Nuclear factor-κB (NF-κB) is tightly modulated by IκB kinases and IκBα in the cytoplasm. On stimulation, NF-κB translocates into the nucleus to initiate transcription; however, regulation of its transcriptional activity remains obscure. Here, we show that protein kinase C (PKC) δ controls the main subunit of NF-κB, RelA/p65. On exposure to tumor necrosis factor-α (TNF-α), the expression of RelA/p65 target genes such as IκBα, RelB, and p100/p52 is up-regulated in a PKCδ-dependent manner. The results also show that PKCδ is targeted to the nucleus and forms a complex with RelA/p65 following TNF-α exposure. Importantly, kinase activity of PKCδ is required for RelA/p65 transactivation. In concert with these results, PKCδ activates RelA/p65 for its occupancy to target-gene promoters, including IκBα and p100/p52. Moreover, functional analyses show that inhibition of PKCδ is associated with substantial attenuation of NF-κB activity in response to TNF-α. These findings provide evidence that PKCδ orchestrates RelA/p65 transactivation, a requisite for NF-κB signaling pathway in the nucleus. [Cancer Res 2009;69(14):5927–35]
The protein kinase C (PKC) family represents serine and threonine kinases that are responsible for a variety of cellular responses such as growth, proliferation, transformation, and cell death ( 1, 2). The PKC family is subdivided into three categories: conventional, novel, and atypical PKCs ( 1, 2). Accumulating lines of evidence have revealed that PKCδ, a novel PKC, plays a crucial role in the cellular response to genotoxic stress ( 3– 5). On exposure to DNA damage, PKCδ is activated and cleaved by caspase-3 to form a 40 kDa catalytically active fragment. Overexpression of the PKCδ catalytic fragment induces chromatin condensation and DNA fragmentation, which supports a role for PKCδ cleavage in the induction of apoptosis ( 6). Other studies have shown that PKCδ interacts with the c-Abl tyrosine kinase ( 7). c-Abl is a proapoptotic tyrosine kinase that targets to the nucleus following genotoxic stress ( 8– 10). Importantly, c-Abl-mediated phosphorylation activates PKCδ and induces its translocation to the nucleus ( 7). Consistent with these findings, tyrosine phosphorylation of PKCδ is necessary for its nuclear translocation and subsequent caspase-dependent cleavage ( 2, 11, 12). Previous studies have also shown that the nuclear complex of c-Abl and Lyn tyrosine kinases includes the protein tyrosine phosphatase SHPTP1 ( 13, 14) and that PKCδ phosphorylates and inactivates SHPTP1 in response to genotoxic stress ( 15). Another study showed that cells derived from PKCδ-null transgenic mice were defective in mitochondria-dependent apoptosis induced by DNA damage ( 16). We have recently shown that PKCδ phosphorylates the p53 tumor suppressor to induce apoptotic cell death ( 17). Furthermore, recent studies have shown that PKCδ interacts with and phosphorylates Rad9, a key factor involved in checkpoint regulation of the DNA damage responses ( 18, 19). Inhibition of PKCδ attenuates Rad9-mediated apoptosis. These findings collectively support an essential role for PKCδ in the induction of apoptosis in the genotoxic stress response ( 2). By contrast, several lines of studies showed a pivotal role for PKCδ in antiapoptotic function in response to cytokines, including tumor necrosis factor-α (TNF-α; refs. 20– 22). Expression with a PKCδ kinase-dead mutant or with a small interfering RNA (siRNA) targeting PKCδ increased the apoptotic effect of TNF-related apoptosis-inducing ligand, whereas overexpression of PKCδ decreased it ( 22). Intriguingly, phosphorylation of PKCδ on Tyr155 was required for its cleavage in response to TNF-related apoptosis-inducing ligand. In addition, cleavage of PKCδ by caspases was essential for its protective effect because overexpression of a caspase-resistant mutant did not protect glioma cells from TNF-related apoptosis-inducing ligand-induced apoptosis ( 22). Another study showed that inhibition of PKCδ attenuated TNF-α–induced nuclear factor-κB (NF-κB) activation in human neutrophils ( 21). A further study suggested that PKCδ depletion by PKCδ siRNA resulted in inhibition of TNF-mediated extracellular signal-regulated kinase 1/2 activation, which is involved in an essential component of TNF-α–mediated signaling ( 20). These findings collectively imply a protective role for PKCδ in TNF-α–induced apoptosis. Precise mechanisms in which PKCδ controls signaling pathways to protect cells from apoptosis, however, remain obscure.
NF-κB is an inducible transcription factor that controls the expression of several proteins involved in the regulation of cell survival and immune response ( 23). NF-κB is a dimer formed from a multisubset family consisting of RelA/p65, RelB, c-Rel, p105/p50 (NF-κB1), and p100/p52 (NF-κB2). NF-κB is activated by a bewildering array of stimuli, including biological agents such as TNF-α, interleukin (IL)-1, bacterial endotoxin, and phorbol esters and cytotoxic stimuli such as chemotherapeutic agents, ultraviolet light, oxidative stress, and ionizing radiation ( 24, 25). Activation of NF-κB is regulated by multiple distinct signaling cascades including inhibitors of the NF-κB (IκB) kinase (IKK) signalosome ( 26, 27). IKK phosphorylates IκBα at Ser32 and Ser36 in response to a variety of stimuli, resulting in its ubiquitination and subsequent proteasomal degradation ( 26, 27). The released NF-κB targets to the nucleus and thereby induces the expression of specific target genes. In addition to nuclear translocation of the NF-κB complex, previous studies have shown that a subunit of NF-κB, RelA/p65, is post-translationally modified such as phosphorylation or acetylation, and those changes influence its transcriptional activity. However, recent data showing a role for Ser536 phosphorylation by IKKs on RelA/p65 activation in response to TNF-α remain controversial ( 28– 33). In this context, mechanisms for RelA/p65 transactivation following nuclear translocation are largely unclear.
In this study, we show that inhibition of PKCδ is associated with attenuation of RelA/p65 transactivation in response to TNF-α. The results show that PKCδ translocates from the cytoplasm into the nucleus after TNF-α exposure. Importantly, kinase activity of PKCδ is required for its nuclear targeting and RelA/p65 transactivation. However, phosphorylation of RelA/p65 is not involved in PKCδ-mediated activation of RelA/p65. These findings collectively support a novel mechanism in which regulation of RelA/p65 by PKCδ contributes to TNF-α–induced activation of NF-κB signaling pathway.
Materials and Methods
Cell culture. U2OS (human osteosarcoma) cells were cultured in RPMI 1640 supplemented with 10% heat-inactivated fetal bovine serum, 100units/mL penicillin, 100 μg/mL streptomycin, and 2 mmol/L l-glutamine. 293T (human embryonal kidney) cells were grown in DMEM containing 10% fetal bovine serum and antibiotics. Cells were treated with 20 ng/mL TNF-α (human TNF-α; PeproTech), 5 μmol/L rottlerin (Sigma-Aldrich), or 100 ng/mL NF-κB inhibitor (Santa Cruz Biotechnology).
siRNA transfection. siRNA duplexes (siRNAs) targeting for PKCδ were synthesized and purified by Invitrogen (Stealth RNAi). Transfection of siRNAs was done using Lipofectamine RNAi Max (Invitrogen) according to the manufacturer's protocol.
Immunoprecipitation and immunoblot analysis. Cell lysates were prepared as described elsewhere ( 14, 34). Soluble proteins were incubated with anti-PKCδ (Santa Cruz Biotechnology) or anti-RelA/p65 (Santa Cruz Biotechnology) antibodies for 2 h at 4°C followed by a 1 h incubation with protein A/G (Santa Cruz Biotechnology) Sepharose beads. Cell lysates or immunoprecipitates were separated by SDS-PAGE and transferred to nitrocellulose filters, which were then incubated with anti-IκBα (Santa Cruz Biotechnology), anti-RelA/p65, anti-p50 (Santa Cruz Biotechnology), anti-RelB (Santa Cruz Biotechnology), anti-p52 (Santa Cruz Biotechnology), anti-PKCδ, anti-phospho-RelA/p65 (Cell Signaling), anti-PCNA (Santa Cruz Biotechnology), or anti-tubulin (Sigma-Aldrich). After washing, the membranes were incubated with anti-rabbit or anti-mouse IgG-peroxidase conjugate (Santa Cruz Biotechnology). The antigen-antibody complexes were visualized by chemiluminescence (Perkin-Elmer).
Immunofluorescence assay. Cells cultured in chamber slides were fixed in methanol for 5 min, permeabilized in 1% Triton X-100 for 15 min, washed with PBS, and blocked with 10% goat serum in PBS for 1 h. After washing with PBS, the cells were immunostained with anti-RelA/p65 or anti-PKCδ followed by reaction with FITC- or TRITC-conjugated secondary antibodies. Nuclei were stained with 4′,6-diamidino-2-phenylindole.
Electrophoretic mobility shift assay. Electrophoretic mobility shift assay was done using LightShift Chemiluminescent EMSA Kit (Pierce) according to the manufacturer's instructions. For each reaction, 1 μg nuclear extract was incubated with biotin end-labeled κB oligonucleotide probes. The probe sequence is as follows: 5′-AGTTGAGGGGACTTTCCCAGGC-3′.
Chromatin immunoprecipitation assay. Chromatin immunoprecipitation and re-chromatin immunoprecipitation assays were done as described previously ( 35). PCR amplification was done in chromatin immunoprecipitated fragments using the following oligonucleotide pairs: IκBα (5′-GACGACCCCAATTCAAATCG-3′ and 5′-TCAGGCTCGGGGAATTTCC-3′) and p100/p52 (5′-GTGAAAGACCCTCCTGTTCCCT-3′ and 5′-GTGGAGAGCGAGATCCGGAGTT-3′).
Reporter gene assays. 293 cells stably transfected with pNF-κB-luc and pTK-hyg (Panomics) were treated with TNF-α. The luciferase activity was determined by the Bright-Glo Luciferase Assay System (Promega) according to the manufacturer's protocol. The relative fold increase in activity compared with untreated cells was determined ( 36). The data represent mean ± SD from at least three to four independent experiments, each done in triplicate.
IL-6 production. Cells were plated onto 24-well plates and stimulated with TNF-α. After 24 h, the culture supernatants were assayed for IL-6 production using OptEIA human IL-6 ELISA kit II (BD Pharmingen) according to the manufacturer's instruction.
Apoptosis assay. The apoptotic effect was measured after 24 h using the DeadEnd Fluorometric TUNEL System (Promega).
PKCδ is involved in the activation of RelA/p65 transcription function in response to TNF-α. Previous studies showed that treatment of cells with TNF-α induces IκBα degradation, resulting in nuclear translocation and activation of NF-κB ( 37). To determine if PKCδ is involved in TNF-α–induced NF-κB activation, 293T cells were treated with TNF-α in the presence or absence of the specific PKCδ inhibitor rottlerin. Subcellular fractionation assays revealed that nuclear targeting of NF-κB including RelA/p65 and p105/p50 is independent of PKCδ activity ( Figs. 1A and 4A, nuclear lysates). However, resynthesis of IκBα after TNF-α exposure, which is transcriptionally induced by RelA/p65 ( 38), was diminished in rottlerin-treated cells, suggesting that PKCδ activity is associated with the activation of NF-κB transcription function ( Fig 1A, cytoplasmic lysates). Moreover, on exposure to TNF-α, increased expression of RelB and p100/p52, both are also the transcriptional targets of RelA/p65 ( 39, 40), significantly reduced in cells pretreated with rottlerin ( Fig. 1, whole-cell lysates). Similar results were obtained in U2OS cells ( Fig. 1C). To further define the involvement of PKCδ in transcriptional activity of RelA/p65, reverse transcription and subsequent PCR assays were done to monitor transcription of RelA/p65 target genes. The results showed that increased expression of RelB and p100/p52 attenuated in cells pretreated with rottlerin ( Fig. 1B). Similar findings were obtained in U2OS cells (data not shown). To confirm the requirement of PKCδ in RelA/p65 activation, U2OS cells were transfected with scramble siRNA or PKCδ-specific siRNA followed by treatment with TNF-α. As shown for pretreatment with rottlerin ( Fig. 1A-C), the status of PKCδ expression was not associated with nuclear translocation of RelA/p65 and p105/p50 ( Fig. 1D, nuclear lysates). In contrast, TNF-α–induced expression of RelB and p100/p52 was markedly attenuated in cells silenced for PKCδ ( Fig. 1D, whole-cell lysates). Taken together, these results show that PKCδ induces the activation of RelA/p65 transcription function in response to TNF-α.
Nuclear targeting of PKCδ triggers TNF-α–induced RelA/p65 activation. To explore precise mechanisms in which PKCδ control RelA/p65, we first examined PKCδ phosphorylation of RelA/p65 because our previous study showed that PKCδ activates IKKα in response to oxidative stress ( 41). Other studies showed that IKK phosphorylates RelA/p65 at Ser536 to induce its transactivation ( 31– 33). Hence, to assess the possibility that PKCδ activates IKKs that subsequently phosphorylate Ser536, 293T cells were treated with TNF-α in the presence or absence of rottlerin. Immunoblot analysis with anti-phospho-Ser536 showed no remarkable effect on Ser536 phosphorylation, indicating that IKK phosphorylation of RelA/p65 is irrelevant to PKCδ on exposure to TNF-α ( Fig. 2A ). Other studies have shown that phosphorylation of RelA/p65 at Ser276 is essential for its activity after TNF-α stimulation ( 29, 42). To examine the involvement of PKCδ on Ser276 phosphorylation, 293T cells were treated with TNF-α in the presence or absence of rottlerin. The results showed that PKCδ activity is scarcely involved in Ser276 phosphorylation following TNF-α exposure ( Fig. 2A). Another study has shown that phosphorylation of RelA/p65 at Ser529 is essential for its activity after TNF-α stimulation ( 43). To determine if PKCδ is associated with Ser529 phosphorylation, 293T cells were left untreated or treated with TNF-α in the presence or absence of rottlerin. Ser529 was constitutively phosphorylated, and as shown for phosphorylation of Ser276 or Ser536, there was no significant difference on Ser529 phosphorylation with impairment of PKCδ activity after TNF-α exposure ( Fig. 2A). These findings collectively indicate that PKCδ affects RelA/p65 in a phosphorylation-independent manner. Our previous studies also showed that PKCδ rapidly moves to the nucleus after genotoxic stress and this nuclear targeting depends on its kinase activity ( 19). These findings led us to examine whether TNF-α induces nuclear translocation of PKCδ. Subcellular fractionation assays clearly showed that cytolasmic PKCδ immediately moved into the nucleus ( Fig. 2B). Importantly, inhibition of PKCδ activity impeded its nuclear translocation following TNF-α treatment ( Fig. 2B), suggesting the possibility that nuclear targeting of PKCδ affects regulation of TNF-α–induced RelA/p65 activation. To determine if nuclear targeting of PKCδ is dependent on RelA/p65, cells were pretreated with NF-κB inhibitor followed by treatment with TNF-α. The results showed that there is little, if any, effect of NF-κB activity on TNF-α–induced nuclear translocation of PKCδ (Supplementary Fig. S1). To convince nuclear targeting of PKCδ in response to TNF-α, we performed immunofluorescent staining. Nuclear PKCδ was increased after TNF-α stimulation ( Fig. 2C and D). Moreover, in concert with the immunoblot analysis, inhibition of PKCδ activity by rottlerin diminished its nuclear translocation in response to TNF-α, confirming that kinase activity is required for nuclear targeting of PKCδ ( Fig. 2C and D). We also confirmed that TNF-α–induced nuclear translocation of RelA/p65 is independent of PKCδ activity ( Fig. 2C).
PKCδ interacts with RelA/p65 after TNF-α exposure. The findings that both PKCδ and RelA/p65 move into the nucleus following TNF-α treatment provided a conceivable model in which PKCδ associates with RelA/p65. To address this possibility, 293T cells were left untreated or treated with TNF-α; then, cell lysates were subjected to immunoprecipitation with anti-RelA/p65 and subsequent immunoblotting with anti-PKCδ. PKCδ was coimmunoprecipitated with RelA/p65 in TNF-α–treated cells but not untreated cells ( Fig. 3A ). Moreover, reciprocal experiments confirmed this interaction ( Fig. 3B). These findings show the inducible interaction of PKCδ with RelA/p65 after TNF-α exposure. To examine whether PKCδ affects NF-κB activity, U2OS cells were left untreated or treated with TNF-α in the presence or absence of rottlerin. Nuclear lysates were then subjected to electrophoretic mobility shift assay analysis. A shifted band was detected when the κB probes were incubated with nuclear lysates from TNF-α–treated cells but not control cells ( Fig. 3C). Moreover, the band disappeared in the presence of rottlerin, suggesting that PKCδ activity is required for the efficient binding of NF-κB to κB elements in response to TNF-α.
PKCδ controls RelA/p65 occupancy to the target-gene promoters. To further define the functional interaction between PKCδ and RelA/p65, we performed chromatin immunoprecipitation assays to clarify the role for PKCδ on promoter binding of RelA/p65. 293T cells were left untreated or treated with TNF-α in the presence or absence of rottlerin. Isolated chromatin was immunoprecipitated with anti-RelA/p65 or control IgG followed by PCR analysis with primers targeted to the κB elements of IκBα or p100/p52 promoter. RelA/p65 occupancy to the IκBα promoter was detectable after TNF-α exposure ( Fig. 4A ). Importantly, inhibition of PKCδ activity abrogated this occupancy ( Fig. 4A). Similar results were obtained with the p100/p52 promoter ( Fig. 4A). To establish the requirement of PKCδ on binding of RelA/p65 to the target-gene promoters, 293T cells were transfected with scramble siRNA or PKCδ-specific siRNA followed by treatment with TNF-α. As shown for rottlerin, chromatin immunoprecipitation analyses revealed that RelA/p65 occupies the κB elements of IκBα promoter in control cells and not PKCδ silencing cells ( Fig. 4B). Similar findings were obtained with the p100/p52 promoter ( Fig. 4B). Taken together, these results show that PKCδ is required for the binding of RelA/p65 to the κB elements in response to TNF-α. The demonstration that PKCδ inducibly interacts with RelA/p65 following TNF-α exposure led us to determine if PKCδ forms a complex with RelA/p65 on the promoters. As expected, chromatin immunoprecipitation analyses with anti-PKCδ immunoprecipitates clearly showed that, on exposure to TNF-α, PKCδ occupies the κB elements of IκBα promoter ( Fig. 4C). More importantly, inhibition of PKCδ activity was associated with complete abrogation of its occupancy ( Fig. 4C). Comparable results were obtained with the p100/p52 promoter ( Fig. 4C). These findings indicate that PKCδ and RelA/p65 form a complex to occupy κB elements on RelA/p65 target promoters in response to TNF-α. Furthermore, activation of PKCδ is necessary to induce the transcription function of RelA/p65. To determine if recruitment of PKCδ to the promoters depends on its interaction with RelA/p65. U2OS cells were transfected with scramble siRNA or RelA/p65 siRNA followed by treatment with TNF-α. Analysis of chromatin immunoprecipitation assays showed that TNF-α–induced PKCδ occupancy to the IκBα promoter was completely abolished in cells silenced for RelA/p65 (Supplementary Fig. S2A). Similar results were obtained with the p100/p52 promoter (Supplementary Fig. S2B). These findings suggest that PKCδ binding to the promoters of the NF-κB target genes is dependent on its interaction with RelA/p65. To address the mechanism of PKCδ-dependent recruitment of RelA/p65 to the promoters of NF-κB genes, we have performed re-chromatin immunoprecipitation assays. Chromatin was immunoprecipitated with anti-PKCδ as the first antibody, and the eluted samples were then immunoprecipitated with anti-RelA/p65 or anti-IgG antibody. Chromatin templates containing κB elements of IκBα promoter, which were associated with PKCδ, were also immunoprecipitated by anti-RelA/p65 but not by anti-IgG after TNF-α exposure ( Fig. 4D). More importantly, this co-occupancy was markedly diminished in the presence of rottlerin ( Fig. 4D). To confirm these results, we performed reciprocal re-chromatin immunoprecipitation assays in which anti-RelA/p65 was used for the first immunoprecipitation and then anti-PKCδ for the second immunoprecipitation. As expected, the results yielded similar conclusions ( Fig. 4D). These findings indicate that, on exposure to TNF-α stimulation, PKCδ may form a complex with RelA/p65 to occupy κB elements and that this activation of PKCδ may be required for the co-occupancy to κB elements.
PKCδ induces transcriptional activity of RelA/p65 following TNF-α exposure. To examine whether PKCδ enhances TNF-α–induced RelA/p65 activation, 293 cells stably transfected with the luciferase-reporter vector-containing κB elements were treated with TNF-α in the presence or absence of rottlerin. Pretreatment with rottlerin significantly attenuated the NF-κB activity in a dose-dependent manner ( Fig. 5A ). By sharp contrast, there was little, if any, effect on pretreatment of classic PKC inhibitor Gö6976 (data not shown). To confirm this finding, cells were transfected with scramble siRNA, PKCδ-specific siRNA, or RelA/p65-specific siRNA. As expected, silencing of RelA/p65 was associated with pronounced inhibition of NF-κB activity in response to TNF-α ( Fig. 5B). Importantly, knocking down PKCδ significantly diminished TNF-α–induced NF-κB activation ( Fig. 5B).
PKCδ affects cellular function of NF-κB by controlling RelA/p65 in response to TNF-α. To examine the biological significance of PKCδ-dependent regulation of RelA/p65 transcriptional activity, we have investigated TNF-α–induced IL-6 production and apoptotic cell death. 293T cells were left untreated or treated with TNF-α in the presence or absence of rottlerin. As expected, TNF-α stimulation substantially imposed IL-6 production ( Fig. 6A, left ). In contrast, inhibition of PKCδ activity by pretreatment with rottlerin impaired production of IL-6 in response to TNF-α ( Fig. 6A, left). Similar results were obtained with U2OS cells silenced for PKCδ ( Fig. 6A, right). These data show that PKCδ induces transcriptional activity of RelA/p65 to produce IL-6 in response to TNF-α. With regard to the apoptotic insults, U2OS cells were left untreated or treated with TNF-α in the presence or absence of rottlerin. Analysis of TUNEL assays revealed that apoptotic cells were slightly increased following TNF-α exposure ( Fig. 6B). Importantly, TNF-α–induced apoptotic induction was significantly enhanced in the presence of rottlerin ( Fig. 6B). Comparable findings were obtained with cells silenced for PKCδ ( Fig. 6B). To further assess TNF-α–induced apoptosis, cells were left untreated or treated with TNF-α in the presence or absence of NF-κB inhibitor. Analysis of TUNEL assays showed that impeding NF-κB activity facilitates apoptotic cell death induced by TNF-α ( Fig. 6B). Similar results were obtained in cells transfected with RelA/p65 siRNA ( Fig. 6B). To substantiate whether the finding that inhibition of PKCδ potentiates TNF-α–induced apoptosis is dependent on NF-κB, NF-κB inhibitor was cotreated together with rottlerin in cells. After TNF-α stimulation, induction of apoptosis by combination of NF-κB inhibitor and rottlerin was comparable with that by NF-κB inhibitor or RelA/p65 siRNA alone ( Fig. 6B). Consistent results were obtained with combination of NF-κB inhibitor and PKCδ siRNA ( Fig. 6B). These results show that abrogation of the PKCδ-NF-κB signaling, at least in part, diminishes the protective effect from TNF-α–induced apoptotic cell death. As a result, cells undergoing apoptosis were significantly increased.
Taken together, these findings thus support a model in which, on exposure to TNF-α, PKCδ is targeted into the nucleus and forms a complex with RelA/p65 to activate transcription function on the promoters ( Fig. 6C). More importantly, PKCδ plays a crucial role in NF-κB activation by controlling RelA/p65 to regulate cellular function and fate in response to TNF-α.
NF-κB is a key transcription factor in cell survival, immunity, development, and many other important biological processes. Understanding regulation of NF-κB thus contributes to elucidation for cellular response to the homeostasis system. Accumulating lines of evidence have revealed that NF-κB is activated by various stimuli such as cytokines, radiation, viral infection, and reactive oxygen species ( 27). In this study, we have focused on TNF-α–induced NF-κB activation. In resting cells, NF-κB is localized in the cytoplasm tethered by IκBα. Once IKK signalosome is activated by various stimuli, IκBα is phosphorylated and degraded to allow nuclear translocation of dimeric NF-κB. Numerous studies have paid much attention on regulation of nuclear targeting of NF-κB in response to diverse stimuli. However, little is known about an activation mechanism of NF-κB in the nucleus. In this regard, the present study shows that PKCδ is involved in RelA/p65 activation in the nucleus. We have shown previously that PKCδ translocates into the nucleus after treatment of cells with 1-β-d-arabinofuranosylcytosine ( 19). Moreover, pretreatment with rottlerin attenuated nuclear targeting of PKCδ ( 19). We also proved that PKCδ activates nuclear substrates by phosphorylation, including Rad9, topoisomerase IIα, and p53, to induce apoptosis in response to DNA damage ( 2). These findings support a model in which, on exposure to genotoxic stress, PKCδ is targeted to the nucleus and phosphorylates several nuclear targets for induction of apoptosis. In the present study, the results indicate TNF-α–induced nuclear translocation of PKCδ and its interaction with RelA/p65. To our best knowledge, this is the first report showing nuclear translocation of PKCδ after TNF-α stimulation. Mechanisms for nuclear targeting of PKCδ remain unclear. Nevertheless, the findings that kinase activity is required for nuclear translocation, as shown similarly in the genotoxic stress response ( 19), suggest a common machinery for its nuclear migration. We also found inducible interaction between PKCδ and RelA/p65 predominantly in the nucleus after TNF-α stimulation ( Figs. 2C and 3A and B). In this context, at least in a physiologic condition, activation of PKCδ would be prerequisite for its nuclear targeting and subsequent binding to RelA/p65. These data thus suggest that both activity of and interaction with PKCδ could be indispensable for TNF-α–induced RelA/p65 activity. The demonstration that inhibition of PKCδ by rottlerin abrogates its interaction with RelA/p65 (data not shown) further supports the involvement for nuclear targeting of PKCδ on exposure to TNF-α. More importantly, rottlerin or siRNA blocked TNF-α–induced IκBα or p100/p52 expression ( Fig. 1), suggesting that inducible nuclear interaction between PKCδ and RelA/p65 is, at least in part, essential for activation of NF-κB signaling pathways in response to TNF-α. In concert with these observations, chromatin immunoprecipitation assays further indicated that both PKCδ and RelA/p65 are detectable on κB elements of IκBα or p100/p52 promoters, and these occupancies are substantially diminished by inhibition of PKCδ activity. These findings thus indicate that PKCδ is associated with the regulation of RelA/p65 transcriptional activity elicited by TNF-α. Moreover, it is conceivable that PKCδ controls RelA/p65 by inducible interaction because PKCδ is not involved in nuclear targeting of RelA/p65 ( Figs. 1A and D, 2C, and 4A). Another issue to be solved is the precise mechanism by which PKCδ regulates RelA/p65 in the nucleus. Given previous findings that PKCδ activates IKKα by oxidative stress and IKKα can phosphorylate and activate RelA/p65 at Ser536, we examined whether PKCδ activates RelA/p65 by IKKα-mediated phosphorylation at Ser536. The present results clearly showed that PKCδ is not involved in Ser536 phosphorylation following TNF-α exposure. We also showed the dispensable role for PKCδ on Ser276 and Ser529 phosphorylation of RelA/p65. It also remains elusive whether PKCδ directly regulates the other NF-κB members, such as RelB or p100/p52, in response to TNF-α. Further studies are needed to clarify this issue and this feasibility is currently under investigation.
In tumor cell lines, the role of PKCδ is a paradox. It can induce both cell survival and apoptosis depending on cell types and its subcellular localization ( 2). For instance, treatment with anticancer agents resulted in a translocation of PKCδ from cytoplasm into nucleus concomitant with induction of apoptosis ( 12, 19, 44). Our recent studies showed PKCδ is involved in phosphorylation of p53 and potentiates p53-dependent apoptosis in response to DNA damage ( 17). PKCδ also induces transcription of the p53 tumor suppressor gene in the apoptotic response to DNA damage ( 35). These results thus indicate that PKCδ functions as a proapoptotic kinase in response to genotoxic stress ( 2). However, in this study, inhibition of PKCδ depressed both DNA binding of RelA/p65 and expression of the antiapoptotic proteins, RelB and p100/p52 ( Fig. 1). In agreement with these results, PKCδ has been identified as a prosurvival factor in human breast tumor cell lines ( 45). Moreover, activation of PKCδ increased expression of the antiapoptotic proteins, FLIP ( 46) and cIAP-2 ( 47). In multiple myeloma cells, down-regulation of PKCδ resulted in apoptosis ( 48). These findings show a role for PKCδ on antiapoptotic regulation in some tumor cells. In this regard, previous studies also suggested an antiapoptotic role for PKCδ in response to TNF-α; however, precise mechanisms remain unknown ( 20– 22). Importantly, numerous studies have shown the association between constitutive NF-κB activity in various types of cancer ( 49). The present study thus shows that PKCδ is a pivotal activator of NF-κB and is essential for its nuclear function in tumors. A thorough understanding of how PKCδ/NF-κB pathway regulates cell fate should help benefit for the cancer therapy.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Grant support: Ministry of Education, Science and Culture of Japan (K. Yoshida and Y. Miki) and Sumitomo Foundation, Mochida Memorial Foundation for Medical and Pharmaceutical Research, Astellas Foundation for Research on Metabolic Disorders, Life Science Foundation of Japan, Uehara Memorial Foundation, Cell Science Research Foundation, and Senri Life Science Foundation (K. Yoshida).
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).
K. Yoshida and Y. Miki share senior authorship.
- Received December 20, 2008.
- Revision received April 16, 2009.
- Accepted May 4, 2009.
- ©2009 American Association for Cancer Research.