BBAMCR-17583; No. of pages: 10; 4C: 4, 5, 7, 9 Biochimica et Biophysica Acta xxx (2015) xxx–xxx

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Lisa Schwind a,1, Nadine Wilhelm a,b,1, Sabine Kartarius a, Mathias Montenarh a, Erwin Gorjup b, Claudia Götz a,⁎

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Article history: Received 13 November 2014 Received in revised form 21 May 2015 Accepted 23 May 2015 Available online xxxx

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Keywords: Stem cells Protein kinase CK2 Adipogenic differentiation Kinase inhibitors

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Medizinische Biochemie und Molekularbiologie, Universität des Saarlandes, Geb. 44, D-66421 Homburg, Germany Fraunhofer-Institut für Biomedizinische Technik IBMT, Ensheimer, Strasse 48, D-66386 St. Ingbert, Germany

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CK2 is a serine/threonine protein kinase, which is so important for many aspects of cellular regulation that life without CK2 is impossible. Here, we analysed CK2 during adipogenic differentiation of human mesenchymal stem cells (hMSCs). With progress of the differentiation CK2 protein level and the kinase activity decreased. Whereas CK2α remained in the nucleus during differentiation, the localization of CK2β showed a dynamic shuttling in the course of differentiation. Over the last years a large number of inhibitors of CK2 kinase activity were generated with the idea to use them in cancer therapy. Our results show that two highly specific inhibitors of CK2, CX-4945 and quinalizarin, reduced its kinase activity in proliferating hMSC with a similar efficiency. CK2 inhibition by quinalizarin resulted in nearly complete inhibition of differentiation whereas, in the presence of CX-4945, differentiation proceeded similar to the controls. In this case, differentiation was accompanied by the loss of CX-4945 inhibitory function. By analysing the subcellular localization of PPARγ2, we found a shift from a nuclear localization at the beginning of differentiation to a more cytoplasmic localization in the presence of quinalizarin. Our data further show for the first time that a certain level of CK2 kinase activity is required for adipogenic stem cell differentiation and that inhibition of CK2 resulted in an altered localization of PPARγ2, an early regulator of differentiation. © 2015 Published by Elsevier B.V.

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Protein kinase CK2 is necessary for the adipogenic differentiation of human mesenchymal stem cells

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1. Introduction

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Adipogenesis denotes the development of differentiated adipocytes from multipotent mesenchymal precursor cells. The multipotent mesenchymal stem cells (MSCs) are adult stem cells with a fibroblastoid shape and a high ability for self-renewal [11]. They can be isolated – among others – from adipose tissue and bone marrow. They are characterized by different surface markers, i.e. they lack the haematopoietic markers CD23 and CD45, but express high levels of CD29, CD44, CD73, CD90, CD105 and CD106 [49]. By diverse extracellular signals MSCs are prompted to differentiate into several lineages like osteoblasts, chondrocytes, myoblasts or adipocytes in vivo and in vitro [60]. Many findings concerning the events during differentiation of stem cells were obtained from model cell lines like the pluripotent stem cell line C3H10T/1/2 [51] or the preadipocyte cell line 3T3-L1 [20,21] which have been proven reliable models for the processes occurring during differentiation. During differentiation hMSCs undergo defined phases which begin with mitotic clonal expansion, commitment to a certain

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⁎ Corresponding author at: Claudia Götz, Medizinische Biochemie und Molekularbiologie, Universität des Saarlandes, Gebäude 44, 66421 Homburg, Germany. E-mail address: [email protected] (C. Götz). 1 Lisa Schwind and Nadine Wilhelm contributed equally to this work.

lineage, lineage progression, terminal differentiation, and finally maturation [6,7]. Lineage determination seems to be regulated by a large network of extracellular signalling factors which exert an impact on the promoters of lineage specific transcription factors. Balancing these different signalling molecules and pathways determines the fate of the MSC ([66] and literature therein). In the late phase of adipogenic differentiation adipocyte specific proteins like perilipin, lipoprotein lipase, glycerol phosphate dehydrogenase, leptin and adiponectin are expressed which support the characteristics of an adipocyte phenotype [44]. The activity of several signalling molecules and transcription factors during differentiation is controlled by phosphorylation [27,33,68]. Also the commitment of human mesenchymal stem cells to an osteogenic or adipogenic lineage is regulated by protein kinases [26,28,34]. One of the kinases which might also be implied in the regulation of differentiation is protein kinase CK2. CK2 is one of the most pleiotropic protein kinases with hundreds of substrates already known [37,52]. It is a ubiquitous heterotetrameric serine/threonine kinase, consisting of two catalytic α- or α′-subunits and two non-catalytic β-subunits, which is deeply implicated in a multitude of cellular processes including embryonal development and differentiation. A lack of the catalytic α- or the regulatory β-subunit is not compatible with life [5,35]. During early embryogenesis of the mouse, catalytic as well as the non-catalytic subunits of CK2 are particularly expressed in neuronal tissues, in later stages CK2 is found everywhere in the body [40]. Similar

http://dx.doi.org/10.1016/j.bbamcr.2015.05.023 0167-4889/© 2015 Published by Elsevier B.V.

Please cite this article as: L. Schwind, et al., Protein kinase CK2 is necessary for the adipogenic differentiation of human mesenchymal stem cells, Biochim. Biophys. Acta (2015), http://dx.doi.org/10.1016/j.bbamcr.2015.05.023

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Human mesenchymal stem cells (hMSCs) were isolated from bone marrow of the caput femoris from patients undergoing hip replacement surgery. Briefly, bone fragments were minced and isolated cells were pooled in α-MEM. Mononuclear cells (MNC) were extracted by ficoll density gradient centrifugation at 350 ×g for 30 min. Collected cells were seeded in complete medium (α-MEM, 15% foetal bovine serum (FBS) and 100 U/ml penicillin and 100 μg/ml streptomycin) and cultured in a humidified atmosphere and 5% CO2 at 37 °C. The medium was changed twice a week. Before reaching confluence, cells were harvested with trypsin/EDTA and seeded at 1 × 103 cells/cm2 for further expansion. Prior to their usage the stem cell potential of hMSC was verified by the expression of cell surface markers CD29, CD44, CD73, CD90, CD105, CD106 and HLA-ABC as well as absence of CD34, CD45, CD133 and HLA-DR and their ability to differentiate adipogenically and osteogenically.

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2.3. Staining of lipid droplets in hMSC

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After 3 weeks of differentiation cells were washed with PBS, fixed in 3.7% formaldehyde in PBS for 15 min at room temperature and then washed again. Cells were permeabilized with 0.2% Triton X-100, 5% bovine serum albumin in PBS for 15 min on ice. Then, cells were washed twice and incubated with the lipid staining dye Bodipy® (493/503) (Invitrogen, Karlsruhe, Germany) (50 μl/ coverslip, 30 μg Bodipy®/ml PBS) for 1 h at room temperature in a humidified chamber in the dark. To stain the nuclei, cells were incubated with 4′,6-diamidino-2phenylindole (DAPI) (50 μl, 5 ng/ml) at 37 °C for 15 min. Finally, cells were washed again and analysed under a fluorescence microscope (excitation wave length: Bodipy®: 488 nm, DAPI: 340 nm). Alternatively, lipids were visualized with OilRedO (Sigma Aldrich, Munich, Germany). After removing the medium and washing with PBS, cells were fixed in 10% formaldehyde in PBS for 30 min at room temperature and then washed twice with deionized H2O. Cells were incubated for 3 min with 60% isopropanol. OilRedO was dissolved in isopropanol (0.3 g/100 ml). A fresh 60% staining solution of OilRedO was prepared with deionized H2O, incubated for 10 min at room temperature and subsequently filtered through a folded filter (3MM, Whatman, United Kingdom). Cells were incubated with the staining solution for 5 min at room temperature. After incubation, the OilRedO solution was removed and cells were washed three times with distilled water. The staining of the lipid droplets was visualized by phase contrast microscopy.

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2.4. Immunofluorescence analysis

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For immunofluorescence analysis hMSCs were seeded in 8 well Permanox slides (Nalge Nunc International Corp., Naperville, USA) at a density of 12,000 cells per well and differentiation was started in the presence or absence of 30 μM quinalizarin or 20 μM CX-4945 when cells had reached confluence. Every third day immunofluorescence analysis was performed as described [15]. For the identification of CK2α we used the mouse monoclonal antibody 1A5 [56], for the identification of CK2β the polyclonal rabbit serum #32 [16]. The PPARγ2 isoform was detected with the anti-PPARγ2 antibody ab45036 (Abcam, Cambridge, UK), the marker perilipin was identified with the specific antibody D418 (Cell Signaling Technology, Schwalbach, Germany ). All primary antibodies were incubated overnight.

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2.5. Extraction of cells

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2.1. Isolation of human mesenchymal stem cells

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Characterized hMSCs were cultivated in complete medium and kept at a maximum confluence of 70–80%. For adipogenic differentiation cells were seeded into six-well plates or in 60 mm culture dishes at a density of 1.5 × 104 cells/cm2. When cells reached confluence the culture medium was replaced by the differentiation mix (α-MEM, 10% heat-inactivated FCS, 500 μM isobutylmethylxanthine (IBMX), 100 nM dexamethasone, 200 μM indomethacin and 100 ng/ml insulin) (corresponds to day 0 of the differentiation). Differentiation medium was replaced twice a week. The CK2 inhibitors CX-4945 (Selleckchem, Munich, Germany) or quinalizarin (1,2,5,8-tetrahydroxyanthraquinone) (Labotest OHG, Niederschöna, Germany) were dissolved in dimethyl sulfoxide (DMSO) to a 20 mM stock solution, which was used to treat the cells with a final concentration of 20 or 30 μM, unless otherwise stated. The inhibitors were added with every change of the differentiation medium. The same volume of the solvent DMSO was used in control experiments without CK2 inhibitors.

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2. Material and methods

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observations have been made in chicken [36] and in the nematode Caenorhabditis elegans [23]. The enhanced expression goes along with an enhancement of the CK2 activity which reaches a maximum at day 11 during mouse embryogenesis and is then subsequently down-regulated [55]. Changes in activity depending on the embryonic stage were confirmed for rat and for C. elegans [22,46]. CK2β is discussed as a positive regulator for the proliferation of neuronal progenitor cells and their multipotency [75]. Knocking out the gene for the regulatory β-subunit in mice leads to a diminished cell proliferation and the development of the murine embryos stops at the blastocyst stage with the resorption of the embryo. CK2β−/− blastocysts are not able to generate an inner cell mass [5]. Mouse embryos with a lack of CK2β in neuronal stem cells show a disturbed cell proliferation and severe defects in brain development [24]. When knocking out the α-subunit of CK2, mouse embryos die during embryogenesis and exhibit structural injuries in the heart and neural tube [12,35]. The lack of the catalytic α′-subunit is compatible with life; however, it is detrimental for spermatogenesis and male α′-knock-out mice are infertile [14,73]. In a number of different genetic experiments it has been demonstrated that mice with combined loss of CK2α and CK2α′ are viable as long as one of the two CK2α alleles is present [31]. Furthermore, these experiments have demonstrated that a combined loss of one CK2α allele or both CK2α′ alleles leads to abnormalities in growth and development of the offspring. In particular, CK2α+/− CK2α′−/− mice differ in weight but not in size compared to their wild-type litter mates. In fact, knock-out mice were found to have about 50% of the body fat compared to mice of other genotypes. There are some observations about a distinct role of CK2 upon signalling during differentiation in several stem cell models for haematopoietic [8,13,65], osteogenic [3,4] or neuronal differentiation [19,70]. In these publications there are examples for a positive as well as a negative role for CK2 in differentiation. There are only few indications that CK2 might be employed in the differentiation of established stem cell or preadipocyte lines to adipocytes [41,72]. In the preadipocyte line 3T3-L1 CK2 is necessary for the early stages of differentiation. Upon inhibition of CK2 preadipocytes fail to differentiate into mature adipocytes [72]. In later phases the expression as well as the activity of CK2 has to be down-regulated to enable a successful differentiation. Over the last 8 years the development of a great number of inhibitors of the kinase activity of CK2 with an increasing specificity has provided new tools for studying the role of CK2 for many biological processes [10,50,53]. Here we used two highly specific CK2 inhibitors – CX4945 [47] and quinalizarin [9] – for the investigation of the fate of human mesenchymal stem cells (hMSCs) with respect to adipogenic differentiation.

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Cells were washed twice with cold PBS, scraped off the plate, 197 centrifuged (250 ×g, 4 °C) and the pellet was extracted with RIPA- 198

Please cite this article as: L. Schwind, et al., Protein kinase CK2 is necessary for the adipogenic differentiation of human mesenchymal stem cells, Biochim. Biophys. Acta (2015), http://dx.doi.org/10.1016/j.bbamcr.2015.05.023

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2.7. CK2 in vitro phosphorylation assay

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To determine the kinase activity of CK2, cells were lysed and the extracts were used in a kinase filter assay. In this assay we measured the incorporation rate of [32P]phosphate into the synthetic CK2 specific substrate peptide with the sequence RRRDDDSDDD [29]. Twenty microlitre kinase buffer (50 mM Tris–HCl, pH 7.5, 100 mM NaCl, 10 mM MgCl2, 1 mM DTT) containing 20 μg proteins was mixed with 30 μl CK2 mix (25 mM Tris–HCl, pH 8.5, 150 mM NaCl, 5 mM MgCl2, 1 mM dithiothreitol (DTT), 50 μM ATP, 0.19 mM substrate peptide, containing 10 μCi/500 μl [32P]γATP) and incubated at 37 °C for 10 min. The mixture was spotted onto a P81 ion exchange paper. The paper was washed with 85 mM H3PO4 for three times. After treatment with ethanol the paper was dried and the Čerenkov-radiation was determined in a scintillation counter.

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2.8. MTT viability assay

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Cell proliferation and viability was determined using a colorimetric MTT-based assay (MTT: 3-[4, 5-dimethylthiazol-2-yl] 2, 5-diphenyl tetrazolium bromide, Sigma-Aldrich, Deisenhofen, Germany) as described in [54].

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3.1. CK2 expression, activity and localization during the adipogenic differentiation of human mesenchymal stem cells

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Proteins were analysed by SDS-polyacrylamide gel electrophoresis according to the procedure of Laemmli [30]. 50 μg extracted proteins were used for the analysis. Proteins dissolved in sample buffer (130 mM Tris–HCl, pH 6.8, 0.02% bromophenol blue (w/v), 10% β-mercaptoethanol, 20% glycerol (v/v), and 4% SDS) were separated on a 12.5% SDS-polyacrylamide gel in electrophoresis buffer (25 mM Tris–HCl, pH 8.8, 192 mM glycine, and 3.5 mM SDS) and transferred onto a PVDF membrane (Roche Diagnostics, Mannheim, Germany) in a buffer containing 20 mM Tris–HCl, 150 mM glycine, pH 8.3. The membrane was blocked with 5% dry milk in PBS-Tween20 for 1 h and then incubated with appropriate primary antibodies diluted in PBS– Tween20 with 1% dry milk (incubation buffer). The membrane was washed twice with the incubation buffer and incubated with the peroxidase-coupled secondary antibody (anti-rabbit 1:30,000 or antimouse 1:10,000 in incubation buffer) for 1 h. Signals were visualized by the Lumilight system of Roche Diagnostics (Mannheim, Germany). For the detection of protein kinase CK2 we used the mouse monoclonal antibodies 1A5 (α-subunit) [56] and 6D5 (β-subunit) [43]. GAPDH was detected with the specific antibody FL-335 (Santa Cruz Biotechnology, Heidelberg, Germany) and perilipin with D418 (Cell Signaling Technology, Schwalbach, Germany). All antibodies were diluted 1:1000 in the incubation buffer and incubated with the membrane for 1 h at room temperature or overnight at 4 °C.

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2.6. SDS-polyacrylamide gel electrophoresis and Western blot analysis

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It is well known that the CK2 protein level, as well as the protein kinase activity is elevated in cancer cells compared to normal cells. Further, inhibition of the kinase activity in cancer cells results in apoptosis

whereas normal cells survive. These observations led to the conclusion that CK2 has anti-apoptotic properties. There are some indications, that CK2 also plays a role in the development of organs and in differentiation. However, nothing is known about its role in the differentiation of stem cells to adipocytes. As adult stem cells like hMSCs play an important role in tissue maintenance or regeneration processes of injured tissue we investigated the role of CK2 in proliferating and differentiating hMSCs. Adipogenic differentiation was induced in hMSCs for 21 days while their morphology and lipid storage were verified by time lapse imaging. During hMSC differentiation cell morphology changed from a fibroblastlike to a rounded shape and lipid droplet formation appeared from day 6 on and increased until day 21 (Fig. 1A). We wondered whether this differentiation process might be accompanied by alteration in the expression of CK2 or/and the protein kinase activity. Therefore, proteins were extracted and subsequently analysed by Western blot analysis for the detection of the catalytic α- and regulatory β-subunits as well as an in vitro phosphorylation assay with a CK2-specific substrate peptide at certain time points. We observed a decrease of the expression of CK2α (Fig. 1B). The expression of the regulatory β-subunit varied in the course of differentiation; however, at the end the expression of the protein was down-regulated to about 80% of the control. Thus, the expression of both subunits is seemingly individually regulated during the differentiation process. The reduction in the expression of the CK2α subunit went along with a reduction in kinase activity (Fig. 1C). By the end of differentiation we noticed only about half of the initial CK2 activity. Beside the detection of lipid droplets in situ, we also analysed a marker protein for differentiation by Western blot, namely perilipin, a protein surrounding the lipid droplets. Perilipin was first detectable at day 12 and its expression increased until the end of differentiation (Fig. 1B). In a next experiment we analysed the subcellular localization of protein kinase CK2. The results of these immunofluorescence studies are shown in Fig. 2. We observed a predominant nuclear staining for the catalytic α-subunit of CK2. Beginning from day 12 the localization of the α-subunit became more and more cytoplasmic. The regulatory β-subunit initially showed an overall staining which became more and more nuclear during differentiation with the most intense nuclear staining at days 6 and 9. Afterwards, the β-subunit was redistributed again in the cytoplasm and nucleus. Thus, CK2, especially the noncatalytic β-subunit showed a dynamic shuttling during the adipogenic differentiation of hMSCs. We also performed an immunofluorescence analysis of the late marker protein perilipin (Fig. 2). Single cells expressed this lipid droplet surrounding protein already at day 6, and its expression was steadily increasing till the end of differentiation.

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buffer (50 mM Tris–HCl, 160 mM NaCl, 0.5% sodium desoxycholate, 1% Triton X-100, 0.1% SDS, pH 8.0) in the presence of protease inhibitors (CompleteTM, Roche Diagnostics, Mannheim, Germany). After 30 min on ice cells were sonicated (3× 30 s). After lysing, the cell debris was removed by centrifugation at 13,000 ×g. The protein content was determined with the BioRad reagent dye (BioRad, Munich, Germany). Protein extracts were immediately used for Western blot analysis or for in vitro phosphorylation.

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3.2. CK2 inhibitors CX-4945 and quinalizarin reduce CK2 activity in 301 non-differentiating human mesenchymal stem cells 302 Over the last few years CK2 has been discovered as therapeutic target for the treatment of cancer and several CK2 inhibitors have been developed which are tested in ongoing clinical trials [61,62]. Although some of the inhibitors – especially CX-4945 – show high bio-availability, good pharmacokinetics and non-cardiac toxicity [64], no one has yet tested the impact of CK2 inhibitors on the fate of adult stem cells. In order to study the inhibition of CK2 in human stem cells, we chose the well-accepted CK2 inhibitor quinalizarin [9] and as a second inhibitor CX-4945, which is currently investigated in clinical phase II trials as a tumour therapeutic drug [61]. As the inhibition of such an essential kinase like CK2 might be fatal for the viability of cells, we first tested different concentrations of the inhibitors in a viability assay. We treated proliferating hMSCs with different concentrations of both inhibitors which formerly have been proven suitable for the treatment of established cell lines in our laboratory. After 24, 48 and 72 hour incubation with the inhibitor we performed an MTT assay where the activity

Please cite this article as: L. Schwind, et al., Protein kinase CK2 is necessary for the adipogenic differentiation of human mesenchymal stem cells, Biochim. Biophys. Acta (2015), http://dx.doi.org/10.1016/j.bbamcr.2015.05.023

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Fig. 1. Lipid droplet formation, CK2 expression and CK2 activity in differentiating hMSCs. Time-lapse images of hMSCs are shown after induction of adipogenic differentiation (A). CK2α- and β-subunits and perilipin in differentiating hMSCs shown by Western blot analysis with specific antibodies over time. GAPDH was used as a loading control. One representative of three Western blots is shown. Bands are quantified against GAPDH (B). CK2 activity [%] during adipogenic differentiation of hMSC over time shown by the incorporation of [32P]phosphate into the CK2 specific peptide RRRDDDSDDD of at least three independent experiments with two technical replicates each (C).

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of mitochondrial dehydrogenases was determined. The viability of treated cells was calculated in reference to a control with the solvent DMSO. As shown in Fig. 3A the relative viability of hMSC was only slightly affected by the addition of 20, 30 or even 40 μM quinalizarin. In contrast, the application of CX-4945 resulted in a drop of viability ranging from 70% viable cells (20 μM, 24 h) to only 35% after 72 h (40 μM, 72 h). We asked next whether these inhibitors were able to reduce CK2 activity in hMSCs. Based on the results of the viability assay we used 30 μM quinalizarin and 20 μM CX-4945. Cells were treated over 24, 48 and 72 h and harvested, proteins were extracted and subjected to an in vitro kinase assay with the CK2-specific substrate peptide. CK2 activity was efficiently

reduced to 10% residual activity with 20 μM CX-4945 already after 24 h of treatment; this reduction was not stronger after longer incubation times (Fig. 3B). Similar results were observed with quinalizarin, where we found a 25% residual activity already after 24 h, which did not decrease further after longer incubation times (Fig. 3C).

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3.3. CK2 inhibitors CX-4945 and quinalizarin have a different impact upon 336 the adipogenic differentiation of human mesenchymal stem cells 337 Next, we investigated the influence of CK2 inhibitors on hMSCs 338 during adipogenic differentiation. We incubated hMSCs with 20 μM 339

Please cite this article as: L. Schwind, et al., Protein kinase CK2 is necessary for the adipogenic differentiation of human mesenchymal stem cells, Biochim. Biophys. Acta (2015), http://dx.doi.org/10.1016/j.bbamcr.2015.05.023

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Fig. 2. Localization of the CK2 subunits during differentiation of hMSCs. Adipogenic differentiation was started when cells reached confluence. Every three days cells were fixed and immunofluorescence was performed. CK2α was detected with the mouse monoclonal antibody 1A5 and labelled by Alexa Fluor 546-coupled secondary antibody (red). CK2β was detected with a rabbit polyclonal antibody and labelled by Alexa Fluor 488-coupled secondary antibody (green). Perilipin was used as a differentiation control, detected with a rabbit polyclonal antibody and labelled by Alexa Fluor 488-coupled secondary antibody (green). The nucleus was stained with DAPI. One representative of two experiments is shown here.

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CX-4945 or 30 μM quinalizarin added to the differentiation mix for 21 days. The state of adipogenic differentiation of inhibitor-treated cells and control cells was evaluated by the appearance of lipid droplets (Fig. 4A and B). In hMSCs treated with the solvent DMSO for control we observed differentiating hMSCs already at day 9 and this population increased further until day 21. After treatment of hMSCs with CX-4945 only single cells with a differentiated phenotype appeared at day 9, but at day 15 the differentiating cell population was significantly increased and at day 21 cells were differentiated similar to the untreated control

cells. In contrast, cells treated with the CK2 inhibitor quinalizarin demonstrated a completely other outcome. At day 9 a small number of cells harbouring lipid droplets had formed, but this population did not increase during the differentiation process. At the end of the differentiation period most of the hMSCs remained undifferentiated. The difference between the three differently treated cell populations was more impressively shown by staining the lipid droplets at day 21 of the differentiation process with the lipophilic dye OilRedO or with the fluorescent dye Bodipy® (Fig. 4B).

Please cite this article as: L. Schwind, et al., Protein kinase CK2 is necessary for the adipogenic differentiation of human mesenchymal stem cells, Biochim. Biophys. Acta (2015), http://dx.doi.org/10.1016/j.bbamcr.2015.05.023

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of CX-4945, differentiating hMSCs turned out to be less sensitive towards the treatment than their proliferating counterparts (60% viable cells vs. 35%). Next, we analysed the consequences of the treatment for the cellular CK2 activity. Surprisingly, in cells treated with CX-4945 (20 μM) the CK2 activity was only reduced to about 40% residual activity after 24 h (Fig. 5B). Moreover, the inhibition of CK2 enzyme activity by CX-4945 decreased again slightly from 24 h to 72 h, to a residual activity of 75% compared to control cells. In contrast, using quinalizarin we observed a down-regulation of CK2 activity to about 25% of the original activity after 24, 48 and 72 hour treatment which corresponded to an inhibiting activity of 75% (Fig. 5C). This corresponded with the inhibiting effect we observed in proliferating cells with quinalizarin. Obviously, both inhibitors differ in their inhibition efficacy towards CK2 in differentiating cells. The different effect on CK2 activity correlates perfectly with the adipogenic differentiation capacity of hMSC when treated with CX-4945 or quinalizarin. This further supports the observation that the CK2 kinase activity is necessary for the adipogenic differentiation process of human stem cells.

Although we have tested both CK2 inhibitors before starting the differentiation process and demonstrated that CK2 kinase activity was inhibited with great efficacy in proliferating hMSCs we wondered whether this inhibition activity is maintained during differentiation. Therefore, we started the differentiation of hMSCs in the presence or in the absence of CK2 inhibitors. After 24, 48 and 72 h we either performed a viability assay or extracted the proteins and subjected the extract to an in vitro phosphorylation of the CK2 specific substrate peptide. Fig. 5A shows the outcome of the viability assay. As for proliferating hMSCs (see Fig. 3A) quinalizarin had no great impact on the viability of differentiating hMSCs. Viability was hardly reduced to 90%. Concerning the effects

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It is known for quite some time that protein kinase CK2 is absolutely necessary for the correct development of organisms and that life without CK2α and/or CK2β is impossible [5,35]. On the other hand, the level and the protein kinase activity are elevated in rapidly proliferating cells [18, 42]. The proliferation promoting activity might also be responsible for cell death in the developing embryo of CK2α knock-out mice. Furthermore, the importance of CK2 for cell growth is supported by the observation that a great number of CK2 substrates are implicated in the regulation of proliferation and growth [37]. Moreover, overexpression of CK2α and c-myc resulted in the formation of leukaemia in mice which supports the idea that at least CK2α might have oncogenic properties [32,58]. These observations have brought CK2 into the focus as a therapeutic target for the treatment of human cancer. A great number of CK2 inhibitors have been developed over the last ten years and at least

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The correct localization of key signal transduction proteins is a prerequisite for a successful differentiation. A key transcription factor during the early stage of adipogenic differentiation is PPARγ, in particular the γ2 isoform [59]. PPARγ is a ligand activated, early stage transcription factor which transactivates further key components leading to the expression of adipocyte specific proteins like the fatty acid binding protein perilipin [39]. From previous experiments (Figs. 1 and 2) we know that lipid droplets appear from day 6 of differentiation onwards. Moreover, we suspect from the CK2 dynamics that an active participation of CK2 in the differentiation takes place at early stages. Thus, we analysed PPARγ2 and perilipin localization until day 9 in the absence and presence of CX-4945 or quinalizarin (Fig. 6). PPARγ2 showed a nuclear localization at the beginning of differentiation. We observed a more intensive nuclear staining when hMSCs proceeded in the adipogenic differentiation. The fatty acid binding protein perilipin was first seen at day 6 and its expression steadily increased. Using CX-4945 as inhibitor essentially led to the same results as the control cells with a little delay in the first appearance of perilipin (day 9 vs. day 6). However, this observation coincided with the delay in the formation of lipid droplets after CX-4945 treatment (Fig. 4A). When analysing the proteins in quinalizarin treated cells we observed a somewhat different outcome. As for the control cells PPARγ2 was first present in the nuclear compartment. During the course of treatment PPARγ2 was not able to increase or maintain the nuclear localization; it became more and more cytoplasmic. In consequence, the expression of the PPARγ2 target perilipin was missing in the quinalizarin treated cells. Thus, upon CK2 inhibition the nuclear translocation of a master transcription factor of adipogenesis is abrogated and consequently, all downstream events are therefore interrupted.

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3.5. Localization of adipocyte marker proteins in differentiating hMSCs

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Fig. 3. Effect of different CK2 inhibitors on cell viability and CK2 activity in proliferating hMSCs. hMSCs were grown under non-differentiating conditions and treated with different concentrations of CX-4945 or quinalizarin. Cell viability was determined using the MTT assay after 24, 48 and 72 h of incubation. Viability was set in reference to the solvent DMSO. The mean and standard deviation of two independent experiments with two technical replicates each are shown (A). To measure the CK2 activity hMSCs were either treated with 20 μM CX-4945 (B) or 30 μM quinalizarin (C) or the vehicle DMSO as a control. After 24, 48 and 72 h cells were harvested, extracted and subjected to an in vitro phosphorylation with the CK2 specific peptide and [ 32 P]γATP. The graph shows the incorporation of [32P]phosphate into the peptide in counts per minute (cpm) of three independent experiments with two technical replicates each.

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Please cite this article as: L. Schwind, et al., Protein kinase CK2 is necessary for the adipogenic differentiation of human mesenchymal stem cells, Biochim. Biophys. Acta (2015), http://dx.doi.org/10.1016/j.bbamcr.2015.05.023

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Fig. 4. Adipogenic differentiation of hMSC in the presence or absence of CK2 inhibitors. hMSCs were seeded into six-well plates, induced to differentiate in the presence or absence of 20 μM CX-4945 or 30 μM quinalizarin.(A). Brightfield images show the lipid droplet formation at days 0, 9, 15 and 21 (B). At day 21 lipid droplets in adipogenic differentiated hMSCs were either stained with OilRedO and analysed by phase contrast microscopy or stained with the lipophilic fluorescent dye Bodipy® and analysed under a fluorescence microscope (excitation wave length: 488 nm).

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two of them are in clinical trials [45,47,62]. Among these inhibitors is CX4945 which has been shown to have anti-angiogenic, anti-proliferative and apoptosis-inducing properties in cancer cells. Apart from its involvement in proliferation there are reports showing that CK2 might also play a

role in the regulation of the carbohydrate metabolism, in regulation of the insulin production in β-cells of the pancreas [38], in information storage, synaptic plasticity, synaptic transmission and development of the brain [2] and also during differentiation [63,72].

Please cite this article as: L. Schwind, et al., Protein kinase CK2 is necessary for the adipogenic differentiation of human mesenchymal stem cells, Biochim. Biophys. Acta (2015), http://dx.doi.org/10.1016/j.bbamcr.2015.05.023

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CK2α and CK2β obviously localize independently to the nucleus. Whereas CK2α showed a mostly nuclear staining, the CK2β subunit represents the more dynamic component of the holoenzyme. It shuttles from the cytoplasm to the nucleus to presumably form an active holoenzyme with the α-subunit. Although, there are few examples that the α-subunit can phosphorylate substrates without its regulatory subunit, most substrates are phosphorylated by the holoenzyme [48]. It is known for tagged CK2 subunits that they undergo a dynamic shuttling and that they enter the nucleus independently [17]. In a recent paper we observed in glucose responsive MIN6 cells a glucose dependent shuttling of CK2 from the cytoplasm into the nucleus [71]. Here, we add another example where endogenous CK2 shows a spatial re-organization during a physiological process. Quinalizarin was tested with 72 kinases and CX-4945 was tested with 235 kinases and both turned out to be highly CK2 specific [1,9]. Using quinalizarin and CX-4945 in proliferating human stem cells we observed an efficient inhibition of the CK2 kinase activity. Although it has been repeatedly observed that every inhibitor has to be tested on a particular cell line and that there are enormous differences in the efficiency of inhibition, the general observation is that CX-4945 is more active than quinalizarin. Also in the present study CX-4945 was more active at lower concentrations than quinalizarin. From the results published so far it also seems that normal non-cancer cells require higher concentrations of CX-4945 than cancer cells to inhibit CK2 [74]. Moreover, normal cell lines seem to be somewhat more resistant towards the anti-proliferative efficacy of CK2 inhibitors [25]. Using quinalizarin as CK2 inhibitor only a few hMSCs differentiate to an adipogenic phenotype. In contrast, CX-4945 treated cells differentiate very efficiently similar to the control cells. However, the differentiation in CX-4945 treated cells is retarded in the initial phase. The analysis of the reason for this discrepancy reveals that the inhibition of the CK2 activity decreases during the differentiation process of stem cells in the case of CX-4945 whereas inhibition of CK2 remains high in the presence of quinalizarin. These results show that the remaining CK2 activity in the presence of CX-4945 is sufficient for adipogenic differentiation of human stem cells, whereas the low activity of CK2 in the presence of quinalizarin is not. Thus, a particular threshold of CK2 kinase activity seems to be required for the adipogenic differentiation of human stem cells. The reason for the failure of CX-4945 to ensure a permanent inhibition of CK2 in these stems cells during differentiation remains to be elucidated. Even higher concentrations of CX-4945 (30 and 40 μM) failed to reduce the kinase activity efficiently (data not shown). It is tempting to speculate that hMSC, which share similarities with cancer cells, develop some kind of drug resistance in response to the CX-4945 treatment as it is known for tumour cells [67,69]. Our present data show that CK2 plays a role in the early phase of differentiation. CK2 activity, expression and localization are necessary for the timely activation of the master transcription factor for adipogenesis, PPARγ2. We know from a recently published paper [57] that the upstream transcription factor C/EBPδ is a substrate for CK2, and that its transactivation efficiency is influenced by CK2 phosphorylation. Furthermore, we have shown that the localization of the C/EBPδ downstream target PPARγ2 changes during differentiation and that its nuclear localization is compromised upon CK2 inhibition. Whether this is due to an indirect impact of CK2 via C/EBPδ or a direct impact of the kinase on PPARγ2 remains to be elucidated. Although in most cases CK2 has to be down-regulated during differentiation, a particular threshold of CK2 activity is required for the adipogenic differentiation of human stem cells.

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Recently, it was found that inhibition of the kinase activity of CK2 by CX-4945 leads to an inhibition of osteoclast and osteoblast differentiation [63]. In line with this observation it was reported in 2012 that CK2 is implicated in the differentiation of preadipocytes into adipocytes in the mouse cell line 3T3-L1 [72]. In the present study we could show for the first time that CK2 plays a role in the differentiation of human stem cells. Upon progression of the differentiation the level of CK2α and CK2β decreased. Surprisingly, the expression of the α- and the β-subunits was not evenly down-regulated. The down-regulation of the α-subunit was more significant than that of the β-subunit (56 vs. 83%). This result is one of the few examples that the subunits have to be considered as individual proteins with distinct functions and a distinct regulation. The decrease in expression was accompanied by a reduction of the protein kinase activity of CK2 which was also seen during the differentiation process of preadipocytes. Another interesting observation was that

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Fig. 5. Effect of CK2 inhibitors on cell viability and CK2 activity in differentiating hMSCs. Confluent hMSCs were incubated with the differentiation mix including given concentrations of CX-4945, quinalizarin or DMSO as control. Cell viability was determined using the MTT assay after 24, 48 and 72 h of incubation. Viability was set in reference with DMSO. The mean and standard deviation of three independent experiments with two technical replicates each are shown (A). To measure the CK2 activity differentiation was started with the differentiation mix and 20 μM CX-4945 (B), 30 μM quinalizarin (C) or with DMSO for control. After 24, 48 and 72 h cells were harvested, extracted and subjected to an in vitro phosphorylation with the CK2 specific peptide and [32P]γATP. The graph shows the incorporation of [32P]phosphate into the peptide in counts per minute (cpm) of three independent experiments with two technical replicates each.

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CK2 activity is high at the beginning of stem cell differentiation. 516 With the progress of differentiation CK2 activity and protein level are 517 down-regulated. Experiments with CK2 inhibitors show that a particular 518

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Fig. 6. Localization of PPARγ2 during differentiation of hMSC in the presence and absence of CX-4945 or quinalizarin. When cells reached confluence differentiation mix including 20 μM CX-4945, 30 μM quinalizarin or DMSO as solvent control was added to start adipogenic differentiation. At given time points cells were fixed and immunofluorescence was performed. PPARγ2 was detected with a rabbit polyclonal antibody and labelled by Alexa Fluor 488-coupled secondary antibody (green). Perilipin was used as a differentiation control, detected with a rabbit polyclonal antibody and labelled by Alexa Fluor 488-coupled secondary antibody (green). The nucleus was stained with DAPI. One representative of two experiments is shown here.

threshold of CK2 activity is required for a successful adipogenic differentiation of stem cells.

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Conflict of interest

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The authors disclose any actual or potential conflict of interest including any financial, personal or other relationships with other people or organizations that could inappropriately influence the work.

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The authors would like to thank Andrea Hecker for hMSC isolation and characterization. This work was supported in part by “Anschubfinanzierung von Forschungsprojekten im HH-Jahr 2013” of the University of the Saarland to CG.

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References

531 532 533 534 535 536 537 538 539 540 541 542 543 544 545 546

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[1] R. Battistutta, G. Cozza, F. Pierre, E. Papinutto, G. Lolli, S. Sarno, S.E. O'Brien, A. Siddiqui-Jain, M. Haddach, K. Anderes, D.M. Ryckman, F. Meggio, L.A. Pinna, Unprecedented selectivity and structural determinants of a new class of protein kinase CK2 inhibitors in clinical trials for the treatment of cancer, Biochemistry 50 (2011) 8478–8488. [2] P.R. Blanquet, Casein kinase 2 as a potentially important enzyme in the nervous system, Prog. Neurobiol. 60 (2000) 211–246. [3] B. Bragdon, S. Thinakaran, O. Moseychuk, L. Gurski, J. Bonor, C. Price, L. Wang, W.G. Beamer, A. Nohe, Casein kinase 2 regulates in vivo bone formation through its interaction with bone morphogenetic protein receptor type Ia, Bone 49 (2011) 944–954. [4] B. Bragdon, S. Thinakaran, O. Moseychuk, D. King, K. Young, D.W. Litchfield, N.O. Petersen, A. Nohe, Casein kinase 2 beta-subunit is a regulator of bone morphogenetic protein 2 signaling, Biophys. J. 99 (2010) 897–904. [5] T. Buchou, M. Vernet, O. Blond, H.H. Jensen, H. Pointu, B.B. Olsen, C. Cochet, O.G. Issinger, B. Boldyreff, Disruption of the regulatory β subunit of protein kinase CK2 in mice leads to a cell-autonomous defect and early embryonic lethality, Mol. Cell. Biol. 23 (2003) 908–915. [6] A.I. Caplan, All MSCs are pericytes? Cell Stem Cell 3 (2008) 229–230.

[7] A.I. Caplan, S.P. Bruder, Mesenchymal stem cells: building blocks for molecular medicine in the 21st century, Trends Mol. Med. 7 (2001) 259–264. [8] P. Cheng, V. Kumar, H. Liu, J.I. Youn, M. Fishman, S. Sherman, D. Gabrilovich, Effects of notch signaling on regulation of myeloid cell differentiation in cancer, Cancer Res. 74 (2014) 141–152. [9] G. Cozza, M. Mazzorana, E. Papinutto, J. Bain, M. Elliott, M.G. Di, A. Gianoncelli, M.A. Pagano, S. Sarno, M. Ruzzene, R. Battistutta, F. Meggio, S. Moro, G. Zagotto, L.A. Pinna, Quinalizarin as a potent, selective and cell-permeable inhibitor of protein kinase CK2, Biochem. J. 421 (2009) 387–395. [10] G. Cozza, F. Meggio, S. Moro, The dark side of protein kinase CK2 inhibition, Curr. Med. Chem. 18 (2011) 2867–2884. [11] M.L. da Silva, A.I. Caplan, N.B. Nardi, In search of the in vivo identity of mesenchymal stem cells, Stem Cells 26 (2008) 2287–2299. [12] I. Dominguez, I.R. Degano, K. Chea, J. Cha, P. Toselli, D.C. Seldin, CK2alpha is essential for embryonic morphogenesis, Mol. Cell. Biochem. 356 (2011) 209–216. [13] S. Dovat, C. Song, K.J. Payne, Z. Li, Ikaros, CK2 kinase, and the road to leukemia, Mol. Cell. Biochem. 356 (2011) 201–207. [14] D. Escalier, D. Silvius, X. Xu, Spermatogenesis of mice lacking CK2α′: failure of germ cell survival and characteristic modifications of the spermatid nucleus, Mol. Reprod. Dev. 66 (2003) 190–201. [15] M. Faust, J. Günther, E. Morgenstern, M. Montenarh, C. Götz, Specific localization of the catalytic subunits of protein kinase CK2 at the centrosomes, Cell. Mol. Life Sci. 59 (2002) 2155–2164. [16] M. Faust, N. Schuster, M. Montenarh, Specific binding of protein kinase CK2 catalytic subunits to tubulin, FEBS Lett. 462 (1999) 51–56. [17] O. Filhol, A. Nueda, V. Martel, D. Gerber-Scokaert, M.J. Benitez, C. Souchier, Y. Saoudi, C. Cochet, Live-cell fluorescence imaging reveals the dynamics of protein kinase CK2 individual Subunits, Mol. Cell. Biol. 23 (2003) 975–987. [18] M. Gabriel, D.W. Litchfield, Protein kinase CK2: at the crossroad of pathways controlling cell proliferation and survival, in: L.A. Pinna (Ed.), Protein Kinase CK2, John Wiley Sons, Inc., Ames, Chichester, Oxford 2013, pp. 169–189. [19] M.O. Gratton, E. Torban, S.B. Jasmin, F.M. Theriault, M.S. German, S. Stifani, Hes6 promotes cortical neurogenesis and inhibits Hes1 transcription repression activity by multiple mechanisms, Mol. Cell. Biol. 23 (2003) 6922–6935. [20] H. Green, O. Kehinde, An established preadipose cell line and its differentiation in culture. II. Factors affecting the adipose conversion, Cell 5 (1975) 19–27. [21] H. Green, M. Meuth, An established pre-adipose cell line and its differentiation in culture, Cell 3 (1974) 127–133. [22] E. Hu, C.S. Rubin, Expression of wild-type and mutated forms of the catalytic (alpha) subunit of Caenorhabditis elegans casein kinase II in Escherichia coli, J. Biol. Chem. 265 (1990) 20609–20615.

Please cite this article as: L. Schwind, et al., Protein kinase CK2 is necessary for the adipogenic differentiation of human mesenchymal stem cells, Biochim. Biophys. Acta (2015), http://dx.doi.org/10.1016/j.bbamcr.2015.05.023

547 548 549 550 551 552 553 554 555 556 557 558 559 560 561 562 563 564 565 566 567 568 569 570 571 572 573 574 575 576 577 578 579 580 581 582 583 584 585 586 587

D

P

R O

O

F

[49] M.F. Pittenger, A.M. Mackay, S.C. Beck, R.K. Jaiswal, R. Douglas, J.D. Mosca, M.A. Moorman, D.W. Simonetti, S. Craig, D.R. Marshak, Multilineage potential of adult human mesenchymal stem cells, Science 284 (1999) 143–147. [50] R. Prudent, C. Cochet, New protein kinase CK2 inhibitors: jumping out of the catalytic box, Chem. Biol. 16 (2009) 112–120. [51] C.A. Reznikoff, D.W. Brankow, C. Heidelberger, Establishment and characterization of a cloned line of C3H mouse embryo cells sensitive to postconfluence inhibition of division, Cancer Res. 33 (1973) 3231–3238. [52] M. Salvi, S. Sarno, L. Cesaro, H. Nakamura, L.A. Pinna, Extraordinary pleiotropy of protein kinase CK2 revealed by weblogo phosphoproteome analysis, Biochim. Biophys. Acta 1793 (2009) 847–859. [53] S. Sarno, E. Papinutto, C. Franchin, J. Bain, M. Elliott, F. Meggio, Z. Kazimierczuk, A. Orzeszko, G. Zanotti, R. Battistutta, L.A. Pinna, ATP site-directed inhibitors of protein kinase CK2: an update, Curr. Top. Med. Chem. 11 (2011) 1340–1351. [54] C.C. Schneider, A. Hessenauer, C. Götz, M. Montenarh, DMAT:an inhibitor of protein kinase CK2 induces reactive oxygen species and DNA double strand breaks, Oncol. Rep. 21 (2009) 1593–1597. [55] H.R. Schneider, G.H. Reichert, O.G. Issinger, Enhanced casein kinase II activity during mouse embryogenesis. Identification of a 110-kDa phosphoprotein as the major phosphorylation product in mouse embryos and Krebs II mouse ascites tumor cells, Eur. J. Biochem. 161 (1986) 733–738. [56] N. Schuster, C. Götz, M. Faust, E. Schneider, A. Prowald, A. Jungbluth, M. Montenarh, Wild-type p53 inhibits protein kinase CK2 activity, J. Cell. Biochem. 81 (2001) 172–181. [57] L. Schwind, A. Zimmer, C. Götz, M. Montenarh, CK2 phosphorylation of C/EBPδ regulates its transcription factor activity, Int. J. Biochem. Cell Biol. 61 (2015) 81–89. [58] D.C. Seldin, P. Leder, Casein kinase II alpha transgene-induced murine lymphoma: relation to theileriosis in cattle, Science 267 (1995) 884–897. [59] J.B. Seo, H.M. Moon, W.S. Kim, Y.S. Lee, H.W. Jeong, E.J. Yoo, J. Ham, H. Kang, M.G. Park, K.R. Steffensen, T.M. Stulnig, J.A. Gustafsson, S.D. Park, J.B. Kim, Activated liver X receptors stimulate adipocyte differentiation through induction of peroxisome proliferator-activated receptor gamma expression, Mol. Cell. Biol. 24 (2004) 3430–3444. [60] P.R. Shepherd, L. Gnudi, E. Tozzo, H. Yang, F. Leach, B.B. Kahn, Adipose cell hyperplasia and enhanced glucose disposal in transgenic mice overexpressing GLUT4 selectively in adipose tissue, J. Biol. Chem. 268 (1993) 22243–22246. [61] A. Siddiqui-Jain, J. Bliesath, D. Macalino, M. Omori, N. Huser, N. Streiner, C.B. Ho, K. Anderes, C. Proffitt, S.E. O'Brien, J.K. Lim, D.D. Von Hoff, D.M. Ryckman, W.G. Rice, D. Drygin, CK2 inhibitor CX-4945 suppresses DNA repair response triggered by DNA-targeted anticancer drugs and augments efficacy: mechanistic rationale for drug combination therapy, Mol. Cancer Ther. 11 (2012) 994–1005. [62] A. Siddiqui-Jain, D. Drygin, N. Streiner, P. Chua, F. Pierre, S.E. O'Brien, J. Bliesath, M. Omori, N. Huser, C. Ho, C. Proffitt, M.K. Schwaebe, D.M. Ryckman, W.G. Rice, K. Anderes, CX-4945, an orally bioavailable selective inhibitor of protein kinase CK2, inhibits prosurvival and angiogenic signaling and exhibits antitumor efficacy, Cancer Res. 70 (2010) 10288–10298. [63] Y.H. Son, S.H. Moon, J. Kim, The protein kinase 2 inhibitor CX-4945 regulates osteoclast and osteoblast differentiation In vitro, Mol. Cells 36 (2013) 417–423. [64] Y.H. Son, J.S. Song, S.H. Kim, J. Kim, Pharmacokinetic characterization of CK2 inhibitor CX-4945, Arch. Pharm. Res. 36 (2013) 840–845. [65] C. Song, Z. Li, A.K. Erbe, A. Savic, S. Dovat, Regulation of Ikaros function by casein kinase 2 and protein phosphatase 1, World J. Biol. Chem. 2 (2011) 126–131. [66] Q.Q. Tang, M.D. Lane, Adipogenesis: from stem cell to adipocyte, Annu. Rev. Biochem. 81 (2012) 715–736. [67] G. Theyer, M. Schirmbock, T. Thalhammer, E.R. Sherwood, G. Baumgartner, G. Hamilton, Role of the MDR-1-encoded multiple drug resistance phenotype in prostate cancer cell lines, J. Urol. 150 (1993) 1544–1547. [68] T.L. Tsai, P.A. Manner, W.J. Li, Regulation of mesenchymal stem cell chondrogenesis by glucose through protein kinase C/transforming growth factor signaling, Osteoarthr. Cartil. 21 (2013) 368–376. [69] J.P. van Brussel, G.J. van Steenbrugge, J.C. Romijn, F.H. Schroder, G.H. Mickisch, Chemosensitivity of prostate cancer cell lines and expression of multidrug resistance-related proteins, Eur. J. Cancer 35 (1999) 664–671. [70] F. Vinals, J. Reiriz, S. Ambrosio, R. Bartrons, J.L. Rosa, F. Ventura, BMP-2 decreases Mash1 stability by increasing Id1 expression, EMBO J. 23 (2004) 3527–3537. [71] S. Welker, C. Götz, C. Servas, M.W. Laschke, M.D. Menger, M. Montenarh, Glucose regulates protein kinase CK2 in pancreatic ß-cells and its interaction with PDX-1, Int. J. Biochem. Cell Biol. 45 (2013) 2786–2795. [72] N. Wilhelm, K. Kostelnik, C. Götz, M. Montenarh, Protein kinase CK2 is implicated in early steps of the differentiation of preadipocytes into adipocytes, Mol. Cell. Biochem. 365 (2012) 37–45. [73] U. Wirkner, W. Pyerin, CK2α loci in the human genome: structure and transcriptional activity, Mol. Cell. Biochem. 191 (1999) 59–64. [74] S. Zanin, C. Borgo, C. Girardi, S.E. O'Brien, Y. Miyata, L.A. Pinna, A. Donella-Deana, M. Ruzzene, Effects of the CK2 Inhibitors CX-4945 and CX-5011 on drug-resistant cells, PLoS One 7 (2012) e49193. [75] L. Ziercher, O. Filhol, B. Laudet, R. Prudent, C. Cochet, T. Buchou, Structure–function analysis of the beta regulatory subunit of protein kinase CK2 by targeting embryonic stem cell, Mol. Cell. Biochem. 356 (2011) 75–81.

N

C

O

R

R

E

C

T

[23] E. Hu, C.S. Rubin, Casein kinase II from Caenorhabditis elegans. Cloning, characterization, and developmental regulation of the gene encoding the beta subunit, J. Biol. Chem. 266 (1991) 19796–19802. [24] E. Huillard, L. Ziercher, O. Blond, M. Wong, J.C. Deloulme, S. Souchelnytskyi, J. Baudier, C. Cochet, T. Buchou, Disruption of CK2beta in embryonic neural stem cells compromises proliferation and oligodendrogenesis in the mouse telencephalon, Mol. Cell. Biol. 30 (2010) 2737–2749. [25] J. Intemann, N.E.B. Saidu, L. Schwind, M. Montenarh, ER stress signaling in ARPE-19 cells after inhibition of protein kinase CK2 by CX-4945, Cell. Signal. 26 (2014) 1567–1575. [26] R.K. Jaiswal, N. Jaiswal, S.P. Bruder, G. Mbalaviele, D.R. Marshak, M.F. Pittenger, Adult human mesenchymal stem cell differentiation to the osteogenic or adipogenic lineage is regulated by mitogen-activated protein kinase, J. Biol. Chem. 275 (2000) 9645–9652. [27] J.W. Kang, Y. Choi, J.H. Park, J.S. Kim, K.D. Park, D.H. Baek, S.K. Seong, K.S. Choi, S.Y. Lim, H.S. Kim, The effects of cyclin-dependent kinase inhibitors on adipogenic differentiation of human mesenchymal stem cells, Biochem. Biophys. Res. Commun. 366 (2008) 624–630. [28] E.K. Kim, S. Lim, J.M. Park, J.K. Seo, J.H. Kim, K.T. Kim, S.H. Ryu, P.G. Suh, Human mesenchymal stem cell differentiation to the osteogenic or adipogenic lineage is regulated by AMP-activated protein kinase, J. Cell. Physiol. 227 (2012) 1680–1687. [29] E.A. Kuenzel, E.G. Krebs, A synthetic peptide substrate specific for casein kinase II, Proc. Natl. Acad. Sci. U. S. A. 82 (1985) 737–741. [30] U.K. Laemmli, Cleavage of structural proteins during the assembly of the head of bacteriophage T 4, Nature 227 (1970) 680–682. [31] E. Landesman-Bollag, A. Belkina, B. Hovey, E. Connors, C. Cox, D.C. Seldin, Developmental and growth defects in mice with combined deficiency of CK2 catalytic genes, Mol. Cell. Biochem. 356 (2011) 227–231. [32] E. Landesman-Bollag, P.L. Channavajhala, R.D. Cardiff, D.C. Seldin, p53 deficiency and misexpression of protein kinase CK2α collaborate in the development of thymic lymphomas in mice, Oncogene 16 (1998) 2965–2974. [33] J.S. Lee, L. Ha, I.K. Kwon, J.Y. Lim, The role of focal adhesion kinase in BMP4 induction of mesenchymal stem cell adipogenesis, Biochem. Biophys. Res. Commun. 435 (2013) 696–701. [34] S. Lee, H.Y. Cho, H. Bui, D. Kang, The osteogenic or adipogenic lineage commitment of human mesenchymal stem cells is determined by protein kinase C delta, BMC Cell Biol. 15 (2014) 42. [35] D.Y. Lou, I. Dominguez, P. Toselli, E. Landesman-Bollag, C. O'Brien, D.C. Seldin, The alpha catalytic subunit of protein kinase CK2 is required for mouse embryonic development, Mol. Cell. Biol. 28 (2008) 131–139. [36] G. Maridor, W. Park, W. Krek, E.A. Nigg, Casein kinase II. cDNA sequences, developmental expression, and tissue distribution of mRNAs for alpha, alpha', and beta subunits of the chicken enzyme, J. Biol. Chem. 266 (1991) 2362–2368. [37] F. Meggio, L.A. Pinna, One-thousand-and-one substrates of protein kinase CK2? FASEB J. 17 (2003) 349–368. [38] R. Meng, F. Al-Quobaili, I. Müller, C. Götz, G. Thiel, M. Montenarh, CK2 phosphorylation of Pdx-1 regulates its transcription factor activity, Cell. Mol. Life Sci. 67 (2010) 2481–2489. [39] A. Menssen, T. Haupl, M. Sittinger, B. Delorme, P. Charbord, J. Ringe, Differential gene expression profiling of human bone marrow-derived mesenchymal stem cells during adipogenic development, BMC Genomics 12 (2011) 461. [40] P. Mestres, B. Boldyreff, C. Ebensperger, H. Hameister, O.-G. Issinger, Expression of casein kinase 2 during mouse embryogenesis, Acta Anat. 149 (1994) 13–20. [41] O. Moseychuk, H. Akkiraju, J. Dutta, A. D'Angelo, B. Bragdon, R.L. Duncan, A. Nohe, Inhibition of CK2 binding to BMPRIa induces C2C12 differentiation into osteoblasts and adipocytes, J. Cell Commun. Signal. 7 (2013) 265–278. [42] U. Münstermann, G. Fritz, G. Seitz, L. Yiping, H.R. Schneider, O.-G. Issinger, Casein kinase II is elevated in human tumours and rapidly proliferating non-neoplastic tissue, Eur. J. Biochem. 189 (1990) 251–257. [43] W. Nastainczyk, I. Schmidt-Spaniol, B. Boldyreff, O.-G. Issinger, Isolation and characterization of a monoclonal anti-protein kinase CK2 β-subunit antibody of the IgG class for the direct detection of CK2 β-subunit in tissue cultures of various mammalian species and human tumors, Hybridoma 14 (1995) 335–339. [44] T.C. Otto, M.D. Lane, Adipose development: from stem cell to adipocyte, Crit. Rev. Biochem. Mol. Biol. 40 (2005) 229–242. [45] S.E. Perea, O. Reyes, I. Baladron, Y. Perera, H. Farina, J. Gil, A. Rodriguez, D. Bacardi, J.L. Marcelo, K. Cosme, M. Cruz, C. Valenzuela, P.A. Lopez-Saura, Y. Puchades, J.M. Serrano, O. Mendoza, L. Castellanos, A. Sanchez, L. Betancourt, V. Besada, R. Silva, E. Lopez, V. Falcon, I. Hernandez, M. Solares, A. Santana, A. Diaz, T. Ramos, C. Lopez, J. Ariosa, L.J. Gonzalez, H. Garay, D. Gomez, R. Gomez, D.F. Alonso, H. Sigman, L. Herrera, B. Acevedo, CIGB-300, a novel proapoptotic peptide that impairs the CK2 phosphorylation and exhibits anticancer properties both in vitro and in vivo, Mol. Cell. Biochem. 316 (2008) 163–167. [46] M. Perez, J. Grande, E. Itarte, Developmental changes in rat hepatic casein kinases 1 and 2, Eur. J. Biochem. 170 (1987) 493–498. [47] 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, Pre-clinical characterization of CX-4945, a potent and selective small molecule inhibitor of CK2 for the treatment of cancer, Mol. Cell. Biochem. 356 (2011) 37–43. [48] L.A. Pinna, The raison D'Etre of constitutively active protein kinases: the lesson of CK2, Acc. Chem. Res. 36 (2003) 378–384.

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Please cite this article as: L. Schwind, et al., Protein kinase CK2 is necessary for the adipogenic differentiation of human mesenchymal stem cells, Biochim. Biophys. Acta (2015), http://dx.doi.org/10.1016/j.bbamcr.2015.05.023

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