mM Tris, 190 mM glycine, and 1 mM EDTA), dried, and autoradiographed.

Western Blot Analysis-- Equal amounts of cytoplasmic extracts were electrophoresed in 10% polyacrylamide-SDS gels and transferred to nitrocellulose membranes (Schleicher & Schuell) (27). The upper half of each membrane was probed with an antibody specific for Ikappa Bbeta (sc-945, Santa Cruz), and the lower half was probed with an antibody specific for Ikappa Balpha (100-4167C, Rockland). Specific proteins were visualized by enhanced chemiluminescence (Amersham Pharmacia Biotech).

Transient Transfections and Luciferase Assays-- Transient transfection of PC12 cells was accomplished using 20 µl/ml LipofectAMINE reagent (Life Technologies, Inc.) and a total of 6 µg of DNA for each sample. The MHC-NF-kappa BLuc plasmid contains three tandem repeats of the kappa B site from the class I MHC enhancer cloned into a luciferase expression vector (44). Luciferase expression vector was a gift of Bill Sugden, University of Wisconsin, Madison. The wild-type PKR expression plasmid (wtPKR) and the catalytically inactive Lys296 right-arrow Arg mutant PKR expression plasmid (mutPKR) were described previously (32). PC12 cells were plated in 60-mm tissue culture plates (7 × 106 cells/plate) the day before transfection. LipofectAMINE-DNA complexes were allowed to form for 30 min in serum-free medium before being added to plates containing cells plus 2 ml of serum-free medium (27). Cells were incubated with the complexes for 7-8 h, the medium was replaced with medium containing 0.5% serum, and 8 h of cytokine treatment began 36 h after the medium change. Cells were washed, collected, resuspended in 2 pellet volumes of 0.25 M Tris pH 7.5, and subjected to three cycles of freeze/thaw. Samples were cleared, and protein concentrations were determined using the Bio-Rad protein assay dye reagent. Luciferase assays were performed in duplicate on equal amounts of protein using 200 µM D-Luciferin as a substrate (Sigma), and relative light units were determined using an AutoLumat LB953 luminometer (Berthold Analytical Instruments, Inc., Nashua, NH).

Stable Transfectants-- PC12 cells stably expressing MHC-NF-kappa BLuc or its mutated counterpart were obtained by LipofectAMINE cotransfections with the reporter plasmid and pcDNA3 (Invitrogen, Carlsbad, CA), which contains the neomycin resistance gene. 2 days post-transfection, the medium was replaced with complete medium containing 600 µg/ml Geneticin/G418 (Life Technologies) for selection purposes. Fresh G418-containing medium was added every 4-5 days for 2 months, allowing pools of cells stably expressing MHC-NF-kappa BLuc to grow out. Stably transfected cells were plated (80% confluent in 60-mm plates) the day before treatment. Where indicated, cells were pretreated for 1 h with 10 mM 2-aminopurine before treatment for 8 h with TNF-alpha and/or IFN-gamma . Cells were collected, lysed, and assayed as described for transient transfections (see above).

    RESULTS

TNF-alpha /IFN-gamma -induced NF-kappa B Nuclear Translocation Is Inhibited by 2-AP-- Previously, we demonstrated that IFN-gamma treatment synergistically enhances TNF-alpha -induced nuclear translocation of NF-kappa B in PC12 cells even though IFN-gamma , by itself, does not induce NF-kappa B in these cells (27). In response to dsRNA treatment but not to TNF-alpha or IL-1beta treatment, the Ser/Thr protein kinase PKR can induce NF-kappa B DNA binding activity, and this may occur following phosphorylation of Ikappa Balpha (32-35). To determine if IFN-gamma -enhanced NF-kappa B activity involves PKR, PC12 cells were pretreated for 30 min with 2-AP, a selective inhibitor of PKR, which can inhibit PKR autophosphorylation and activation (36, 37), and then were treated with TNF-alpha and/or IFN-gamma . Nuclear extracts were prepared and analyzed by EMSA. As described previously (27), there was very little binding to a consensus kappa B site with nuclear extracts from untreated PC12 cells (Fig. 1A, lane 1). Although it has been documented that a 2-h incubation with 10 mM 2-AP can slightly increase NF-kappa B DNA binding in the human promonocytic cell line U937 (45), we do not detect a change in binding in PC12 cells after 1 or 3.5 h of treatment (compare lanes 1-3). Treatment with TNF-alpha alone for 30 min (lane 4) induced binding of one major NF-kappa B-specific complex that was identified previously as p50-p65 (27). By 3 h of TNF-alpha treatment the binding activity was reduced significantly (lane 6) and returned to basal levels by 16 h (data not shown). Pretreatment with 2-AP did not affect the TNF-alpha -induced NF-kappa B DNA binding profile (compare lane 4 with 5 and lane 6 with 7). As expected, treatment with IFN-gamma alone or after pretreatment with 2-AP did not induce binding to the NF-kappa B-specific probe (lanes 8-11). Cotreatment with TNF-alpha and IFN-gamma elicited a striking synergistic effect on kappa B-specific binding activity after 3 h of cotreatment as reported earlier (compare lane 6 with lane 14) (27). Pretreatment with 2-AP completely blocked the TNF-alpha /IFN-gamma -induced synergy (compare lanes 14 and 15). Similar experiments were performed with the central nervous system-derived B12 cell line, and the effects of cytokine treatment with and without 2-AP treatment were nearly indistinguishable from the PC12 cell NF-kappa B activation profiles (data not shown). In the endothelial cell line, EA.hy926, the TNF-alpha /IFN-gamma -induced activation of NF-kappa B was not inhibited by 2-AP but was enhanced slightly (Fig. 1B, compare lanes 3 and 4). Pretreatment with the broad specificity serine/threonine kinase inhibitor staurosporine had no effect on the TNF-alpha /IFN-gamma -induced synergy in either cell type (data not shown). Collectively, the data from these three cell lines indicate that PKR may be involved in the regulatory mechanism for this synergistic response in cells of neural origin.


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Fig. 1.   2-AP inhibits TNF-alpha /IFN-gamma -induced NF-kappa B activation in PC12 cells but not in EA.hy926 cells. EMSAs of equal amounts of protein from nuclear extracts using a probe containing a consensus NF-kappa B binding site are shown. The time and treatment are indicated above each lane (UT is untreated). Where indicated, cells treated with cytokine were preincubated with 10 mM 2-AP for 30 min. Arrows indicate the major NF-kappa B-specific band (p50/p65), a nonspecific band (n.s.), and free probe. Panel A, for PC12 cells, TNF-alpha and IFN-gamma concentrations were 10 ng/ml and 100 units/ml, respectively. As a control, cells not treated with cytokine were incubated with 2-AP for 1 h (lane 2) or 3.5 h (lane 3). Panel B, for EA.hy926 cells, TNF-alpha and IFN-gamma concentrations were 25 pg/ml and 100 units/ml, respectively. As a control, cells not treated with cytokine were incubated with 2-AP for 1.5 h (lane 2).

TNF-alpha /IFN-gamma -induced Degradation of Ikappa Bbeta Is Inhibitied by 2-AP-- Typically, NF-kappa B is retained in the cytoplasm by inhibitory proteins that are collectively referred to as Ikappa B proteins (for review, see Refs. 3 and 4). In general, NF-kappa B-inducing stimuli promote the degradation of specific Ikappa B proteins, which allows the release and nuclear translocation of NF-kappa B subunits. Previously, we demonstrated that costimulation of NF-kappa B by TNF-alpha and IFN-gamma in PC12 cells requires the de novo degradation of Ikappa Bbeta (27). Therefore, we investigated whether 2-AP affects Ikappa Balpha or Ikappa Bbeta protein degradation. Western blot analyses were performed on cytoplasmic extracts collected at the same time as the nuclear extracts that were analyzed for Fig. 1A. Incubation for up to 3 h with 2-AP alone had no effect on either Ikappa Balpha or Ikappa Bbeta protein levels (Fig. 2, compare lanes 1-3). TNF-alpha treatment for 30 min resulted in degradation of Ikappa Balpha but not Ikappa Bbeta protein levels (compare lanes 1 and 4), and 2-AP did not inhibit this degradation (compare lanes 1, 4, and 5). In fact, 2-AP appears to enhance Ikappa Balpha degradation in the presence of TNF-alpha , consistent with the slight increase in DNA binding activity observed in Fig. 1A, lanes 4 and 5. TNF-alpha was able to lead to a modest reduction in Ikappa Bbeta levels after 3 h of stimulation, and this was not affected by 2-AP (Fig. 1A, lanes 6 and 7). As expected, Ikappa Balpha was resynthesized within 3 h because the expression of Ikappa Balpha is transcriptionally regulated by NF-kappa B (lane 6) (for review, see Ref. 4). Treatment with IFN-gamma either alone or after pretreatment with 2-AP also did not change the level of either Ikappa B protein (lanes 8-11). Furthermore, TNF-alpha /IFN-gamma cotreatment caused extensive degradation of Ikappa Bbeta which corresponds to the synergistic activation of NF-kappa B shown in Fig. 1 (Fig. 2, lane 14). Interestingly, the degradation of Ikappa Bbeta was inhibited by 2-AP (compare lanes 14 and 15), which corresponds to the inhibition of NF-kappa B activity shown in Fig. 1. Similar experiments were performed with the central nervous system-derived B12 cell line, and the effect of 2-AP on TNF-alpha /IFN-gamma -induced Ikappa Bbeta degradation was nearly identical (data not shown). We were unable to identify a higher mobility, hypophosphorylated form of Ikappa Bbeta which has been detected following its initial degradation (17). Also, we have not analyzed the potential of TNF-alpha and IFN-gamma to lead to enhanced degradation of other forms of Ikappa B, such as Ikappa Bepsilon . Our data indicate that the degradation of Ikappa Bbeta in response to TNF-alpha /IFN-gamma cotreatment and therefore the synergistic activation of NF-kappa B may be PKR-dependent in cells of neuronal origin.


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Fig. 2.   2-AP inhibits TNF-alpha /IFN-gamma -induced Ikappa Bbeta degradation. Western analyses of equal amounts of protein from PC12 cell cytoplasmic extracts using polyclonal antibodies specific for Ikappa Balpha or Ikappa Bbeta are shown. The time and treatment are indicated above each lane (UT is untreated). TNF-alpha , IFN-gamma , and 2-AP concentrations were 10 ng/ml, 100 units/ml, and 10 mM, respectively. As a control, cells not treated with cytokine were incubated with 2-AP for 1 h (lane 2) or 3.5 h (lane 3). Where indicated, cells treated with cytokine were preincubated with 2-AP for 30 min (lanes 5, 7, 9, 11, 13, and 15). Arrows indicate each specific Ikappa B protein.

PKR May Play a Role in the Synergistic Activation of kappa B-dependent Gene Expression by TNF-alpha /IFN-gamma Cotreatment-- To test whether the synergistic activation of NF-kappa B-dependent transcriptional responses requires PKR activity, we examined the ability of 2-AP to inhibit kappa B-dependent reporter gene expression. We used cells that were stably transfected rather than transiently transfected with a kappa B-dependent reporter because transient expression can be affected by 2-AP (45-47). PC12 cells stably maintaining a luciferase reporter construct containing three kappa B sites cloned in tandem in front of the minimal luciferase promoter (MHC-NF-kappa BLuc) were treated with TNF-alpha and/or IFN-gamma in the presence or absence of 2-AP. The kappa B sites conferred a ~35-fold induction of luciferase activity upon treatment with TNF-alpha , a ~5-fold induction upon treatment with IFN-gamma , and a synergistic ~95-fold induction upon cotreatment (Fig. 3). Preincubation with 2-AP significantly reduced the TNF-alpha /IFN-gamma induction of MHC-NF-kappa BLuc by ~40%, eliminating the IFN-gamma -supplied synergism. 2-AP did not nonspecifically affect gene expression (data not shown).


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Fig. 3.   2-AP inhibits TNF-alpha /IFN-gamma -induced kappa B-specific gene expression. PC12 cells stably expressing a kappa B-dependent luciferase reporter construct (MHC-NF-kappa BLuc) were pretreated for 30 min with 10 mM 2-AP or were left untreated. Subsequently, the cells were treated with 10 ng/ml TNF-alpha , 100 units/ml IFN-gamma , or a combination of both for 7 h. Lysates were assayed in duplicate for luciferase activity, and fold activity was determined by dividing the number of relative light units from treated samples by the number of relative light units from untreated (UT) samples. The data shown are averages of three independent experiments, and the S.E. of the mean are indicated by error bars.

To explore further the requirement for PKR during TNF-alpha /IFN-gamma -induced kappa B-dependent gene expression, we transiently cotransfected PC12 cells with MHC-NF-kappa BLuc plus a plasmid that expresses wtPKR or one that expresses mutPKR. MutPKR contains a Lys296 right-arrow Arg mutation which makes it a catalytically inactive kinase (32). The inactive PKR acts as a dominant negative either by competing for an endogenous PKR activator (48, 49) or by forming inactive dimers with endogenous PKR (50, 51). After transfection, we treated the cells with TNF-alpha and/or IFN-gamma and compared the luciferase activity relative to cells that were transfected with the reporter construct alone. WtPKR had little effect on the increased luciferase activity observed after cytokine treatment; however, mutPKR inhibited TNF-alpha /IFN-gamma -induced MHC-NF-kappa BLuc activity by ~80% (Fig. 4). The expression of mutPKR also decreased TNF-alpha -induced luciferase activity, suggesting that a minor component of TNF-alpha signaling may involve PKR and that its inhibitory effect may be on the ability of NF-kappa B to transactivate gene expression rather than on its ability to translocate to the nucleus. This effect has been documented previously by Kumar et al. (32). In summary, these data indicate that IFN-gamma enhances TNF-alpha -induced NF-kappa B-dependent transcription through PKR.


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Fig. 4.   MutPKR inhibits TNF-alpha /IFN-gamma -induced kappa B-specific gene expression. PC12 cells transiently transfected with a kappa B-dependent luciferase reporter construct (MHC-NF-kappa BLuc) alone or in combination with a plasmid expressing wtPKR or a catalytically inactive mutPKR were treated with 10 ng/ml TNF-alpha , 100 units/ml IFN-gamma , or a combination of both for 7 h. Lysates were assayed in duplicate for luciferase activity, and fold activity was determined by dividing the number of relative light units from treated samples by the number of relative light units from untreated (UT) samples. The data shown are averages of three independent experiments, and the S.E. of the mean are indicated by error bars.


    DISCUSSION

The cooperation between TNF-alpha and IFN-gamma during many biological responses including the regulation of gene expression is well documented (for review, see Refs. 52 and 53), and there are several mechanisms by which these two cytokines can collaborate. For example, cooperation can be achieved by mutual up-regulation of each other's receptors (54-58). In the context of gene expression, the synergy between TNF-alpha and IFN-gamma is thought to be the result of the independent activation of NF-kappa B by TNF-alpha and of IFN regulatory factors or signal transducers and activators of transcription by IFN-gamma which bind to unique promoter sites and synergistically regulate gene expression. Previously, we reported a mechanism by which these two cytokines can synergistically activate gene expression in an endothelial and a preneuronal cell line (27). In our model, IFNs significantly potentiate the TNF-alpha -induced nuclear translocation of NF-kappa B and kappa B-dependent gene expression. The mechanism for this synergy involves the de novo degradation of Ikappa Bbeta . This is a novel mechanism for NF-kappa B activation because IFN-gamma alone does not activate NF-kappa B. The net result is the targeted degradation of both Ikappa Balpha and Ikappa Bbeta which increases the amount of NF-kappa B that is free to translocate into the nucleus and therefore synergistically increases kappa B-dependent gene expression.

What is the signal generated by IFN-gamma binding to its receptor which is responsible for enhanced nuclear translocation of NF-kappa B and the synergistic activation of kappa B-dependent gene expression during TNF-alpha /IFN-gamma cotreatment? Our data indicate that in cells of neural origin, IFN-gamma potentiates the ability of TNF-alpha to induce NF-kappa B activity by targeting the serine/threonine kinase, PKR. This dsRNA-activated, IFN-inducible kinase is best known for its role during antiviral responses where, in response to dsRNA, it homodimerizes and autophosphorylates and then phosphorylates and deactivates its primary target, the translation initiation factor, eIF-2. However, PKR has also been implicated in controlling cell growth, cell differentiation, and tumor supression (for review, see Refs. 29 and 30), and there is evidence that it can become phosphorylated in the absence of viral infection or dsRNA treatment (59). In PC12 and B12 cells, pretreatment with the PKR-specific inhibitor 2-AP completely blocked TNF-alpha /IFN-gamma -induced NF-kappa B nuclear translocation and reduced kappa B-dependent gene expression by at least 40% (Figs. 1 and 3). Because it is possible that 2-AP could affect molecules other than PKR (36), we specifically targeted PKR by transfecting cells with a catalytically inactive form of PKR. This dominant negative PKR effectively reduced the level of TNF-alpha /IFN-gamma -induced kappa B-dependent gene expression to the level observed with TNF-alpha alone (~80% reduction) (Fig. 4).

The requirement for PKR may be cell type-specific because 2-AP completely blocked the TNF-alpha /IFN-gamma -induced activation of NF-kappa B in cells of neural origin but not in endothelial cells (Fig. 1). Petryshyn et al. have shown that IFN-induced PKR activity does not occur until at least 3 h after treatment (60, 61). This might explain the cell type-specific effect of 2-AP because the synergistic activation of NF-kappa B occurs in endothelial cells within 15 min to 1 h post-stimulation, whereas the synergy in neuronal derived cells does not occur until later than 2 h post-stimulation (27). Also, 2-AP did not block NF-kappa B activity induced by TNF-alpha alone, therefore 2-AP is most likely targeting a signal generated by IFN-gamma binding to its receptor.

There is evidence that PKR can affect the activation of NF-kappa B in mouse embryo fibroblasts isolated from the PKR knockout mouse (Pkr0/0 MEFs). In Pkr0/0 MEFs, dsRNA-activated NF-kappa B is reduced compared with levels in wild-type MEFs (Pkr+/+ MEFs), but TNF-alpha -activated NF-kappa B levels are normal (32). Upon pretreatment with IFN-alpha or IFN-gamma , dsRNA-induced NF-kappa B activity is restored to normal. Maran et al. (34) have used an antisense procedure to decrease selectively the level of PKR activity in cells. In these cells, dsRNA could not activate NF-kappa B, but the activation of NF-kappa B by TNF-alpha was unaffected. Recently, several groups have shown that Ikappa Balpha may be phosphorylated by PKR in vitro in response to dsRNA, but it is not clear whether this occurs in vivo (32-35). There is no direct evidence that Ikappa Bbeta can be phosphorylated by PKR; however, Ikappa Bbeta has two serine residues that are analogous to the two serines present in Ikappa Balpha which can be inducibly phosphorylated prior to ubiquitination and degradation (10-12, 14, 18). In an attempt to implicate PKR further during synergistic NF-kappa B activation, we began to examine the effect of TNF-alpha /IFN-gamma cotreatment in Pkr+/+ MEFs and Pkr0/0 MEFs. The Pkr+/+ MEFs did not exhibit synergistic activation of NF-kappa B (at least with the concentrations of TNF-alpha and IFN-gamma used to generate synergism in PC12 cells), therefore we were unable to use this cell model for further studies. These results further strengthen our hypothesis that the mechanisms for the synergistic activation of NF-kappa B will be diverse and cell type-specific.

What is PKR's target during NF-kappa B activation? A major component of the TNF-alpha /IFN-gamma -induced activation of NF-kappa B is the mechanistic switch from Ikappa Balpha degradation to Ikappa Bbeta degradation, leading to persistent activation of NF-kappa B. This is similar to previous documentation where Ikappa Balpha is thought to be involved in the transient activation of NF-kappa B, whereas Ikappa Bbeta is targeted during the persistent activation of NF-kappa B (14-17) Also, it has been proposed that Ikappa Bbeta can act either as an inhibitor or as a chaperone-like protein. As a chaperone, Ikappa Bbeta could protect NF-kappa B from the inhibitory properties of Ikappa Balpha , and this mechanism may be explained by the differential phosphorylation of Ikappa Bbeta (17). In our system, using 2-AP to block PKR activity inhibits the de novo degradation of Ikappa Bbeta during costimulation but does not affect the pattern of Ikappa Balpha degradation. Blocking Ikappa Bbeta degradation is concomitant with inhibiting the synergistic and prolonged activation of NF-kappa B. This leads to a model whereby signals generated from TNF-alpha /IFN-gamma cotreatment activate PKR, which in turn either directly or indirectly causes the induced phosphorylation and degradation of Ikappa Bbeta and consequently the synergistic activation of NF-kappa B.

There are several possible mechanisms by which TNF-alpha /IFN-gamma cotreatment could target PKR. First, PKR protein levels could be up-regulated by IFN-gamma or by TNF-alpha (62; and for review, see Refs. 29 and 30). However, our previous work using cycloheximide demonstrates that protein synthesis is not required for this TNF-alpha /IFN-gamma synergy, therefore an increase in PKR protein levels cannot account for this response. Second, IFN-gamma may lead to an increased activity of PKR, but signals generated by the presence of both cytokines may be required to target the NF-kappa B·Ikappa Bbeta complex. Third, signals generated by cotreatment could induce the synthesis of or change the structure of a cellular dsRNA or other PKR activator, which could then activate PKR (59). Also, there is evidence for endogenous proteins that act as cellular PKR inhibitors (63, 64); therefore cotreatment could generate signals that could counteract the inhibitory roles of these proteins. Another possibility is that IFN-gamma and/or TNF-alpha may generate signals that induce phosphorylation and activation of PKR. PKR could then directly phosphorylate Ikappa B proteins and target them for degradation or activate a kinase that is responsible for Ikappa B phosphorylation. Recently, two subunits of the multiprotein complex that forms a functional IKK have been isolated and characterized. Both IKKalpha and IKKbeta are TNF-alpha -inducible and specifically phosphorylate both Ikappa Balpha and Ikappa Bbeta on critical serine residues (6-9). It has been reported that IKK itself may require phosphorylation for activation, although the required kinase(s) has not been identified (7). Potentially, there could be distinct IKKs that are specific for individual Ikappa B proteins in vivo, and an IKK which specifically targets Ikappa Bbeta could be a substrate for PKR. It is also likely that the strength of the signal determines the extent to which NF-kappa B will be activated. For example, TNF-alpha can activate IKK but maybe only to limited levels. However, in the presence of signals generated by IFN-gamma (such as increased PKR activity) the activity of IKK could be elevated and subsequently increase and/or prolong the activity of NF-kappa B.

The results presented here are directed at elucidating the mechanisms(s) whereby IFNs impinge on TNF-alpha -induced activation of NF-kappa B. Our data indicate a role for the IFN-inducible kinase PKR in this response in cells of neuronal origin. Clearly there are implications that the synergistic activation of NF-kappa B in neural derived cells may be important in suppressing an apoptotic mechanism. TNF-alpha and IFN-gamma are both potentially apoptotic agents; however, together they potentiate the activation of the anti-apoptotic activity of NF-kappa B. Although PKR has been implicated in a mechanism for stress-induced apoptosis (65, 66), the net cellular response to TNF-alpha and IFN-gamma may depend on the source(s) and/or strength of the signals that are generated. Therefore, the synergistic activation of NF-kappa B by TNF-alpha and IFN-gamma during an inflammation response could protect cells of neural origin from death.

    ACKNOWLEDGEMENTS

We thank William E. Miller for insightful discussions and for critical reading of the manuscript.

    FOOTNOTES

* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ Supported by National Institutes of Health/National Research Service Award Fellowship 1 F32 AG05745. Present address: Bayer Corp., R&D Pathogen Safety Research, Research Triangle Park, NC 27709.

parallel Supported by National Institutes of Health Grant AI34039.

** Supported by National Institutes of Health Grant AI35098. To whom correspondence should be addressed: Lineberger Comprehensive Cancer Center, CB 7295, University of North Carolina School of Medicine, Chapel Hill, NC 27599. Fax: 919-966-0444.

    ABBREVIATIONS

The abbreviations used are: NF-kappa B, nuclear factor kappa B; Ikappa B, inhibitor of kappa B; IKK, Ikappa B kinase; TNF-alpha , tumor necrosis factor-alpha ; IFN, interferon; MHC, major histocompatibility complex; PKR, double-stranded RNA-activated protein kinase; dsRNA, double-stranded RNA; eIF-2, eukaryotic initiation factor-2; 2-AP, 2-aminopurine; EMSA, electrophoretic mobility shift analysis; wtPKR, wild-type PKR, mutPKR, mutant PKR; Pkr0/0 MEFs, mouse embryo fibroblasts devoid of functional PKR; Pkr +/+ MEFs, wild-type mouse embryo fibroblasts.

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