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CK2 regulates ATF4 and CHOP transcription within the cellular stress response signalling pathway
Cellular Signalling 24 (2012) 1797–1802
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CK2 regulates ATF4 and CHOP transcription within the cellular stress response signalling pathway Carolin C. Schneider ⁎, Emmanuel Ampofo, Mathias Montenarh Medizinische Biochemie und Molekularbiologie und Kompetenzzentrum Molekulare Medizin (KOMM), Universität des Saarlandes, Homburg, Saarland 66424, Germany
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Article history: Received 14 April 2012 Accepted 9 May 2012 Available online 16 May 2012 Keywords: Protein kinase CK2 Activating transcription factor 4 ER stress CHOP promoter ATF4 promoter Prostate cancer
a b s t r a c t Protein kinase CK2 is an ubiquitously expressed serine/threonine kinase. The protein levels along with CK2 activity are highly elevated in tumour cells where it protects cells from apoptosis. Accordingly, inhibition of CK2 is known to induce programmed cell death, making it a promising target for cancer therapy. Analysis of the different behaviour of hormone sensitive LNCaP cells and hormone refractory PC-3 cells after CK2 inhibition revealed CHOP ((C/EBP)-homologous protein) induction and therefore probably ER stress as crucial for apoptosis in the LNCaP cells. In the present study we investigated which promoter element of the CHOP promoter is responsible for its induction. ER stress can be generated by the accumulation of unfolded proteins, by depletion of amino acids or by oxidative stress. ER stress induces specific signalling pathways. In order to analyse which pathway might be activated by CK2 inhibition we started to analyse the activation of the different CHOP promoter elements. By using mutated reporter constructs of the CHOP promoter, it turned out that the amino acid response element (AARE) is the most prominent element for CHOP induction after CK2 inhibition. The ER stress element, however, proves to be less crucial, and along with the AP-1 binding site, they do not seem to play any role. Further we found an up-regulation of the transcription factor ATF4 after CK2 inhibition. ATF4 is involved in ER stress signalling through the AARE, which further supports our finding that CK2 inhibition provokes an amino acid induced response pathway. © 2012 Elsevier Inc. All rights reserved.
1. Introduction Protein kinase CK2 (formerly known as casein kinase CK2) is a pleiotropic protein kinase which is implicated in the decision of life and death of a cell. The CK2 protein level and activity is elevated in tumour cells compared to normal cells [1]. It was shown that CK2 has strong anti-apoptotic properties and inhibition of CK2 activity in tumour cells rapidly induces apoptosis (for review see: [2]). Most of these experiments were performed in human prostate cancer cells, because cell culture models representing different progression states of prostate cancer are well characterized. Development and progression of prostate cancers are dependent on androgen receptor signalling. Therefore hormone ablation strategies are often used to treat hormone sensitive prostate carcinoma cells. Upon treatment, prostate cancers develop a hormone refractory type of cancer which is insensitive to further treatment [3]. Therefore, new alternative strategies for the treatment of prostate cancer are urgently needed. It was
Abbreviations: AARE, amino acid response element; ATF4, activating transcription factor 4; AP-1, activating protein-1; C/EBP, CCAAT/enhancer binding protein; CHOP, C/EBP homologous protein; CK2, protein kinase CK2 (casein kinase CK2); ER, endoplasmic reticulum; ERSE, ER stress response element; GADD153, growth arrest and DNA damage inducible gene 153; TBB, 4,5,6,7-tetrabromobenzotriazole; TG, thapsigargin. ⁎ Corresponding author. Tel.: + 49 6841 1626518; fax: + 49 6841 1626027. E-mail address: [email protected] (C.C. Schneider). 0898-6568/$ – see front matter © 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.cellsig.2012.05.006
shown that CK2α overexpression rescued hormone refractory PC-3 cells from apoptosis [4]. In another study it was recently shown that inhibition of CK2 led to apoptosis in hormone sensitive prostate carcinoma cells (LNCaP). Hormone refractory PC-3 cells however do not show any loss of cell viability, although the intrinsic apoptotic pathway is induced [5,6]. The analysis of the different behaviour of hormone sensitive LNCaP cells and hormone refractory PC-3 cells after CK2 inhibition revealed some indication for the induction of endoplasmic reticulum stress (ER stress) pathway with a strong induction of the transcription factor CHOP in LNCaP cells, whereas in PC-3 cells, CHOP was undetectable [6]. Furthermore, overexpression of CHOP in PC-3 cells provoked apoptosis, supporting the idea that CHOP activation might be crucial for the induction of apoptosis after CK2 inhibition at least in prostate cancer cells [6]. CHOP, also called GADD153, encodes a small nuclear protein that regulates certain aspects of the cell response to stress [7,8]. The CHOP protein belongs to the CCAAT/enhancer-binding protein (C/ EBP) family of transcription factors that have been implicated in the regulation of energy metabolism, cellular proliferation, differentiation and the expression of cell type-specific genes (for review see: [9]). By the formation of heterodimers with other members of the C/EBP family, CHOP can influence gene expression. One of the down-stream targets of CHOP for the induction of apoptosis is bcl-2 [10]. However, the upstream regulation which leads to CHOP expression after inhibition of CK2 is not known. It is known that the CHOP gene can be activated
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through the ER stress response elements (ERSE) in response to cellular stress [11,12], the amino acid response element (AARE) in response to amino acid depletion [13,14] and ER stress, and the AP-1 element in response to oxidative stress [15]. In the present study we used these different promoter elements of the CHOP promoter in order to define the pathways that are implicated in the regulation of CHOP expression after CK2 inhibition. We show for the first time that CK2 inhibition activates the amino acid response element of the CHOP promoter. Moreover, we found an activation of the transcription factor ATF4 which is an upstream transcription factor for CHOP activation. 2. Material and methods 2.1. Cell culture LNCaP cells (ATCC: CRL-1740) are androgen-sensitive prostate cancer cells, which were established from a lymph-node metastasis [16]. Cells were maintained at 37 °C in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 10% fetal calf serum (FCS) in an atmosphere enriched with 5% CO2. The CK2 inhibitor TBB (Calbiochem, Merck KGaA, Darmstadt, Germany) as well as thapsigargin (Enzo Life Sciences GmbH, Lörrach, Germany) were dissolved in dimethyl sulfoxide (DMSO) to a 10 mM stock solution for TBB and a 100 μM stock solution for thapsigargin. Transfection of cells was performed by using the “Effectene Transfection Reagent” (Qiagen, Hilden, Germany) according to the manufacturer's instructions. 2.2. Extraction of proteins For harvesting, cells were scraped off the plate with a rubber policeman and sedimented together with floating cells by centrifugation (7 min, 4 °C, 400 x g). Cells were washed with cold phosphate buffered saline (PBS) and lysed with the double volume of RIPA buffer (50 mM Tris/HCl, pH 8.0, 150 mM NaCl, 0.5% sodium desoxycholate, 1% Triton X-100, 0.1% sodium dodecylsulfate) supplemented with the Protease Inhibitor Cocktail Complete according to the instructions of the manufacturer (Roche Diagnostics, Mannheim, Germany). After lysis, cell debris was removed by centrifugation. The protein content was determined according to a modified Bradford method with the BioRad reagent dye (BioRad, München, Germany). Protein extracts were immediately used for Western blot analysis. 2.3. Western blot analysis Proteins were separated by SDS polyacrylamide gel electrophoresis according to the procedure of Laemmli [17]. For Western blot analysis, proteins were transferred to a PVDF membrane by tank blotting with 20 mM Tris/HCl, pH 8.7, 150 mM glycine as transfer buffer. Membranes were blocked in PBS with 0.1% Tween20 (PBS-T) and 5% dry milk for 1 h at room temperature. The membrane was incubated with the primary antibody (usually in a dilution of 1:1000) in PBS-T with 1% dry milk over night. ATF4 (sc-200) and GAPDH antibodies (FL-335) were obtained from Santa Cruz (Heidelberg, Germany). The membrane was washed with PBS-T three times before incubating with the peroxidase coupled secondary antibody in a dilution of 1:30,000 in PBS-T with 1% dry milk. Signals were developed and visualized by the Lumilight system of Roche Diagnostics (Mannheim, Germany).
after transfection, cells were treated with 50 μM TBB or thapsigargin for another 24 h. After harvest and lysis of the cells, the luciferase activity was measured using the luciferase assay system from Promega GmbH (Mannheim, Germany). In case of co-transfection with pcDNA3.1/ Hygro/lacZ (Invitrogen GmbH, Darmstadt, Germany) encoding βgalactosidase, the β-galactosidase staining was performed as described in [18]. The reporter gene vectors used were kindly provided by Arie B. Vaandrager (University of Utrecht, The Netherlands) [19] and by Alain Bruhat (INRA de Theix, Saint Genès Champanelle, France) [13]. The ATF4 cDNA was cloned into the 3xFLAG expression vector by the use of the EcoRI and BamHI restriction sites. The expression vector for the ATF4 dominant-negative mutant was kindly provided by Jawed Alam, New Orleans, USA [20]. The luciferase reporter gene 5'UTR and AUG-luc (21850) for measuring the ATF4 promotor activity was obtained from Addgene (Cambridge, USA). 3. Results 3.1. CK2 inhibition induces the transcription factor CHOP We have recently shown that inhibition of CK2 kinase activity resulted in apoptosis in hormone sensitive LNCaP cells. In addition inhibition of CK2 kinase activity led to an induction of the transcription factor CHOP. A number of different stress induced signalling pathways converge on CHOP. So far, nothing is known about the stress induced pathways after CK2 inhibition. Therefore, in the present study we addressed the question, which are the upstream elements that might be responsible for the CHOP induction after CK2 inhibition. Endoplasmic reticulum stress can be induced by thapsigargin (TG) which is a potent non-competitive inhibitor of the sarco/endoplasmic reticulum Ca 2+ ATPase (SERCA). It raises the cytosolic Ca 2+-ion concentration by blocking the ability of the cell to pump Ca 2+-ions into the ER, which causes ER stress [21]. Cells were treated with 1 μM thapsigargin (TG) for 24 h. Cell extracts were analysed on a 10% SDS-polyacrylamide gel. CHOP was detected with a CHOP specific antibody. As shown in Fig. 1A we found an increase in the level of CHOP protein after treatment of the cells with thapsigargin whereas the solvent DMSO alone did not increase the level of CHOP. In the next step we wanted to know whether inhibition of CK2 might have the same effect on the level of CHOP. Therefore, LNCaP cells were treated with the widely used CK2 inhibitor TBB (50 μM). After 24 h treatment the cell extract was analysed for CHOP on the same SDSpolyacrylamide gel. As shown in Fig. 1A we found nearly the same level of CHOP protein after inhibition of CK2 kinase activity and after treatment of the cells with thapsigargin. The expression of CHOP is regulated by the binding of different transcription factors to specific promoter elements. In order to analyse whether the CHOP promoter is activated after inhibition of CK2 in comparison to thapsigargin treatment of the cells we used the wt −442 CHOP promoter construct (Fig. 1B) which contains the C/EBPATF composite site within the AARE, the AP-1 binding site and the ERSE elements in front of the luciferase gene. Twenty‐four hours after CK2 inhibition by TBB following transfection, we detected an up-regulation of the transcription of CHOP (4.8 fold). We found an even higher up-regulation after treatment of LNCaP cells with thapsigargin (18 fold) (Fig. 1C). These results showed that the CHOP promoter is turned on by inhibition of the CK2 kinase activity although not to the same extent as after thapsigargin induced ER stress. 3.2. CHOP is mainly induced via the C/EBP-ATF element in its promoter
2.4. Transfection and reporter gene assay For transient transfection with the reporter gene vectors (2×105/ well) cells were seeded into a 6 well plate and allowed to adhere overnight. Cells were transfected with 1 μg DNA per well using Effectene Transfection Reagent (Qiagen, Hilden, Germany). Twenty‐four hours
Next, we raised the question which element within the CHOP promoter is responsible for the CHOP induction after CK2 inhibition. In addition to the wt −442 CHOP promoter construct, we used constructs where one of the three promoter elements is mutated within the core sequence (Fig. 2A). The mutation leads to the inactivity of that element. In order to
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Fig. 1. Induction of CHOP by CK2 inhibition. (A) LNCaP cells were treated with 50 μM TBB or 1 μM thapsigargin (TG) for 24 h. Whole cell extracts were separated through a 10% SDSpolyacrylamide gel and transferred onto a PVDF membrane. CHOP and GAPDH were detected with the antibodies sc-22800 and FL-335 from Santa Cruz by enhanced chemiluminescence. (B) Reporter gene construct for wt −442 CHOP promoter with the C/EBP-ATF composite site, the AP-1 binding element and the ERSE [19]. (C) Cells were transfected with the reporter gene vectors for 24 h and then treated with 50 μM TBB or 1 μM thapsigargin (TG) or with DMSO as a control for another 24 h. The promoter activity was detected with a luciferase assay from Promega and presented as fold induction of control.
study the influence of CK2 inhibition on these different promoter elements, LNCaP cells were transfected with the reporter gene constructs and then subsequently CK2 was inhibited by TBB or cells were treated with the solvent as a control (Fig. 2B). After treatment for 24 h, luciferase activity was measured. The transactivation is shown as fold induction compared to the luciferase activity in solvent treated cells. We found that the mutation within the AP-1 binding site has no effect on the reporter activity after CK2 inhibition. The mutation within the ER stress element (ERSE) slightly attenuated the induction of transcription. The induction is 20% lower than that of the wt −442 CHOP promoter construct. The strongest reduction was, however, detected for the promoter construct containing the inactive C/EBP-ATF composite site. Here, the activity is reduced for more than 50% compared to the wt −442 CHOP promoter construct. These results indicate that the C/EBP-ATF composite site is the most crucial promoter element for CHOP induction after CK2 inhibition in LNCaP cells. For comparison we induced ER stress by treating the cells with thapsigargin and analysed the requirements of the promoter elements under the same experimental conditions as described for CK2 inhibition (Fig. 2C). The luciferase activity measured for the wt −442 CHOP promoter construct was abolished when the ERSE or the C/EBP-ATF composite site is inactive. The mutation in those elements reduces the activity for more than 75%. The AP-1 binding site in contrast does not seem to play any role in the CHOP induction after thapsigargin treatment as the luciferase activity was not altered by the mutation. We found that the C/EBP-ATF composite site is crucial for CHOP induction after CK2 inhibition and also after thapsigargin treatment of the cells. Thus, it seems that CK2 inhibition induces CHOP partially by affecting the ER stress signalling pathway in LNCaP cells. 3.3. Dominant negative mutant of ATF4 blocks CHOP induction after CK2 inhibition In the next step we wanted to analyse the up-stream element which might be responsible for the induction of CHOP. ATF4 is a member of the ER stress response signalling pathways that activates CHOP by binding to the C/EBP-ATF composite site within the AARE-
Fig. 2. Identification of the active element in the CHOP promoter during CK2 inhibition. (A) Reporter constructs used for the identification of the active element [19]. (B and C) Cells were transfected with the reporter gene vectors for 24 h and then treated with 50 μM TBB (B) or 1 μM thapsigargin (TG, C) or as a control with DMSO for another 24 h. The promoter activity was detected with a luciferase assay from Promega, normalized to β-galactosidase activity and presented as fold induction of control.
element [22]. Therefore, it was an obvious question whether ATF4 is implicated in the activation of CHOP after CK2 inhibition. We transfected the wt −442 CHOP promoter construct or a construct containing a duplicate of the C/EBP-ATF composite site of the CHOP promoter in front of a luciferase gene as a reporter into LNCaP cells (Fig. 3A). In addition cells were co-transfected with a dominant negative mutant of ATF4 (dn) or as a control with an empty vector. Furthermore, we treated the cells with the CK2 inhibitor TBB for 24 h or with the solvent as a control. The activity was presented as fold induction compared to the solvent control. As shown in Fig. 3B, CK2 inhibition resulted in a strong stimulation of the AARE promoter construct as well as of the wt − 442 CHOP promoter construct. The AARE promoter construct shows only half of the activity in the presence of the dominant negative mutant of ATF4. The activation of the wt − 442 CHOP promoter construct was reduced to nearly control level when the dominant negative mutant of ATF4 was cotransfected. These results indicate that ATF4 indeed seems to be at least one of the up-stream factors that is responsible for CHOP induction. In order to support the findings that ATF4 is indeed responsible for the induction of these two promoter constructs, we transfected the LNCaP cells with one of the reporter constructs and an ATF4 expression vector. Half of the cell population was in addition transfected with the dominant negative mutant of ATF4 (dn) or in the absence of this mutant. As a control, cells were only transfected with an empty vector. The reporter activity was measured after 48 h of transfection. As shown in Fig. 3C, the expression of ATF4 induced both the
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Fig. 4. Induction of ATF4 by CK2 inhibition. (A) Whole cell extracts of treated cells (50 μM TBB or 1 μM thapsigargin (TG), or DMSO as a control, 24 h) were separated on a 10% SDS-polyacrylamide gel and transferred onto a PVDF membrane. ATF4 and GAPDH were detected with polyclonal rabbit antibodies by enhanced chemiluminescence. (B) The reporter gene vector for the ATF4 promoter activity was transfected for 24 h. Cells were treated for another 24 h with 50 μM TBB, 1 μM TG or DMSO as a control and the activity was detected with a luciferase assay from Promega and presented as fold induction of control.
Fig. 3. Identification of ATF4 as the inducing transcription factor. (A) Reporter constructs used for the identification of the inducing transcription factor. The reporter gene constructs were transfected for 24 h either together with the expression vector for the dominant negative mutant of ATF4 (+dn) or an empty vector as a control (−dn). (B) After transfection, the cells were treated with 50 μM TBB or solvent as a control for 24 h. The promoter activity was detected with a luciferase assay from Promega and presented as fold induction of control. (C) Additionally to the reporter gene constructs and the expression vector for the dominant negative mutant of ATF4 (+dn) or an empty vector as a control (−dn), we transfected an expression vector for ATF4 wt into the LNCaP cells. Cells transfected with empty vectors served as a control. The promoter activity was detected with a luciferase assay from Promega and presented as fold induction of control.
AARE promoter construct as well as the wt −442 CHOP promoter construct. In the presence of the dominant negative ATF4 mutant, transcription was considerably reduced. For the AARE promoter construct, we detected a reduction of more than 50%. The wt −442 CHOP promoter construct showed no activity anymore when ATF4 is sequestered by its dominant negative mutant. Since this result is quite similar to the one that was obtained after CK2 inhibition by TBB, we conclude that ATF4 is indeed implicated in the CHOP induction after CK2 inhibition. 3.4. CK2 inhibition leads to increased ATF4 protein level As it is known that not only is ATF4 regulated transcriptionally but also on the level of translation or posttranslationally [23–25], we next determined the ATF4 protein level after CK2 inhibition and compared the level to thapsigargin treated or completely untreated cells. As shown in Fig. 4A, there is an elevated level of ATF4 in cells treated with the CK2 inhibitor TBB and also after thapsigargin treatment. To further analyse these findings, we asked whether the transcription of the ATF4 gene was influenced by CK2 inhibition. Therefore, we transfected a reporter construct with the ATF4 promoter into LNCaP cells for 24 h. Then, cells were treated with the CK2 inhibitor TBB, with thapsigargin or with the solvent as a control and the reporter activity was measured after 24 h of treatment. The results of the reporter assay shown in Fig. 4B illustrate that there was no increase in ATF4 transcription after CK2 inhibition, whereas, thapsigargin treatment of cells led to a strong transcriptional up-regulation of ATF4. Thus, we have to conclude that the up-regulation of ATF4 after CK2 inhibition
in contrast to its up-regulation by ER stress is a posttranscriptional event. 4. Discussion It is known for quite some time that human prostate cancer cells express high levels and activity of protein kinase CK2 [26]. Inhibition of the kinase activity of CK2 results in growth arrest and apoptosis [27–30]. Using different CK2 inhibitors, we observed that the hormone sensitive LNCaP cells always respond with apoptosis to CK2 inhibition. In contrast, hormone refractory PC-3 cells do not show any loss in cell viability although the intrinsic apoptotic pathway is induced [5,6]. One major difference between the two types of prostate cancer was the induction of CHOP which was found in LNCaP cells but not in PC-3 cells [6]. CHOP encodes a nuclear, proapoptotic bzip transcription factor of the CCAAT/enhancer binding protein (C/EBP) family which is often induced by cellular stress [7,31,32]. Furthermore, expression of CHOP in hormone refractory cancer cells PC-3 led to the induction of apoptosis supporting the idea that CHOP is crucial for apoptosis induction after CK2 inhibition. Many different cellular stresses, for example, impaired redox balances, amino acid depletion and deficiencies in proteasome function can lead to ER stress. This ER stress results in disturbance of protein quality control which triggers a cellular response called unfolded protein response (UPR). There are three different signalling pathways which are activated by ER stress. One pathway is the PERK/eIF2α/ATF4 cascade, the second one is the IRE1/INK cascade and the third one is the ATF6 pathway. Since all three pathways converge on CHOP it was an interesting question which might be regulated by CK2. One approach to elucidate the CK2 contribution to these pathways was the analysis of the activation of different promotor elements within the CHOP promoter. It was shown that CHOP expression after various kinds of stress is regulated through promoter sequences between nucleotide position − 954 and + 91 of the 5’ upstream region of the CHOP gene [11,33]. Detailed analysis of the promoter region revealed that the region between nucleotide −442 and −75 contains three different elements such as the amino acid response element (AARE) with the C/EBP-ATF composite site, the AP-1 binding site as well as the ER stress element
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(ERSE) [19]. The AP-1 binding site is activated by oxidative stress. The AARE responds to amino acid limitation as well as to ER stress. During ER stress the ERSE is also activated [34]. We used different promoter constructs to analyse which element is essential for CHOP induction by CK2 inhibition and which signalling pathway could be involved in this induction. Therefore, we compared a totally functional CHOP promoter construct (−442 wt) with constructs which were inactive for one binding site. We found that CK2 inhibition led to an activation of the wt −442 CHOP promoter construct containing the above mentioned elements as well as the promoter element with the mutation in the AP-1 binding site. This indicates that the AP-1 element is not required for CHOP induction by CK2 inhibition. Thus, we can mainly exclude oxidative stress as a trigger for the CHOP induction after CK2 inhibition by TBB. That fits with some former results where reactive oxygen species after CK2 inhibition by TBB were not detected [35]. As we found the CHOP induction only slightly reduced by the inactivation of the ERSE, we conclude that this element is only partially crucial for CHOP induction by CK2 inhibition which is in contrast to ER stress by thapsigargin treatment. Mutation and thereby inactivation of the AARE, however, led to the strongest reduction of the CHOP promoter activity after CK2 inhibition. These results demonstrate that this element is mainly implicated in the regulation of the CHOP expression after CK2 inhibition. As it is known that the AARE is also implicated in the ER stress signalling, CK2 inhibition by TBB affects these signalling pathways although different from the ER stress signalling induced by thapsigargin. The AARE contains a C/EBP-ATF composite site that is composed of a half-site for the C/EBP family of transcription factors and a half-site for the ATF family of transcription factors [22]. One of the transcription factors of the ATF family that triggers increased transcription by binding to this element is ATF4 [22]. Using wild-type ATF4 and a dominant negative mutant of ATF4 [36], we showed here that ATF4 is indeed responsible for the regulation of the AARE promoter construct as well as of the wt −442 CHOP promoter construct. This is because the dominant negative mutant of ATF4 blocks the activation of both promoter constructs after CK2 inhibition by TBB or transfection with ATF4 wt. ATF4 is regulated transcriptionally as well as posttranscriptionally. We demonstrated that CK2 inhibition does not induce ATF4 transcription; however, we found an elevated level of the ATF4 protein after CK2 inhibition. It is known that ATF4 is a phosphoprotein and that the phosphorylation of ATF4 is crucial for its stability [37,38]. Therefore, the up-regulation of ATF4 is probably due to stabilization as a phosphorylation by CK2 and therefore the degradation of ATF4 is inhibited. In contrast, the ER stress inducing agent thapsigargin led to an induction of transcription of the ATF4 promoter and therefore to an increased ATF4 protein level. In a very recent paper Manni et al. reported that CK2 protects multiple myeloma cells from ER stress induced apoptosis. They also found the activation of the UPR and an induction of the signalling cascade including PERK and eIF2α [39]. These two kinases are upstream factors of ATF4 and therefore these data support our finding about a contribution of ATF4 to this signalling pathway. They also presented data on the contribution of IRE1α, Bip and HSP90 to the signalling converging on CHOP [39]. Our present results demonstrate that inhibition of CK2 by TBB induces an up-regulation of the protein level of ATF4. ATF4 then transactivates CHOP expression by binding mainly to the C/EBP-ATF composite site within the AARE triggering apoptosis at least in hormone sensitive prostate cancer cells. Our present data together with the data of Manni et al. clearly demonstrate an active and central role of CK2 in cellular-stress induced signalling pathways. 5. Concluding remarks Inhibition of the kinase activity of CK2 leads to apoptosis in hormone sensitive prostate cancer cells. Here we showed for the first time that inhibition of CK2 activates the amino acid induced stress pathway in these prostate cancer cells. Furthermore, up-regulation of the expression of the transcription factor ATF4 identified ATF4 as
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a new factor in the signalling pathway after CK2 inhibition. The identification of CK2 regulated factors in the cellular stress response pathway improved the understanding of the role of CK2 as an anti-cancer drug target at least in prostate cancer cells. Acknowledgement We thank A. B. Vandraager, University of Utrecht, The Netherlands, for the gift of the reporter gene construct wt −442 CHOP and the constructs with the mutations in the core sequences of the regulatory elements. Furthermore, we thank A. Bruhat, INRA de Theix, Saint Genès Champanelle, France, for kindly providing the AARE construct and we thank J. Alam, New Orleans, USA, for the kind gift of the expression vector for the ATF4 dominant-negative mutant. We also thank Nathaniel Saidu for carefully reading the manuscript. References [1] B. Guerra, O.G. Issinger, Electrophoresis 20 (1999) 391–408. [2] K.A. Ahmad, G. Wang, G. Unger, J. Slaton, K. Ahmed, Advances in Enzyme Regulation 48 (2008) 179–187. [3] G. Wang, K.A. Ahmad, G. Unger, J.W. Slaton, K. Ahmed, Journal of Cellular Biochemistry 99 (2006) 382–391. [4] H.M. Wang, A. Davis, S.H. Yu, K. Ahmed, Molecular and Cellular Biochemistry 227 (2001) 167–174. [5] A. Hessenauer, M. Montenarh, C. Götz, International Journal of Oncology 22 (2003) 1263–1270. [6] A. Hessenauer, C.C. Schneider, C. Götz, M. Montenarh, Cellular Signalling 23 (2011) 145–151. [7] H. Zinszner, M. Kuroda, X. Wang, N. Batchvarova, R.T. Lightfoot, H. Remotti, J.L. Stevens, D. Ron, Genes & Development 12 (1998) 982–995. [8] I. Tabas, D. Ron, Nature Cell Biology 13 (2011) 184–190. [9] J. Tsukada, Y. Yoshida, Y. Kominato, P.E. Auron, Cytokine 54 (2011) 6–19. [10] K.D. McCullough, J.L. Martindale, L.O. Klotz, T.Y. Aw, N.J. Holbrook, Molecular and Cellular Biology 21 (2001) 1249–1259. [11] M. Ubeda, J.F. Habener, Nucleic Acids Research 28 (12-15-2000) 4987–4997. [12] H. Yoshida, T. Okada, K. Haze, H. Yanagi, T. Yura, M. Negishi, K. Mori, Molecular and Cellular Biology 20 (2000) 6755–6767. [13] A. Bruhat, C. Jousse, V. Carraro, A.M. Reimold, M. Ferrara, P. Fafournoux, Molecular and Cellular Biology 20 (2000) 7192–7204. [14] Y. Ma, J.W. Brewer, J.A. Diehl, L.M. Hendershot, Journal of Molecular Biology 318 (2002) 1351–1365. [15] K.Z. Guyton, Q. Xu, N.J. Holbrook, Biochemical Journal 314 (Pt 2) (1996) 547–554. [16] J.S. Horoszewicz, S.S. Leong, E. Kawinski, J.P. Karr, H. Rosenthal, T.M. Chu, E.A. Mirand, G.P. Murphy, Cancer Research 43 (1983) 1809–1818. [17] U.K. Laemmli, Nature 227 (1970) 680–682. [18] T. Maniatis, E.F. Fritsch, J. Sambrook, Sambrook, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1982, book 3, 16.66–16.67. [19] M.H. van der Sanden, H. Meems, M. Houweling, J.B. Helms, A.B. Vaandrager, Journal of Biological Chemistry 279 (2004) 52007–52015. [20] C.H. He, P. Gong, B. Hu, D. Stewart, M.E. Choi, A.M. Choi, J. Alam, Journal of Biological Chemistry 276 (2001) 20858–20865. [21] F. Qi, M. Carbone, H. Yang, G. Gaudino, Expert Review of Respiratory Medicine 5 (2011) 683–697. [22] T.W. Fawcett, J.L. Martindale, K.Z. Guyton, T. Hai, N.J. Holbrook, Biochemical Journal 339 (1999) 135–141. [23] F. Siu, P.J. Bain, R. LeBlanc-Chaffin, H. Chen, M.S. Kilberg, Journal of Biological Chemistry 277 (2002) 24120–24127. [24] H.P. Harding, I. Novoa, Y. Zhang, H. Zeng, R. Wek, M. Schapira, D. Ron, Molecular Cell 6 (2000) 1099–1108. [25] K.M. Vattem, R.C. Wek, Proceedings of the National Academy of Sciences of the United States of America 101 (2004) 11269–11274. [26] S. Tawfic, S. Yu, H. Wang, R. Faust, A. Davis, K. Ahmed, Histology and Histopathology 16 (2001) 573–582. [27] F. Pierre, P.C. Chua, S.E. O'Brien, A. Siddiqui-Jain, P. Bourbon, M. Haddach, J. Michaux, J. Nagasawa, M.K. Schwaebe, E. Stefan, A. Vialettes, J.P. Whitten, T.K. Chen, L. Darjania, R. Stansfield, J. Bliesath, D. Drygin, C. Ho, M. Omori, C. Proffitt, N. Streiner, W.G. Rice, D.M. Ryckman, K. Anderes, Molecular and Cellular Biochemistry 356 (2011) 37–43. [28] G. Wang, G. Unger, K.A. Ahmad, J.W. Slaton, K. Ahmed, Molecular and Cellular Biochemistry 274 (2005) 77–84. [29] J.H. Trembley, Z. Chen, G. Unger, J. Slaton, B.T. Kren, Van WaesC. , K. Ahmed, Biofactors 36 (2010) 187–195. [30] N.A. St-Denis, D.W. Litchfield, Cellular and Molecular Life Sciences 66 (2009) 1817–1829. [31] D. Ron, J.F. Habener, Genes & Development 6 (1992) 439–453. [32] X.Z. Wang, M. Kuroda, J. Sok, N. Batchvarova, R. Kimmel, P. Chung, H. Zinszner, D. Ron, EMBO Journal 17 (1998) 3619–3630. [33] C. Jousse, A. Bruhat, H.P. Harding, M. Ferrara, D. Ron, P. Fafournoux, FEBS Letters 448 (1999) 211–216. [34] S.C. Kwok, I. Daskal, Molecular and Cellular Biochemistry 319 (2008) 203–208.
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[35] C.C. Schneider, A. Hessenauer, C. Götz, M. Montenarh, Oncology Reports 21 (2009) 1593–1597. [36] Y. Makino, K. Omichi, Bioscience, Biotechnology, and Biochemistry 70 (2006) 907–915. [37] C.L. Frank, X. Ge, Z. Xie, Y. Zhou, L.H. Tsai, Journal of Biological Chemistry 285 (2010) 33324–33337.
[38] I. Lassot, E. Segeral, C. Berlioz-Torrent, H. Durand, L. Groussin, T. Hai, R. Benarous, F. Margottin-Goguet, Molecular and Cellular Biology 21 (2001) 2192–2202. [39] S. Manni, A. Brancalion, L.Q. Tubi, A. Colpo, L. Pavan, A. Cabrelle, E. Ave, F. Zaffino, G. Di Maira, M. Ruzzene, F. Adami, R. Zambello, M.R. Pitari, P. Tassone, L.A. Pinna, C. Gurrieri, G. Semenzato, F. Piazza, Clinical Cancer Research 18 (2012) 1888–1900.
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CK2 regulates ATF4 and CHOP transcription within the cellular stress response signalling pathway Carolin C. Schneider ⁎, Emmanuel Ampofo, Mathias Montenarh Medizinische Biochemie und Molekularbiologie und Kompetenzzentrum Molekulare Medizin (KOMM), Universität des Saarlandes, Homburg, Saarland 66424, Germany
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Article history: Received 14 April 2012 Accepted 9 May 2012 Available online 16 May 2012 Keywords: Protein kinase CK2 Activating transcription factor 4 ER stress CHOP promoter ATF4 promoter Prostate cancer
a b s t r a c t Protein kinase CK2 is an ubiquitously expressed serine/threonine kinase. The protein levels along with CK2 activity are highly elevated in tumour cells where it protects cells from apoptosis. Accordingly, inhibition of CK2 is known to induce programmed cell death, making it a promising target for cancer therapy. Analysis of the different behaviour of hormone sensitive LNCaP cells and hormone refractory PC-3 cells after CK2 inhibition revealed CHOP ((C/EBP)-homologous protein) induction and therefore probably ER stress as crucial for apoptosis in the LNCaP cells. In the present study we investigated which promoter element of the CHOP promoter is responsible for its induction. ER stress can be generated by the accumulation of unfolded proteins, by depletion of amino acids or by oxidative stress. ER stress induces specific signalling pathways. In order to analyse which pathway might be activated by CK2 inhibition we started to analyse the activation of the different CHOP promoter elements. By using mutated reporter constructs of the CHOP promoter, it turned out that the amino acid response element (AARE) is the most prominent element for CHOP induction after CK2 inhibition. The ER stress element, however, proves to be less crucial, and along with the AP-1 binding site, they do not seem to play any role. Further we found an up-regulation of the transcription factor ATF4 after CK2 inhibition. ATF4 is involved in ER stress signalling through the AARE, which further supports our finding that CK2 inhibition provokes an amino acid induced response pathway. © 2012 Elsevier Inc. All rights reserved.
1. Introduction Protein kinase CK2 (formerly known as casein kinase CK2) is a pleiotropic protein kinase which is implicated in the decision of life and death of a cell. The CK2 protein level and activity is elevated in tumour cells compared to normal cells [1]. It was shown that CK2 has strong anti-apoptotic properties and inhibition of CK2 activity in tumour cells rapidly induces apoptosis (for review see: [2]). Most of these experiments were performed in human prostate cancer cells, because cell culture models representing different progression states of prostate cancer are well characterized. Development and progression of prostate cancers are dependent on androgen receptor signalling. Therefore hormone ablation strategies are often used to treat hormone sensitive prostate carcinoma cells. Upon treatment, prostate cancers develop a hormone refractory type of cancer which is insensitive to further treatment [3]. Therefore, new alternative strategies for the treatment of prostate cancer are urgently needed. It was
Abbreviations: AARE, amino acid response element; ATF4, activating transcription factor 4; AP-1, activating protein-1; C/EBP, CCAAT/enhancer binding protein; CHOP, C/EBP homologous protein; CK2, protein kinase CK2 (casein kinase CK2); ER, endoplasmic reticulum; ERSE, ER stress response element; GADD153, growth arrest and DNA damage inducible gene 153; TBB, 4,5,6,7-tetrabromobenzotriazole; TG, thapsigargin. ⁎ Corresponding author. Tel.: + 49 6841 1626518; fax: + 49 6841 1626027. E-mail address: [email protected] (C.C. Schneider). 0898-6568/$ – see front matter © 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.cellsig.2012.05.006
shown that CK2α overexpression rescued hormone refractory PC-3 cells from apoptosis [4]. In another study it was recently shown that inhibition of CK2 led to apoptosis in hormone sensitive prostate carcinoma cells (LNCaP). Hormone refractory PC-3 cells however do not show any loss of cell viability, although the intrinsic apoptotic pathway is induced [5,6]. The analysis of the different behaviour of hormone sensitive LNCaP cells and hormone refractory PC-3 cells after CK2 inhibition revealed some indication for the induction of endoplasmic reticulum stress (ER stress) pathway with a strong induction of the transcription factor CHOP in LNCaP cells, whereas in PC-3 cells, CHOP was undetectable [6]. Furthermore, overexpression of CHOP in PC-3 cells provoked apoptosis, supporting the idea that CHOP activation might be crucial for the induction of apoptosis after CK2 inhibition at least in prostate cancer cells [6]. CHOP, also called GADD153, encodes a small nuclear protein that regulates certain aspects of the cell response to stress [7,8]. The CHOP protein belongs to the CCAAT/enhancer-binding protein (C/ EBP) family of transcription factors that have been implicated in the regulation of energy metabolism, cellular proliferation, differentiation and the expression of cell type-specific genes (for review see: [9]). By the formation of heterodimers with other members of the C/EBP family, CHOP can influence gene expression. One of the down-stream targets of CHOP for the induction of apoptosis is bcl-2 [10]. However, the upstream regulation which leads to CHOP expression after inhibition of CK2 is not known. It is known that the CHOP gene can be activated
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through the ER stress response elements (ERSE) in response to cellular stress [11,12], the amino acid response element (AARE) in response to amino acid depletion [13,14] and ER stress, and the AP-1 element in response to oxidative stress [15]. In the present study we used these different promoter elements of the CHOP promoter in order to define the pathways that are implicated in the regulation of CHOP expression after CK2 inhibition. We show for the first time that CK2 inhibition activates the amino acid response element of the CHOP promoter. Moreover, we found an activation of the transcription factor ATF4 which is an upstream transcription factor for CHOP activation. 2. Material and methods 2.1. Cell culture LNCaP cells (ATCC: CRL-1740) are androgen-sensitive prostate cancer cells, which were established from a lymph-node metastasis [16]. Cells were maintained at 37 °C in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 10% fetal calf serum (FCS) in an atmosphere enriched with 5% CO2. The CK2 inhibitor TBB (Calbiochem, Merck KGaA, Darmstadt, Germany) as well as thapsigargin (Enzo Life Sciences GmbH, Lörrach, Germany) were dissolved in dimethyl sulfoxide (DMSO) to a 10 mM stock solution for TBB and a 100 μM stock solution for thapsigargin. Transfection of cells was performed by using the “Effectene Transfection Reagent” (Qiagen, Hilden, Germany) according to the manufacturer's instructions. 2.2. Extraction of proteins For harvesting, cells were scraped off the plate with a rubber policeman and sedimented together with floating cells by centrifugation (7 min, 4 °C, 400 x g). Cells were washed with cold phosphate buffered saline (PBS) and lysed with the double volume of RIPA buffer (50 mM Tris/HCl, pH 8.0, 150 mM NaCl, 0.5% sodium desoxycholate, 1% Triton X-100, 0.1% sodium dodecylsulfate) supplemented with the Protease Inhibitor Cocktail Complete according to the instructions of the manufacturer (Roche Diagnostics, Mannheim, Germany). After lysis, cell debris was removed by centrifugation. The protein content was determined according to a modified Bradford method with the BioRad reagent dye (BioRad, München, Germany). Protein extracts were immediately used for Western blot analysis. 2.3. Western blot analysis Proteins were separated by SDS polyacrylamide gel electrophoresis according to the procedure of Laemmli [17]. For Western blot analysis, proteins were transferred to a PVDF membrane by tank blotting with 20 mM Tris/HCl, pH 8.7, 150 mM glycine as transfer buffer. Membranes were blocked in PBS with 0.1% Tween20 (PBS-T) and 5% dry milk for 1 h at room temperature. The membrane was incubated with the primary antibody (usually in a dilution of 1:1000) in PBS-T with 1% dry milk over night. ATF4 (sc-200) and GAPDH antibodies (FL-335) were obtained from Santa Cruz (Heidelberg, Germany). The membrane was washed with PBS-T three times before incubating with the peroxidase coupled secondary antibody in a dilution of 1:30,000 in PBS-T with 1% dry milk. Signals were developed and visualized by the Lumilight system of Roche Diagnostics (Mannheim, Germany).
after transfection, cells were treated with 50 μM TBB or thapsigargin for another 24 h. After harvest and lysis of the cells, the luciferase activity was measured using the luciferase assay system from Promega GmbH (Mannheim, Germany). In case of co-transfection with pcDNA3.1/ Hygro/lacZ (Invitrogen GmbH, Darmstadt, Germany) encoding βgalactosidase, the β-galactosidase staining was performed as described in [18]. The reporter gene vectors used were kindly provided by Arie B. Vaandrager (University of Utrecht, The Netherlands) [19] and by Alain Bruhat (INRA de Theix, Saint Genès Champanelle, France) [13]. The ATF4 cDNA was cloned into the 3xFLAG expression vector by the use of the EcoRI and BamHI restriction sites. The expression vector for the ATF4 dominant-negative mutant was kindly provided by Jawed Alam, New Orleans, USA [20]. The luciferase reporter gene 5'UTR and AUG-luc (21850) for measuring the ATF4 promotor activity was obtained from Addgene (Cambridge, USA). 3. Results 3.1. CK2 inhibition induces the transcription factor CHOP We have recently shown that inhibition of CK2 kinase activity resulted in apoptosis in hormone sensitive LNCaP cells. In addition inhibition of CK2 kinase activity led to an induction of the transcription factor CHOP. A number of different stress induced signalling pathways converge on CHOP. So far, nothing is known about the stress induced pathways after CK2 inhibition. Therefore, in the present study we addressed the question, which are the upstream elements that might be responsible for the CHOP induction after CK2 inhibition. Endoplasmic reticulum stress can be induced by thapsigargin (TG) which is a potent non-competitive inhibitor of the sarco/endoplasmic reticulum Ca 2+ ATPase (SERCA). It raises the cytosolic Ca 2+-ion concentration by blocking the ability of the cell to pump Ca 2+-ions into the ER, which causes ER stress [21]. Cells were treated with 1 μM thapsigargin (TG) for 24 h. Cell extracts were analysed on a 10% SDS-polyacrylamide gel. CHOP was detected with a CHOP specific antibody. As shown in Fig. 1A we found an increase in the level of CHOP protein after treatment of the cells with thapsigargin whereas the solvent DMSO alone did not increase the level of CHOP. In the next step we wanted to know whether inhibition of CK2 might have the same effect on the level of CHOP. Therefore, LNCaP cells were treated with the widely used CK2 inhibitor TBB (50 μM). After 24 h treatment the cell extract was analysed for CHOP on the same SDSpolyacrylamide gel. As shown in Fig. 1A we found nearly the same level of CHOP protein after inhibition of CK2 kinase activity and after treatment of the cells with thapsigargin. The expression of CHOP is regulated by the binding of different transcription factors to specific promoter elements. In order to analyse whether the CHOP promoter is activated after inhibition of CK2 in comparison to thapsigargin treatment of the cells we used the wt −442 CHOP promoter construct (Fig. 1B) which contains the C/EBPATF composite site within the AARE, the AP-1 binding site and the ERSE elements in front of the luciferase gene. Twenty‐four hours after CK2 inhibition by TBB following transfection, we detected an up-regulation of the transcription of CHOP (4.8 fold). We found an even higher up-regulation after treatment of LNCaP cells with thapsigargin (18 fold) (Fig. 1C). These results showed that the CHOP promoter is turned on by inhibition of the CK2 kinase activity although not to the same extent as after thapsigargin induced ER stress. 3.2. CHOP is mainly induced via the C/EBP-ATF element in its promoter
2.4. Transfection and reporter gene assay For transient transfection with the reporter gene vectors (2×105/ well) cells were seeded into a 6 well plate and allowed to adhere overnight. Cells were transfected with 1 μg DNA per well using Effectene Transfection Reagent (Qiagen, Hilden, Germany). Twenty‐four hours
Next, we raised the question which element within the CHOP promoter is responsible for the CHOP induction after CK2 inhibition. In addition to the wt −442 CHOP promoter construct, we used constructs where one of the three promoter elements is mutated within the core sequence (Fig. 2A). The mutation leads to the inactivity of that element. In order to
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Fig. 1. Induction of CHOP by CK2 inhibition. (A) LNCaP cells were treated with 50 μM TBB or 1 μM thapsigargin (TG) for 24 h. Whole cell extracts were separated through a 10% SDSpolyacrylamide gel and transferred onto a PVDF membrane. CHOP and GAPDH were detected with the antibodies sc-22800 and FL-335 from Santa Cruz by enhanced chemiluminescence. (B) Reporter gene construct for wt −442 CHOP promoter with the C/EBP-ATF composite site, the AP-1 binding element and the ERSE [19]. (C) Cells were transfected with the reporter gene vectors for 24 h and then treated with 50 μM TBB or 1 μM thapsigargin (TG) or with DMSO as a control for another 24 h. The promoter activity was detected with a luciferase assay from Promega and presented as fold induction of control.
study the influence of CK2 inhibition on these different promoter elements, LNCaP cells were transfected with the reporter gene constructs and then subsequently CK2 was inhibited by TBB or cells were treated with the solvent as a control (Fig. 2B). After treatment for 24 h, luciferase activity was measured. The transactivation is shown as fold induction compared to the luciferase activity in solvent treated cells. We found that the mutation within the AP-1 binding site has no effect on the reporter activity after CK2 inhibition. The mutation within the ER stress element (ERSE) slightly attenuated the induction of transcription. The induction is 20% lower than that of the wt −442 CHOP promoter construct. The strongest reduction was, however, detected for the promoter construct containing the inactive C/EBP-ATF composite site. Here, the activity is reduced for more than 50% compared to the wt −442 CHOP promoter construct. These results indicate that the C/EBP-ATF composite site is the most crucial promoter element for CHOP induction after CK2 inhibition in LNCaP cells. For comparison we induced ER stress by treating the cells with thapsigargin and analysed the requirements of the promoter elements under the same experimental conditions as described for CK2 inhibition (Fig. 2C). The luciferase activity measured for the wt −442 CHOP promoter construct was abolished when the ERSE or the C/EBP-ATF composite site is inactive. The mutation in those elements reduces the activity for more than 75%. The AP-1 binding site in contrast does not seem to play any role in the CHOP induction after thapsigargin treatment as the luciferase activity was not altered by the mutation. We found that the C/EBP-ATF composite site is crucial for CHOP induction after CK2 inhibition and also after thapsigargin treatment of the cells. Thus, it seems that CK2 inhibition induces CHOP partially by affecting the ER stress signalling pathway in LNCaP cells. 3.3. Dominant negative mutant of ATF4 blocks CHOP induction after CK2 inhibition In the next step we wanted to analyse the up-stream element which might be responsible for the induction of CHOP. ATF4 is a member of the ER stress response signalling pathways that activates CHOP by binding to the C/EBP-ATF composite site within the AARE-
Fig. 2. Identification of the active element in the CHOP promoter during CK2 inhibition. (A) Reporter constructs used for the identification of the active element [19]. (B and C) Cells were transfected with the reporter gene vectors for 24 h and then treated with 50 μM TBB (B) or 1 μM thapsigargin (TG, C) or as a control with DMSO for another 24 h. The promoter activity was detected with a luciferase assay from Promega, normalized to β-galactosidase activity and presented as fold induction of control.
element [22]. Therefore, it was an obvious question whether ATF4 is implicated in the activation of CHOP after CK2 inhibition. We transfected the wt −442 CHOP promoter construct or a construct containing a duplicate of the C/EBP-ATF composite site of the CHOP promoter in front of a luciferase gene as a reporter into LNCaP cells (Fig. 3A). In addition cells were co-transfected with a dominant negative mutant of ATF4 (dn) or as a control with an empty vector. Furthermore, we treated the cells with the CK2 inhibitor TBB for 24 h or with the solvent as a control. The activity was presented as fold induction compared to the solvent control. As shown in Fig. 3B, CK2 inhibition resulted in a strong stimulation of the AARE promoter construct as well as of the wt − 442 CHOP promoter construct. The AARE promoter construct shows only half of the activity in the presence of the dominant negative mutant of ATF4. The activation of the wt − 442 CHOP promoter construct was reduced to nearly control level when the dominant negative mutant of ATF4 was cotransfected. These results indicate that ATF4 indeed seems to be at least one of the up-stream factors that is responsible for CHOP induction. In order to support the findings that ATF4 is indeed responsible for the induction of these two promoter constructs, we transfected the LNCaP cells with one of the reporter constructs and an ATF4 expression vector. Half of the cell population was in addition transfected with the dominant negative mutant of ATF4 (dn) or in the absence of this mutant. As a control, cells were only transfected with an empty vector. The reporter activity was measured after 48 h of transfection. As shown in Fig. 3C, the expression of ATF4 induced both the
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Fig. 4. Induction of ATF4 by CK2 inhibition. (A) Whole cell extracts of treated cells (50 μM TBB or 1 μM thapsigargin (TG), or DMSO as a control, 24 h) were separated on a 10% SDS-polyacrylamide gel and transferred onto a PVDF membrane. ATF4 and GAPDH were detected with polyclonal rabbit antibodies by enhanced chemiluminescence. (B) The reporter gene vector for the ATF4 promoter activity was transfected for 24 h. Cells were treated for another 24 h with 50 μM TBB, 1 μM TG or DMSO as a control and the activity was detected with a luciferase assay from Promega and presented as fold induction of control.
Fig. 3. Identification of ATF4 as the inducing transcription factor. (A) Reporter constructs used for the identification of the inducing transcription factor. The reporter gene constructs were transfected for 24 h either together with the expression vector for the dominant negative mutant of ATF4 (+dn) or an empty vector as a control (−dn). (B) After transfection, the cells were treated with 50 μM TBB or solvent as a control for 24 h. The promoter activity was detected with a luciferase assay from Promega and presented as fold induction of control. (C) Additionally to the reporter gene constructs and the expression vector for the dominant negative mutant of ATF4 (+dn) or an empty vector as a control (−dn), we transfected an expression vector for ATF4 wt into the LNCaP cells. Cells transfected with empty vectors served as a control. The promoter activity was detected with a luciferase assay from Promega and presented as fold induction of control.
AARE promoter construct as well as the wt −442 CHOP promoter construct. In the presence of the dominant negative ATF4 mutant, transcription was considerably reduced. For the AARE promoter construct, we detected a reduction of more than 50%. The wt −442 CHOP promoter construct showed no activity anymore when ATF4 is sequestered by its dominant negative mutant. Since this result is quite similar to the one that was obtained after CK2 inhibition by TBB, we conclude that ATF4 is indeed implicated in the CHOP induction after CK2 inhibition. 3.4. CK2 inhibition leads to increased ATF4 protein level As it is known that not only is ATF4 regulated transcriptionally but also on the level of translation or posttranslationally [23–25], we next determined the ATF4 protein level after CK2 inhibition and compared the level to thapsigargin treated or completely untreated cells. As shown in Fig. 4A, there is an elevated level of ATF4 in cells treated with the CK2 inhibitor TBB and also after thapsigargin treatment. To further analyse these findings, we asked whether the transcription of the ATF4 gene was influenced by CK2 inhibition. Therefore, we transfected a reporter construct with the ATF4 promoter into LNCaP cells for 24 h. Then, cells were treated with the CK2 inhibitor TBB, with thapsigargin or with the solvent as a control and the reporter activity was measured after 24 h of treatment. The results of the reporter assay shown in Fig. 4B illustrate that there was no increase in ATF4 transcription after CK2 inhibition, whereas, thapsigargin treatment of cells led to a strong transcriptional up-regulation of ATF4. Thus, we have to conclude that the up-regulation of ATF4 after CK2 inhibition
in contrast to its up-regulation by ER stress is a posttranscriptional event. 4. Discussion It is known for quite some time that human prostate cancer cells express high levels and activity of protein kinase CK2 [26]. Inhibition of the kinase activity of CK2 results in growth arrest and apoptosis [27–30]. Using different CK2 inhibitors, we observed that the hormone sensitive LNCaP cells always respond with apoptosis to CK2 inhibition. In contrast, hormone refractory PC-3 cells do not show any loss in cell viability although the intrinsic apoptotic pathway is induced [5,6]. One major difference between the two types of prostate cancer was the induction of CHOP which was found in LNCaP cells but not in PC-3 cells [6]. CHOP encodes a nuclear, proapoptotic bzip transcription factor of the CCAAT/enhancer binding protein (C/EBP) family which is often induced by cellular stress [7,31,32]. Furthermore, expression of CHOP in hormone refractory cancer cells PC-3 led to the induction of apoptosis supporting the idea that CHOP is crucial for apoptosis induction after CK2 inhibition. Many different cellular stresses, for example, impaired redox balances, amino acid depletion and deficiencies in proteasome function can lead to ER stress. This ER stress results in disturbance of protein quality control which triggers a cellular response called unfolded protein response (UPR). There are three different signalling pathways which are activated by ER stress. One pathway is the PERK/eIF2α/ATF4 cascade, the second one is the IRE1/INK cascade and the third one is the ATF6 pathway. Since all three pathways converge on CHOP it was an interesting question which might be regulated by CK2. One approach to elucidate the CK2 contribution to these pathways was the analysis of the activation of different promotor elements within the CHOP promoter. It was shown that CHOP expression after various kinds of stress is regulated through promoter sequences between nucleotide position − 954 and + 91 of the 5’ upstream region of the CHOP gene [11,33]. Detailed analysis of the promoter region revealed that the region between nucleotide −442 and −75 contains three different elements such as the amino acid response element (AARE) with the C/EBP-ATF composite site, the AP-1 binding site as well as the ER stress element
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(ERSE) [19]. The AP-1 binding site is activated by oxidative stress. The AARE responds to amino acid limitation as well as to ER stress. During ER stress the ERSE is also activated [34]. We used different promoter constructs to analyse which element is essential for CHOP induction by CK2 inhibition and which signalling pathway could be involved in this induction. Therefore, we compared a totally functional CHOP promoter construct (−442 wt) with constructs which were inactive for one binding site. We found that CK2 inhibition led to an activation of the wt −442 CHOP promoter construct containing the above mentioned elements as well as the promoter element with the mutation in the AP-1 binding site. This indicates that the AP-1 element is not required for CHOP induction by CK2 inhibition. Thus, we can mainly exclude oxidative stress as a trigger for the CHOP induction after CK2 inhibition by TBB. That fits with some former results where reactive oxygen species after CK2 inhibition by TBB were not detected [35]. As we found the CHOP induction only slightly reduced by the inactivation of the ERSE, we conclude that this element is only partially crucial for CHOP induction by CK2 inhibition which is in contrast to ER stress by thapsigargin treatment. Mutation and thereby inactivation of the AARE, however, led to the strongest reduction of the CHOP promoter activity after CK2 inhibition. These results demonstrate that this element is mainly implicated in the regulation of the CHOP expression after CK2 inhibition. As it is known that the AARE is also implicated in the ER stress signalling, CK2 inhibition by TBB affects these signalling pathways although different from the ER stress signalling induced by thapsigargin. The AARE contains a C/EBP-ATF composite site that is composed of a half-site for the C/EBP family of transcription factors and a half-site for the ATF family of transcription factors [22]. One of the transcription factors of the ATF family that triggers increased transcription by binding to this element is ATF4 [22]. Using wild-type ATF4 and a dominant negative mutant of ATF4 [36], we showed here that ATF4 is indeed responsible for the regulation of the AARE promoter construct as well as of the wt −442 CHOP promoter construct. This is because the dominant negative mutant of ATF4 blocks the activation of both promoter constructs after CK2 inhibition by TBB or transfection with ATF4 wt. ATF4 is regulated transcriptionally as well as posttranscriptionally. We demonstrated that CK2 inhibition does not induce ATF4 transcription; however, we found an elevated level of the ATF4 protein after CK2 inhibition. It is known that ATF4 is a phosphoprotein and that the phosphorylation of ATF4 is crucial for its stability [37,38]. Therefore, the up-regulation of ATF4 is probably due to stabilization as a phosphorylation by CK2 and therefore the degradation of ATF4 is inhibited. In contrast, the ER stress inducing agent thapsigargin led to an induction of transcription of the ATF4 promoter and therefore to an increased ATF4 protein level. In a very recent paper Manni et al. reported that CK2 protects multiple myeloma cells from ER stress induced apoptosis. They also found the activation of the UPR and an induction of the signalling cascade including PERK and eIF2α [39]. These two kinases are upstream factors of ATF4 and therefore these data support our finding about a contribution of ATF4 to this signalling pathway. They also presented data on the contribution of IRE1α, Bip and HSP90 to the signalling converging on CHOP [39]. Our present results demonstrate that inhibition of CK2 by TBB induces an up-regulation of the protein level of ATF4. ATF4 then transactivates CHOP expression by binding mainly to the C/EBP-ATF composite site within the AARE triggering apoptosis at least in hormone sensitive prostate cancer cells. Our present data together with the data of Manni et al. clearly demonstrate an active and central role of CK2 in cellular-stress induced signalling pathways. 5. Concluding remarks Inhibition of the kinase activity of CK2 leads to apoptosis in hormone sensitive prostate cancer cells. Here we showed for the first time that inhibition of CK2 activates the amino acid induced stress pathway in these prostate cancer cells. Furthermore, up-regulation of the expression of the transcription factor ATF4 identified ATF4 as
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a new factor in the signalling pathway after CK2 inhibition. The identification of CK2 regulated factors in the cellular stress response pathway improved the understanding of the role of CK2 as an anti-cancer drug target at least in prostate cancer cells. Acknowledgement We thank A. B. Vandraager, University of Utrecht, The Netherlands, for the gift of the reporter gene construct wt −442 CHOP and the constructs with the mutations in the core sequences of the regulatory elements. Furthermore, we thank A. Bruhat, INRA de Theix, Saint Genès Champanelle, France, for kindly providing the AARE construct and we thank J. Alam, New Orleans, USA, for the kind gift of the expression vector for the ATF4 dominant-negative mutant. We also thank Nathaniel Saidu for carefully reading the manuscript. References [1] B. Guerra, O.G. Issinger, Electrophoresis 20 (1999) 391–408. [2] K.A. Ahmad, G. Wang, G. Unger, J. Slaton, K. Ahmed, Advances in Enzyme Regulation 48 (2008) 179–187. [3] G. Wang, K.A. Ahmad, G. Unger, J.W. Slaton, K. Ahmed, Journal of Cellular Biochemistry 99 (2006) 382–391. [4] H.M. Wang, A. Davis, S.H. Yu, K. Ahmed, Molecular and Cellular Biochemistry 227 (2001) 167–174. [5] A. Hessenauer, M. Montenarh, C. Götz, International Journal of Oncology 22 (2003) 1263–1270. [6] A. Hessenauer, C.C. Schneider, C. Götz, M. Montenarh, Cellular Signalling 23 (2011) 145–151. [7] H. Zinszner, M. Kuroda, X. Wang, N. Batchvarova, R.T. Lightfoot, H. Remotti, J.L. Stevens, D. Ron, Genes & Development 12 (1998) 982–995. [8] I. Tabas, D. Ron, Nature Cell Biology 13 (2011) 184–190. [9] J. Tsukada, Y. Yoshida, Y. Kominato, P.E. Auron, Cytokine 54 (2011) 6–19. [10] K.D. McCullough, J.L. Martindale, L.O. Klotz, T.Y. Aw, N.J. Holbrook, Molecular and Cellular Biology 21 (2001) 1249–1259. [11] M. Ubeda, J.F. Habener, Nucleic Acids Research 28 (12-15-2000) 4987–4997. [12] H. Yoshida, T. Okada, K. Haze, H. Yanagi, T. Yura, M. Negishi, K. Mori, Molecular and Cellular Biology 20 (2000) 6755–6767. [13] A. Bruhat, C. Jousse, V. Carraro, A.M. Reimold, M. Ferrara, P. Fafournoux, Molecular and Cellular Biology 20 (2000) 7192–7204. [14] Y. Ma, J.W. Brewer, J.A. Diehl, L.M. Hendershot, Journal of Molecular Biology 318 (2002) 1351–1365. [15] K.Z. Guyton, Q. Xu, N.J. Holbrook, Biochemical Journal 314 (Pt 2) (1996) 547–554. [16] J.S. Horoszewicz, S.S. Leong, E. Kawinski, J.P. Karr, H. Rosenthal, T.M. Chu, E.A. Mirand, G.P. Murphy, Cancer Research 43 (1983) 1809–1818. [17] U.K. Laemmli, Nature 227 (1970) 680–682. [18] T. Maniatis, E.F. Fritsch, J. Sambrook, Sambrook, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1982, book 3, 16.66–16.67. [19] M.H. van der Sanden, H. Meems, M. Houweling, J.B. Helms, A.B. Vaandrager, Journal of Biological Chemistry 279 (2004) 52007–52015. [20] C.H. He, P. Gong, B. Hu, D. Stewart, M.E. Choi, A.M. Choi, J. Alam, Journal of Biological Chemistry 276 (2001) 20858–20865. [21] F. Qi, M. Carbone, H. Yang, G. Gaudino, Expert Review of Respiratory Medicine 5 (2011) 683–697. [22] T.W. Fawcett, J.L. Martindale, K.Z. Guyton, T. Hai, N.J. Holbrook, Biochemical Journal 339 (1999) 135–141. [23] F. Siu, P.J. Bain, R. LeBlanc-Chaffin, H. Chen, M.S. Kilberg, Journal of Biological Chemistry 277 (2002) 24120–24127. [24] H.P. Harding, I. Novoa, Y. Zhang, H. Zeng, R. Wek, M. Schapira, D. Ron, Molecular Cell 6 (2000) 1099–1108. [25] K.M. Vattem, R.C. Wek, Proceedings of the National Academy of Sciences of the United States of America 101 (2004) 11269–11274. [26] S. Tawfic, S. Yu, H. Wang, R. Faust, A. Davis, K. Ahmed, Histology and Histopathology 16 (2001) 573–582. [27] F. Pierre, P.C. Chua, S.E. O'Brien, A. Siddiqui-Jain, P. Bourbon, M. Haddach, J. Michaux, J. Nagasawa, M.K. Schwaebe, E. Stefan, A. Vialettes, J.P. Whitten, T.K. Chen, L. Darjania, R. Stansfield, J. Bliesath, D. Drygin, C. Ho, M. Omori, C. Proffitt, N. Streiner, W.G. Rice, D.M. Ryckman, K. Anderes, Molecular and Cellular Biochemistry 356 (2011) 37–43. [28] G. Wang, G. Unger, K.A. Ahmad, J.W. Slaton, K. Ahmed, Molecular and Cellular Biochemistry 274 (2005) 77–84. [29] J.H. Trembley, Z. Chen, G. Unger, J. Slaton, B.T. Kren, Van WaesC. , K. Ahmed, Biofactors 36 (2010) 187–195. [30] N.A. St-Denis, D.W. Litchfield, Cellular and Molecular Life Sciences 66 (2009) 1817–1829. [31] D. Ron, J.F. Habener, Genes & Development 6 (1992) 439–453. [32] X.Z. Wang, M. Kuroda, J. Sok, N. Batchvarova, R. Kimmel, P. Chung, H. Zinszner, D. Ron, EMBO Journal 17 (1998) 3619–3630. [33] C. Jousse, A. Bruhat, H.P. Harding, M. Ferrara, D. Ron, P. Fafournoux, FEBS Letters 448 (1999) 211–216. [34] S.C. Kwok, I. Daskal, Molecular and Cellular Biochemistry 319 (2008) 203–208.
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C.C. Schneider et al. / Cellular Signalling 24 (2012) 1797–1802
[35] C.C. Schneider, A. Hessenauer, C. Götz, M. Montenarh, Oncology Reports 21 (2009) 1593–1597. [36] Y. Makino, K. Omichi, Bioscience, Biotechnology, and Biochemistry 70 (2006) 907–915. [37] C.L. Frank, X. Ge, Z. Xie, Y. Zhou, L.H. Tsai, Journal of Biological Chemistry 285 (2010) 33324–33337.
[38] I. Lassot, E. Segeral, C. Berlioz-Torrent, H. Durand, L. Groussin, T. Hai, R. Benarous, F. Margottin-Goguet, Molecular and Cellular Biology 21 (2001) 2192–2202. [39] S. Manni, A. Brancalion, L.Q. Tubi, A. Colpo, L. Pavan, A. Cabrelle, E. Ave, F. Zaffino, G. Di Maira, M. Ruzzene, F. Adami, R. Zambello, M.R. Pitari, P. Tassone, L.A. Pinna, C. Gurrieri, G. Semenzato, F. Piazza, Clinical Cancer Research 18 (2012) 1888–1900.