Biochemical and Biophysical Research Communications 412 (2011) 313–317
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Protein kinase Cg activates NF-jB in response to camptothecin-induced DNA damage Hadas Raveh-Amit a,1, Naama Hai a,1, Noa Rotem-Dai a, Galit Shahaf a, Jacob Gopas a,b, Etta Livneh a,⇑ a b
The Shraga Segal Department of Microbiology and Immunology, Faculty of Health Sciences, The Cancer Research Center, Ben-Gurion University of the Negev, Israel The Department of Oncology, Soroka University Medical Center, Beer-Sheva 84105, Israel
a r t i c l e
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Article history: Received 18 July 2011 Available online 28 July 2011 Keywords: Nuclear factor jB transactivation RelA/P65 Protein kinase C DNA damage Camptothecin
a b s t r a c t The nuclear factor jB (NF-jB) family of transcription factors participates in the regulation of genes involved in innate- and adaptive-immune responses, cell death and inflammation. The involvement of the Protein Kinase C (PKC) family in the regulation of NF-jB in inflammation and immune-related signaling has been extensively studied. However, not much is known on the role of PKC in NF-jB regulation in response to DNA damage. Here we demonstrate for the first time that PKC-eta (PKCg) regulates NF-jB upstream signaling by activating the IjB kinase (IKK) and the degradation of IjB. Furthermore, PKCg enhances the nuclear translocation and transactivation of NF-jB under non-stressed conditions and in response to the anticancer drug camptothecin. We and others have previously shown that PKCg confers protection against DNA damage-induced apoptosis. Our present study suggests that PKCg is involved in NF-jB signaling leading to drug resistance. Ó 2011 Elsevier Inc. All rights reserved.
1. Introduction The activity of nuclear factor jB (NF-jB)/Rel family of transcription factors is deregulated in many diseases, including inflammatory and autoimmune diseases, viral infections and cancers [1]. In cancer, NF-jB affects the transcriptional activation of genes associated with cell proliferation, angiogenesis, metastasis, tumor promotion, inflammation and suppression of apoptosis [2]. Since tumor cells often use the NF-jB pathway to acquire resistance to anticancer drugs and radiation, the inhibition of NF-jB activation appears as a promising solution for improving the efficacy of conventional anti-cancer therapies [3,4]. In its inactive state, NF-jB resides in the cytoplasm in association with proteins known as inhibitors of NF-jB (IjB). In response to diverse extracellular signals, such as proinflammatory cytokines or DNA damaging agents, IjBa becomes rapidly phosphorylated by IjB kinases (IKKs), leading to IjB ubiquitination and subsequent degradation by the proteasome. The release of NF-jB from IjB allows its translocation to the nucleus where it binds to the jB consensus DNA sequences and regulates transcription of downstream target genes, such as Bcl-2, Bcl-xl and XIAP [5].
⇑ Corresponding author. Address: Department of Microbiology and Immunology, Ben-Gurion University of the Negev, P.O. Box 653, Beer-Sheva 84105, Israel. Fax: +972 8 647 7626. E-mail address:
[email protected] (E. Livneh). 1 These authors contributed equally to this work. 0006-291X/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2011.07.090
The involvement of the Protein Kinase C (PKC) family in the regulation of NF-jB in inflammation and immune-related signaling has been extensively studied. However, much less is known with respect to the role of PKC in NF-jB regulation in response to DNA damage. Since different PKC isoforms often exhibit opposite effects on NF-jB activation it is important to elucidate the specific role of each PKC member. PKC is a family of serine and threonine kinases affecting a variety of cellular responses, such as growth, proliferation, transformation and cell death [6]. Based on their primary structures, cofactors and enzymatic properties, PKC members are divided into three sub-groups: conventional PKCs, novel PKCs and atypical PKCs [7,8]. Recent studies suggest that PKCg, of the novel PKCs, confers protection against apoptosis and drug resistance. Its knockdown sensitizes cells towards radiation and anticancer drugs in various cell types. These include camptothecin (CPT) and doxorubicin in Hodgkin’s lymphoma lines [9], CPT and UV-C radiation in breast adenocarcinoma MCF7 cells [10], vincristine and paclitaxel in A549 lung cancer cells [11], UV-C and c-irradiation-induced apoptosis in glioblastoma cells [12] and UV-C in normal human keratinocytes [13]. The molecular mechanisms by which PKCg exerts its role in apoptosis and drug resistance are largely unknown and require further investigation. Given the anti-apoptotic roles of both NF-jB and PKCg in response to DNA damage, our aim was to determine whether PKCg is involved in NF-jB activation and signaling. Here we show that PKCg enhances the nuclear translocation and transactivation of NF-jB in non-stressed conditions and in response to the anticancer
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drug camptothecin. Furthermore, PKCg regulates the NF-jB upstream signaling by activating IjB kinase and degradation of IjB. 2. Materials and methods 2.1. Generation of PKCg-shRNA-expressing stable cell lines PKCg-shRNA-expressing stable cell lines were generated using the SureSilencing pre-designed PKCg short hairpin RNA vector (sh PKCg 2–2 and sh PKCg 3–5) or a scrambled vector (sh scramble 5–3) (SA Biosciences) in MCF-7 cells. In order to validate the silencing of PKCg expression, its levels were examined using qRT-PCR and immunoblotting in stable G418 resistant cells. 2.2. Cell lines and treatments
anti-IKKa/b, anti-IjB-a, anti-PKCg, anti-Bcl-xl, anti-lamin B, and anti-b-Tubulin (Santa Cruz Biotechnology), anti-HA (Covance), anti-b-actin (ICN Biomedicals), anti-bcl-2 (Calbiochem Biochemicals, kindly provided by V. Shoshan-Baramatz, Ben-Gurion University of the Negev, IL), and anti-XIAP (BD Biosciences, kindly provided by A. Kimchi, The Weizmann Institute, IL). 2.6. Immunofluorescence For the localization of RelA/P65, MCF-7 cells were seeded onto glass coverslips in 6-well plates (2 104 cells/well). The next day, cells were transfected with GFP-RelA/P65 alone or together with pHACE or pHA-PKCg plasmids. Two days later, cells were fixed, stained with the primary antibody anti-HA and detected using fluorescent microscopy as described [10].
PKCg-inducibly-expressed MCF-7 cells (MCF21.5) [14], or PKCg-knocked-down MCF-7 cells were maintained as previously described [10,14]. Cells were treated with CPT (10 lM) (Sigma–Aldrich) for the indicated times (1–3 h) with or without the PKCg pseudosubstrate inhibitor (10 lM) (Calbiochem Biochemicals).
2.7. Statistical analysis
2.3. Plasmids
3. Results and discussion
The pNF-jB-luc plasmid was purchased from Clontech Laboratories. The pGL3-promoter and pRL (Renilla) plasmids were purchased from Promega Corporation. The GFP-RelA/P65 plasmid was a gift from Dr. Rainer de Martin (Dept. of Vascular Biology and Thrombosis Research, Medical University of Vienna, AU). The pHACE plasmid was kindly provided by the Dr. JW Soh (Laboratory of the late Prof. IB Weinstein, Herbert Irving Comprehensive Cancer Center, Columbia University, New York, NY). The HA-tagged PKCgexpressing plasmid (pHA-PKCg) was generated by inserting the human cDNA of PKCg in the EcoRI restriction sites of the pHACE plasmid. The HA-tagged dominant negative PKCg-expressing plasmid (pHA-DN-PKCg) was generated by introducing the K384A and K387R substitutions at the ATP binding site of PKCg by PCR-based mutagenesis [15].
3.1. PKCg activates NF-jB in MCF-7 cells
2.4. Transfection and NF-jB activity reporter gene assay MCF-7 cells or PKCg-knocked-down cells were seeded in 24well plates (1 105 cells/well) 24 h prior to transfection. Cells were transiently co-transfected with the pNF-jB-luc and the pRL plasmids using the jetPEI™ kit (Poly transfection, France). The pRL plasmid served as a control for the efficiency of transfection. The pHA-PKCg, pHA-PKCg-DN and pHACE plasmids were added to the transfection reaction where indicated. As varied levels of pHA-PKCg or pHA-PKCg-DN were used, pHACE (empty vector) was added to ensure equal amounts of DNA. Fourty-eight hours post transfection, the cells were treated with camptothecin or left untreated. Cells were harvested and luciferase activities were measured using the Dual Luciferase Reporter assay (Promega) as previously described [16]. 2.5. Cell lysis, nuclear and cytoplasmatic fractionation and immunoblotting Cell lysates were prepared as previously described [17]. Nuclear and cytoplasmic protein lysates (5 106 cells per sample) were prepared using the NucBuster kit (Novagen, USA) according to manufacturer’s instructions. Immunoblotting was preformed as described in [16] using the following antibodies: anti-phosphoNF-jB RelA/P65 (Ser 536), anti-NF-jB RelA/P65, anti-NF-jB p50, anti-RelB, anti-NF-jB p52, anti-phospho-IKKa/b (Ser 180/181),
Results are expressed as means ± standard deviations of at least three independent experiments. Statistical analysis was performed with a t-test with the level of significance set at P < 0.05.
To explore the effect of PKCg on the basal activity of NF-jB, the luciferase reporter pNF-jB-luc and the pRL plasmids were co-transfected into MCF-7 cells with increasing amounts of an HA-tagged PKCg-expressing vector (pHA-PKCg) (Fig. 1A) or an HA-tagged dominant negative PKCg-expressing vector (pHA-PKCg-DN) (Fig. 1B). Overexpression of PKCg enhanced the activity of NF-jB, whereas the dominant-negative form of PKCg attenuated its activity in a dose dependent manner. To confirm these results, pNF-jBluc and pRL plasmids were transfected into PKCg-knocked-down MCF-7 cells (sh PKCg 2–2 and sh PKCg 3–5) and into shRNA scrambled control cells (sh scramble 5–3) (Fig. 1C). Silencing the expression of PKCg significantly attenuated NF-jB activity (2–4-fold reduction). ShRNA scrambled control cells exhibited similar NF-jB activity when compared to MCF-7 cells. To further examine the functional involvement of PKCg in the NF-jB signaling pathway, we investigated the effect of PKCg on the expression of NF-jB target genes: Bcl-2, Bcl-xl and XIAP (Fig. 1D) [5]. Silencing the expression of PKCg inhibited the basal levels of Bcl-2 and XIAP, while Bcl-xl levels were unaffected. Taken together, our results suggest that PKCg enhances the basal activity of NF-jB and specifically upregulates the expression of Bcl-2 and XIAP. 3.2. PKCg enhances the nuclear localization of RelA/P65 The mammalian NF-jB family consists of five members that reside in the cytoplasm in an inactive form. Upon activation, NF-jB translocate to the nucleus to induce the expression of their target genes [5]. We next examined whether PKCg affects the localization of NF-jB. Sub-cellular fractionation assays revealed that silencing the expression of PKCg (sh PKCg 2–2 and 3–5), specifically reduced the levels of NF-jB RelA/P65 in the nucleus. In contrast, the nuclear levels of P50, RelB and P52 were not altered under the same conditions (Fig. 2A). To further confirm the effect of PKCg on the nuclear translocation of RelA/P65, we examined the sub-cellular localization of GFPtagged RelA/P65 in the presence or absence of PKCg (pHA-PKCg or pHACE plasmids) in MCF-7 cells. GFP-RelA/P65 localization was monitored using fluorescent microscopy and scored to whether it
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Fig. 1. PKCg is an upstream regulator of NF-jB. (A and B), MCF-7 cells were transiently co-transfected with the pNF-jB-luc and the pRL plasmids. The pHA-PKCg, pHAPKCg-DN and pHACE plasmids were added to the transfection reaction where indicated. Cells were harvested and luciferase activities were measured using the Dual Luciferase Reporter assay as described in Materials and Methods. (C), MCF-7, shRNA control cells (sh scramble 5–3) and PKCg knockdown cells (sh PKCg 2–2 and sh PKCg 3– 5) were co-transfected with pNF-jB-luc and pRL and the luciferase activities were determined. (D), Whole cell lysates were prepared from shRNA control cells (sh scramble 5–3) and PKCg knockdown cells (sh PKCg 2–2) and subjected to immunoblotting with the indicated antibodies. P value of 0.05 was considered statistically significant (⁄).
Fig. 2. PKCg enhances the nuclear localization of RelA/P65 and its phosphorylation on Ser 536. (A), Nuclear and cytoplasmic fractions prepared from shRNA control cells (sh scramble 5–3) and PKCg knockdown cells (sh PKCg 2–2 and sh PKCg 3–5) were immunoblotted with the indicated antibodies. Densitometry is depicted by a bar diagram that represents the nuclear RelA/P65 levels normalized to lamin B. (B), MCF-7 cells were transfected with pGFP-RelA/P65 together with pHACE or pHA-PKCg. Two days after transfection, cells were fixed and stained with anti-HA or anti-PKCg. Sub-cellular localization of RelA/P65 was scored for cytosolic or nuclear localization or both (n > 50). Representative images are shown (100). Transfected cells were validated for the expression of exogenous PKCg by immunoblotting with anti-HA antibodies (lower panel).
was mainly localized in the cytosol, in the nucleus or in both. As shown in Fig. 2B, most of the cells transfected with an empty vector (pHACE) exhibited both nuclear and cytosolic localization of RelA/P65. The introduction of PKCg into MCF-7 cells (pHA-PKCg)
increased RelA/P65 nuclear localization and reduced its cytosolic and cytosolic plus nuclear localization. Taken together, our results suggest that PKCg specifically enhances the translocation of RelA/ P65 into the nucleus. The distinct DNA binding specificity of NF-jB
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is attributed to the formation of RelA/P65 homo- and hetero- dimers with the NF-jB members [18]. Thus, it is possible that the differential effect of PKCg on NF-jB target genes (Fig. 1D) could be explained by its specific effect on RelA/P65.
3.3. PKCg activates NF-jB in response to DNA damage Previous studies from our laboratory and others showed that PKCg has an anti-apoptotic role in various cell types and contributes to their resistance to DNA damage [9,10]. NF-jB is activated in response to DNA damage and mediates cell survival and chemoresistance [5]. We therefore hypothesized that PKCg may regulate NF-jB signaling in response to DNA damage. MCF-7 cells co-transfected with the pNF-jB-luc and the pRL plasmids were treated with the DNA damage inducer, camptothecin (CPT) a type I topoisomerase inhibitor, in the presence or absence of the PKCg pseudosubstrate inhibitor. Pretreatment with the PKCg inhibitor significantly reduced NF-jB basal activity (Fig. 3A, white bars). As expected, NF-jB activity was elevated in response to CPT treatment [19]. More important, pretreatment with the PKCg inhibitor (Pseudosubstrate, PS) prior to the CPT treatment abrogated the DNA damage-induced NF-jB activation (Fig. 3A, dark bars). To further confirm the involvement of PKCg in the DNA damage-induced NF-jB activation, PKCg knocked-down cells (sh PKCg 2–2) and control cells (sh scramble 5–3) were co-transfected with the pNF-jB-luc and pRL plasmids and treated with CPT. NF-jB activity was enhanced by CPT treatment. The basal as well as the
Fig. 3. PKCg is required for NF-jB transactivation in response to CPT-induced DNA damage. (A), MCF-7 cells were co-transfected with pNF-jB-luc and pRL. Fourtyeight hours post transfection, cells were treated with or without the PKCg pseudosubstrate (PS) inhibitor (10 lM) for 30 min and then subjected (or not) to DNA damage by CPT (10 lM) for 3 h. The luciferase activities were measured as described in Materials and Methods. (B), shRNA control cells (sh scramble 5–3) and PKCg knockdown cells (sh PKCg 2–2) were co-transfected with pNF-jB-luc and pRL. Forty eight hours post transfection, cells were treated (or not) with CPT for 3 h. (C), Whole cell lysates prepared from shRNA control cells (sh scramble 5–3) and PKCg knockdown cells (sh PKCg 2–2) were subjected to immunoblotting with the indicated antibodies. P value of 0.05 was considered statistically significant (⁄).
DNA damage-induced activaties of NF-jB were reduced in PKCg knocked-down cells (Fig. 3B). The CPT-Topoisomerase I interaction initiates a signaling cascade involving ATM that is transmitted to the cytoplasm resulting in nuclear translocation and activation of NF-jB [19]. Here we demonstrate a role for PKCg in response to CPT-induced DNA damage leading to activation of NF-jB (Fig. 3). These results are consistent with our recently published findings that PKCg confers protection against DNA damage induced cell death and that it is part of the stress response to DNA damage [9,10,20]. Furthermore, we have shown that the protein shuttles between the cytosol and nucleus and is tethered to the nuclear envelope in response to DNA damage [20]. 3.4. PKCg promotes IKK activation and IjB degradation in response to DNA damage In response to DNA damage, the release of NF-jB from its inhibitor IjB is an important prerequisite for its transcriptional activity. IjB phosphorylation is achieved primarily by IKK leading to its degradation [5]. To explore whether PKCg affects the DNA damage-induced IKK/IjB pathway, IKK activation and IjB degradation were examined in PKCg knockdown cells (sh PKCg 2–2) and control cells (sh scramble 5–3) subjected to CPT treatments. Silencing of PKCg reduced the phosphorylation of IKK in the activation loop (Ser180/181) and reduced IjB degradation (Fig. 4A). Consistent with these results, overexpression of PKCg (under the response of a tetracycline promoter, -TET cells [15]) enhanced the degradation of IjB in response to CPT in a time dependent manner (Fig. 4B). These results demonstrate that upon DNA damage, PKCg
Fig. 4. PKCg activates IKK activation and promotes IjB degradation in response to CPT-induced DNA damage. (A), Whole cell lysates were prepared from shRNA control cells (sh scramble 5–3) and PKCg knockdown cells (sh PKCg 2–2) treated with or without CPT (10 lM) for 1 and 3 h were subjected to immunoblotting. (B), PKCg expression was induced by the removal of tetracycline from the growth medium ( TET). Whole cell lysates were prepared from control MCF21.5 (+TET) or over-expressing PKCg ( TET) cells (as described in Section 2) and treated with or without CPT (10 lM) for 1 and 3 h were immunoblotted using the indicated antibodies.
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enhanced the activation of NF-jB by promoting the activation of the IKK complex and the degradation of IjB. It remains to be determined whether PKCg acts directly on IKK or on upstream signaling molecules. NF-jB has been proposed as a potential therapeutic target in cancer [4]. Its role in cancer development and progression is attributed to the effects of NF-jB on all hallmarks of cancer through the transcription of genes involved in cell proliferation, angiogenesis, metastasis, inflammation and suppression of apoptosis [21,22]. Here we show that PKCg promotes the nuclear translocation and transactivation of RelA/P65 leading to the induction of specific anti-apoptotic genes, including Bcl-2 (Fig. 1D). JNK was previously shown to phosphorylate Bcl-2, resulting in its inhibition [23]. We have recently published that PKCg inhibits the pro-apoptotic activity of c-Jun N-terminal kinase (JNK) [10]. Thus, in addition to its role at the transcriptional level, PKCg may also indirectly affect the activation of Bcl-2. Overall, these findings can explain, at least in part, the anti-apoptotic role of PKCg in response to DNA damaging agents. Acknowledgments This work was supported by The Israel Science Foundation (grant No. 1413/10). References [1] B.B. Aggarwal, Nuclear factor-kappaB: the enemy within, Cancer Cell 6 (2004) 203–208. [2] R.O. Escarcega, S. Fuentes-Alexandro, M. Garcia-Carrasco, A. Gatica, A. Zamora, The transcription factor nuclear factor-kappa B and cancer, Clin. Oncol. (R Coll. Radiol.) 19 (2007) 154–161. [3] C. Nakanishi, M. Toi, Nuclear factor-kappaB inhibitors as sensitizers to anticancer drugs, Nat. Rev. Cancer 5 (2005) 297–309. [4] V. Baud, M. Karin, Is NF-kappaB a good target for cancer therapy? Hopes and pitfalls, Nat. Rev. Drug Discov. 8 (2009) 33–40. [5] S. Janssens, J. Tschopp, Signals from within: the DNA-damage-induced NFkappaB response, Cell Death Differ. 13 (2006) 773–784. [6] H.J. Mackay, C.J. Twelves, Targeting the protein kinase C family: are we there yet?, Nat Rev. 7 (2007) 554–562. [7] H. Mellor, P.J. Parker, The extended protein kinase C superfamily, Biochem. J. 332 (1998) 281–292.
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