Quinalizarin inhibits adipogenesis through down-regulation of transcription factors and microRNA modulation

Accepted Manuscript Quinalizarin inhibits adipogenesis through down-regulation of transcription factors and microRNA modulation

Lisa Schwind, Lisa Nalbach, Andreas D. Zimmer, Katja B. Kostelnik, Jennifer Menegatti, Friedrich Grässer, Claudia Götz, Mathias Montenarh PII: DOI: Reference:

S0304-4165(17)30318-5 doi:10.1016/j.bbagen.2017.09.018 BBAGEN 28951

To appear in: Received date: Revised date: Accepted date:

28 March 2017 14 September 2017 26 September 2017

Please cite this article as: Lisa Schwind, Lisa Nalbach, Andreas D. Zimmer, Katja B. Kostelnik, Jennifer Menegatti, Friedrich Grässer, Claudia Götz, Mathias Montenarh , Quinalizarin inhibits adipogenesis through down-regulation of transcription factors and microRNA modulation. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Bbagen(2017), doi:10.1016/ j.bbagen.2017.09.018

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ACCEPTED MANUSCRIPT

Quinalizarin inhibits adipogenesis through down-regulation of transcription factors and microRNA modulation

Lisa Schwind1, Lisa Nalbach1, Andreas D. Zimmer1, Katja B. Kostelnik1, Jennifer

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Menegatti2, Friedrich Grässer2, Claudia Götz and Mathias Montenarh*

Medical Biochemistry and Molecular Biology and 2Institute of Virology

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1

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Saarland University

66424 Homburg Germany

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Buildings 44 and 47

(49) 6841 - 162 6501

Fax:

(49) 6841 - 162 6027

e-mail:

[email protected]

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Tel.:

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* corresponding author

ACCEPTED MANUSCRIPT 2 Abstract Background: Protein kinase CK2 is induced early in adipogenesis whereas later on, this kinase seems to be dispensable. Here, we have analysed how CK2 might be involved in early steps of differentiation of 3T3-L1 cells. Methods: 3T3-L1 cells were differentiated to adipocytes in the absence or presence

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of quinalizarin. The expression and localization of important transcription factors was

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analysed by Western blot and immunofluorescence. DNA binding capacity and

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transactivation was analysed with pull-down assays and with luciferase reporter experiments, respectively. mRNA was detected with qRT-PCR, miRNAs with

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Northern hybridization and qRT-PCR.

Results: We show that clonal expansion was considerably repressed upon inhibition

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of CK2 with quinalizarin. Moreover, to prevent adipogenesis CK2 inhibition had to take place before day 4 of differentiation. Neither the expression at the protein or at

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the RNA level nor the subcellular localization of the transcription factors C/EBP and

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C/EBP was affected by CK2 inhibition. There was, however, a drastic reduction in the mRNA and protein levels of C/EBP and PPAR2. Upon inhibition of CK2, we

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found a significant up-regulation of the level of the microRNAs miR-27a and miR-27b,

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which are known to target PPAR mRNA. Conclusions: Time course experiments revealed that CK2 seems to be required at early time points after the induction of differentiation. One important target of CK2 was identified as PPAR, which is down-regulated after inhibition of CK2. General Significance: This is the first report about i) cellular targets of CK2 during adipogenesis and ii) a role of CK2 in microRNA regulation.

Key words: adipogenesis, protein kinase CK2, transcription factors, microRNAs

ACCEPTED MANUSCRIPT 3 1. Introduction Obesity is a severe and prevalent health hazard world-wide [13]. Although adipocytes derive from multipotent mesenchymal stem cells, most of our knowledge about adipogenesis comes from studies with pre-adipocyte mouse cell lines such as 3T3-L1 or 3T3-F442A, which were isolated from Swiss 3T3 cells derived from 17- to 19-days

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old mouse embryos. The first comprehensive characterisation of those two cell lines

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started in the early seventies of the last century [11,12,22]. Differentiation of pre-

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adipocytes can be induced and accelerated by a mix of dexamethasone, isobutylmethylxanthine (IBMX) and insulin. During adipocyte differentiation, the

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shape of the cells converts from a fibroblastic to a spherical one. This conversion in the phenotype and morphology also involves a temporally regulated set of gene

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expression directed by several transcription factors [38]. The cascade of induced transcription factors includes members of the C/EBP family such as C/EBP,

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C/EBP, C/EBP and CHOP. Each C/EBP isoform shows a distinct temporal and

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spatial expression pattern and homo- and hetero-dimerization. In early stages of differentiation, C/EBP is kept in an inactive state by hetero-dimerization with CHOP

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[37]. Upon degradation of CHOP, C/EBP forms heterodimers with C/EBP Both,

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C/EBP and C/EBP, lead to an induction of C/EBPand PPAR2. There are two isoforms of PPAR, namely PPAR1 and PPAR2, which are generated by alternative splicing and promoter usage. In 3T3-L1 cells PPAR2 is crucial for differentiation and for the maintenance of the differentiated state [36].

CK2 is a serine/threonine protein kinase composed of two catalytic - or ’- subunits and two regulatory subunits. Its expression level and its kinase activity is often associated with cell proliferation [27], angiogenesis [31], DNA damage and repair

ACCEPTED MANUSCRIPT 4 processes [32], ER stress [10] and regulation of the nervous system [2]. Furthermore, CK2 plays a key role in embryogenesis and cell differentiation [1,3,7,29]. Most of these results were either obtained by the use of inhibitors of the protein kinase activity or by knock-out experiments. Over the last 40 years 3T3-L1 cells were used to study the differentiation of pre-adipocytes into adipocytes. Sommercorn and Krebs

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found a transient increase of the CK2 kinase activity with a maximum at day 4 after

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start of differentiation. This increased activity was accompanied by an elevated level

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of CK2 [40]. After day 4 CK2 activity decreased until the end of the differentiation process. Inhibition of CK2 in 3T3-L1 cells by two different CK2 inhibitors, 2(DMAT)

and

1,2,5,8-

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dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole

tetrahydroxyanthraquinone (quinalizarin), revealed that CK2 seems to be essential

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for early steps in adipogenesis, whereas it is dispensable at late stages of differentiation [44]. Furthermore, it was shown that the inhibition of CK2 kinase

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activity prevented adipogenesis. How CK2 regulates early events at the beginning of

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differentiation is still an enigma. Therefore, in the present study we have analysed the role of CK2 at early stages of differentiation. We found that inhibition of CK2 by

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quinalizarin i) inhibited the mitotic clonal expansion of cells, ii) did neither influence the subcellular localization of C/EBP and C/EBP nor their DNA binding activity to

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the PPAR2 promoter, iii) reduced the expression of C/EBP and PPAR2 and, iv) this reduction is mediated by the regulation of microRNAs miR-27a and miR-27b.

2. Materials and Methods 2.1 Cell culture, differentiation and treatment of cells The 3T3-L1 cell line (ATCC: CRL-173) is a derivative of the Swiss 3T3 M line. 3T3-L1 cells are pre-adipocytes, which can be induced to terminally differentiate into

ACCEPTED MANUSCRIPT 5 adipocytes by the addition of different hormones or chemical agents. Cells are maintained in Dulbecco’s modified Eagle’s medium (DMEM; Thermo Fisher Scientific, St. Leon-Rot, Germany) containing 2 mM L-glutamine supplemented with 10% (v/v) foetal calf serum (FCS) in a humidified atmosphere containing 5% CO 2 at 37°C.

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To differentiate 3T3-L1 cells into adipocytes, cells were seeded in a concentration of

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1x104 cells/cm2. When cells were confluent, medium was removed and substituted by

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the differentiation mix I (DMEM + 10% FCS, 0.5 mM IBMX, 0.25 µM dexamethasone, 5 µg/ml insulin) (corresponds to day 1 of the differentiation). At day 3, mix I was

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replaced by fresh medium. From day 6, cells were incubated with differentiation mix II (DMEM + 10% FCS, 5 µg/ml insulin), which was replaced every three days.

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The CK2 inhibitor quinalizarin (1,2,5,8-tetrahydroxyanthraquinone) (Labotest OHG, Niederschöna, Germany) [5] was dissolved in dimethyl sulfoxide (DMSO) to a 20 mM

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stock solution, which was used to treat the cells with a final concentration of 30 µM

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throughout the entire differentiation. In control experiments we used the same volume

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of the solvent DMSO alone.

2.2 Determination of proliferation

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Cells were seeded in 24-well plates. At confluence, differentiation was induced in the presence of DMSO or 30 µM quinalizarin. Cells were counted in triplicates using a Neubauer chamber (factor: 104) at 0, 24 (day 2) and 48 h (day 3) after the start of differentiation. Cells were trypsinized and re-suspended in a small amount of cell culture medium. A small amount of the cell suspension was mixed with an equal amount of the diazo dye trypan blue to stain dead cells. To determine the proliferation rate only living cells were counted. The cell number determined for 0 h, that ranged between 5-7x104, was set to 1.

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2.3 MTT assay At given time points the DMSO- or quinalizarin-treated cells were incubated with 3[4,5-dimethylthiazol-2-yl] 2,5-diphenyl tetrazolium bromide (MTT) reagent (1 mg/ml solved in PBS) for 30 min, lysed in solubilisation solution (10% SDS, 0.6% acetic acid

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in DMSO) and absorption was measured at 595 nm. Results were normalized to day

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0.

2.4 Staining of lipid droplets

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Lipids were visualized with the lipophilic dye Oil Red O (Sigma Aldrich, Munich, Germany). Cells were differentiated in 24-well plates for 12 days as described above.

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After removing the medium, cells were washed twice with PBS and subsequently fixed using 3.7% formaldehyde in PBS for 2 min at room temperature. The fixed cells

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were washed two times with PBS and incubated with Oil Red O solution (0.5 ml/well)

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for one hour at room temperature. Cells were washed twice with deionized water and the staining of the lipid droplets was analysed by phase contrast microscopy followed

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by extraction of the dye with isopropanol and measurement of its absorption at 515 nm. To prepare the Oil Red O solution, Oil Red O was dissolved in isopropanol (0.5

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g/100 ml). This solution of Oil Red O was diluted 3:2 with deionized water, incubated for 10 min at room temperature and subsequently filtered through a folded filter (3MM, Whatman, Sigma Aldrich, Munich, Germany).

2.5 Immunofluorescence 3T3-L1 cells were seeded in six-well plates on poly-L-lysine treated coverslips. Immunofluorescence analysis was performed as described earlier [8], 24 h after start of differentiation. For the detection of C/EBP and C/EBP the specific antibodies

ACCEPTED MANUSCRIPT 7 #3087 (Cell Signaling Technology, Frankfurt, Germany) and sc-151 (Santa Cruz Biotechnology Inc., Heidelberg, Germany) were used.

2.6 Transient transfection, cell lysis and luciferase assay Transfection of cells was performed by using the Viafect™ transfection reagent

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(Promega, Mannheim, Germany) according to the manufacturer’s instructions. For

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the luciferase reporter assay, 3T3-L1 cells were seeded in 24-well plates (2x104

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cells/well) in a total volume of 0.5 ml/well of cell culture medium and cultured for 24 h. Subsequently, cells were transfected with 0.5 µg of plasmid DNA (transfection

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mixture per well: 0.45 µg of pGL4 basic vector or pGL4-PPARγ2 [39], 0.05 µg of Renilla luciferase control plasmid, 1.5 µl Viafect™ transfection reagent and 50 µl cell

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culture medium without FCS). Sixteen hours after transfection, medium was replaced by differentiation mix I containing DMSO or quinalizarin. To measure luciferase

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activities, cells were collected at given time points after start of differentiation by

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lysing in passive lysis buffer (Promega) and measured with the Dual-Luciferase Reporter Assay System (Promega) following the manufacturer’s recommendations.

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Firefly luciferase activity was normalized to Renilla luciferase activity and the

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normalized activity of the DMSO sample was set to 1.

2.7 Protein extraction Cells were washed with cold PBS, scraped off the plate and centrifuged (250xg, 4°C). To prepare whole cell extracts, the pellet was extracted with RIPA-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) and phosphatase inhibitors (PhosSTOP, Roche Diagnostics, Mannheim, Germany). After 30 min on ice, cells were sonicated (3 x 30s). After

ACCEPTED MANUSCRIPT 8 lysing, the cell debris was removed by centrifugation at 13.000xg. To prepare cytoplasmic and nuclear extracts, the pellet was re-suspended in buffer A (10 mM HEPES, pH 7.9, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM DTT, CompleteTM) and incubated on ice for 20 min. After centrifugation the supernatant containing the cytoplasmic proteins was transferred to a fresh tube and the pellet was resolved in

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buffer C (20 mM HEPES, pH 7.9, 450 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5

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mM DTT, CompleteTM). Nuclear proteins were isolated by pressing the suspension

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through a 21G and 23G cannula and centrifugation. The protein content was

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determined with the BioRad reagent dye (BioRad, Munich, Germany).

2.8 SDS–polyacrylamide gel electrophoresis and Western blot analysis

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Proteins were analysed by SDS– polyacrylamide gel electrophoresis according to the procedure of Laemmli [23]. Proteins were dissolved in sample buffer (130 mM Tris-

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HCl, pH 6.8, 0.02% bromophenol blue (w/v), 10% β-mercaptoethanol, 20% glycerol

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(v/v), and 4% SDS) and 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).

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Proteins were transferred onto a PVDF membrane (Roche Diagnostics, Mannheim, Germany) in a buffer containing 20 mM Tris-HCl, 150 mM glycine, pH 8.3. The

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membrane was blocked with 5% dry milk in TBS-Tween20 for one hour and then incubated with appropriate primary antibodies diluted in incubation buffer according to the manufacturer’s instructions. The membrane was washed twice with the incubation buffer and incubated with the peroxidase-coupled secondary antibody (anti-rabbit 1:30.000 or anti-mouse 1:10.000 in incubation buffer) for 1 hour. Signals were visualized by the Lumilight system of Roche Diagnostics (Mannheim, Germany).

ACCEPTED MANUSCRIPT 9 For the detection of phospho-S10-histone H3 (#3377), histone H3 (#9715), CREB (#9197), C/EBPβ (#3087), phospho-T235-C/EBPβ (#3084) and PPAR (#2430) specific antibodies from Cell Signaling Technology (New England Biolabs GmbH, Frankfurt, Germany) were used. The C/EBP- (sc-151) and GAPDH- (sc-25778) specific antibodies were purchased from Santa Cruz Biotechnology Inc. (Heidelberg,

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Germany) and the C/EBP- (ab40764) specific antibody from Abcam (Cambridge,

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UK). The phospho-CREB-1 (#05-807) specific antibody was from Merck Millipore

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(Darmstadt, Germany), the CHOP- (#3588-1) specific antibody from Epitomics, Inc. (Burlingame, U.S.A) and the -tubulin-specific antibody (T9026) from Sigma-Aldrich

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(Munich, Germany). HSP70 (SM5066) was detected with a specific antibody from Acris Antibodies GmbH (Herford, Germany) and nucleolin with the rabbit polyclonal

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serum #36 described in [9].

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2.9 Streptavidin-agarose pull-down assay

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To analyse the binding of C/EBP and C/EBP proteins to the PPAR2 promoter, we used the μMacs™ Streptavidin Kit (Miltenyi Biotec, Gladbach, Germany).

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Amplification of the PPAR2 promoter probe, spanning the two C/EBP binding sites

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[4,46], was performed using the forward primer 5’-GAACAGTGAATGTGTGGGTC-3’ and the biotinylated reverse primer 5’-biotin-ATATGCTAATAAGTCAAGACACCAG3’. The plasmid pGL4-PPAR2 served as DNA template. The pull-down assay was performed as described earlier [41]. Briefly, 1 mg of nuclear cell extract was incubated with 1 µg of the PPAR2 promoter probe. The promoter probe and bound proteins were isolated via a streptavidin-conjugated μMacs™-column. Subsequently, proteins were eluted, separated on a 12.5% SDS-polyacrylamide gel and analysed by Western Blot with specific antibodies.

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2.10 RNA extraction Extraction of RNA was accomplished using the peqGOLD TriFast™ reagent (Peqlab Biotechnology GmbH, Erlangen, Germany) according to the manufacturer’s instructions (phenol/chloroform extraction). Briefly, cells were harvested with

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TriFast™ reagent, phases were separated by the addition of chloroform, vigorous

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shaking and centrifugation. The upper phase was transferred to a new tube and the

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RNA was precipitated for 10 minutes (qRT-PCR) or 30 minutes (miRNA detection) using isopropanol followed by centrifugation. For qRT-PCR, RNA was subsequently

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washed twice with 70% ethanol. After drying, RNA was re-suspended in RNAse-free water and the concentration was measured using an Infinite M200 Pro TECAN

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Reader (Tecan Group Ltd., Männedorf, Switzerland).

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2.11 Quantitative Reverse Transcriptase (qRT) real time PCR

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To measure the gene expression of C/EBP, ,  and PPAR2 the QuantiTect® SYBR® Green RT-PCR Kit (Qiagen GmbH, Hilden, Germany) was used according to

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the manufacturer’s manual. Briefly, 50 ng of total RNA per reaction were reverse transcribed and detected in a one-step reaction using the primer combinations shown

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in table 1. 18 S rRNA served as endogenous control.

Table 1: Primer pairs applied in qRT-PCR 18 S rRNA forward

5’-GTA ACC CGT TGA ACC CCA TT-3’

18 S rRNA reverse

5’-CCA TCC AAT CGG TAG TAG CGA-3’

C/EBP forward

5’-GAC TTC TAC GAG GTG GAG C-3’

C/EBP reverse

5’-TCG ATG TAG GCG CTG ATG TC-3’

C/EBP forward

5’-CAA CTT CTA CTA CGA GCC CGA C-3’

C/EBP reverse

5’-GGA GAG GAA GTC GTG GTG C-3‘

ACCEPTED MANUSCRIPT 11 5‘-AGA CAG TGG TGA GCT TGG C-3’

C/EBP reverse

5’-TCT GCT GCA TCT CCT GGT TG-3’

PPAR2 forward

5’-TCC TGT TGA CCC AGA GCA TG-3’

PPAR2 reverse

5’-CAT CAC GGA GAG GTC CAC AG-3’

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C/EBP forward

To measure the amount of mature miRNAs miR-27a-3p, miR-27b-3p, miR-130a-3p

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and miR-130b-3p the miScript PCR System (Qiagen GmbH, Hilden, Germany) was

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used according to the manufacturer’s instructions. Briefly, 500 ng of total RNA was reverse transcribed using the miScript II RT Kit. Subsequently, 1 ng of the RT

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reaction was used for quantitative real time PCR using the miScript SYBR ® Green PCR Kit and appropriate miScript Primer Assays to detect the miRNAs (technical

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replicates: 2). The miScript PCR control snoRD68 was used for normalization.

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2.12 Urea-polyacrylamide gel electrophoresis and Northern Blot analysis At given time points during differentiation, total RNA was extracted and 15-20 µg were separated in a 12% denaturing urea-polyacrylamide gel. The separated RNA

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was stained with ethidium bromide, documented as loading control and subsequently

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transferred to a nylon membrane (Hybond N, Amersham, Freiburg, Germany) by semi-dry electro blotting. The transferred RNA was cross-linked chemically to the membrane and incubated overnight with the specific [32P]UTP-radio-labelled probe according to table 2. The next day, the membrane was washed twice for 15 min with high salt buffer (5xSSC, 1% SDS) and twice for 15 min with low salt buffer (1xSSC, 1% SDS) and the signal was detected using a storage phosphor screen and a Typhoon Scanner 9410 (GE Healthcare, Munich, Germany). The miRNA pairs miR27a/miR-130b and miR-130a/miR-27b were detected on the same membrane after stripping.

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Table 2: Radiolabelled probes used for detection of miRNAs 5’-UUC ACA GUG GCU AAG UUC CGC CCU GUC UC-3’

miR-27b-3p

5’-UUC ACA GUG GCU AAG UUC UGC CCU GUC UC-3’

miR-130a-3p

5’-CAG UGC AAU GUU AAA AGG GCA UCC UGU CUC-3’

miR-130b-3p

5’-CAG UGC AAU GAU GAA AGG GCA UCC UGU CUC-3’

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miR-27a-3p

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2.13 Statistics

Microsoft Excel 2010 software was used to analyse the data. Results were expressed

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as arithmetic mean ± SD. Differences between the experimental groups were analysed using Student’s t-test (two-tail, unpaired). The following statistical significant

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differences were chosen: ***p < 0.001, **p < 0.01 or *p < 0.05.

3. Results

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In a previous publication [43] we reported that inhibition of CK2 using DMAT and

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quinalizarin prevented adipogenesis in 3T3-L1 cells when the inhibitors were applied at the beginning of the differentiation. The questions at which stages of differentiation CK2 is most important and how CK2 regulates the differentiation process, however, remained open. Hence, in the present study we have analysed the role of CK2 during the differentiation using quinalizarin, which was recently shown to be one of the most selective inhibitors of CK2 [6]. In order to narrow down the time when CK2 is required for differentiation, we started the differentiation process by adding the differentiation mix. The CK2 inhibitor

ACCEPTED MANUSCRIPT 13 quinalizarin was either added together with the differentiation mix (day 0) or at days 2, 3, 4, 5, 6, or 8 after the start of differentiation. Adipocytes were stained with Oil Red O at day 12 of differentiation and the staining was quantified. As shown in Fig. 1 A, addition of quinalizarin together with the differentiation mix or at day 2 or 3 nearly completely inhibited differentiation, whereas from day 4 onwards there was an

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increasing amount of Oil Red O stained cells indicating that differentiation was less

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inhibited by quinalizarin after day 4.

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CK2 activity is usually elevated in rapidly proliferating cells such as cancer cells [34]. 3T3-L1 cells also perform two sequential rounds of mitosis after the start of

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differentiation called mitotic clonal expansion (MCE) [43]. In order to analyse whether inhibition of CK2 might influence the proliferation of 3T3-L1 cells during MCE, cells

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were induced to differentiate in the presence of DMSO or quinalizarin. Cell numbers were determined at the start of differentiation (0 h) as well as at day 2 (24 h) and at

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day 3 (48 h). Fig. 1 B shows that proliferation of 3T3-L1 cells was retarded in the

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presence of 30 µM quinalizarin compared to cells which were treated with the solvent DMSO alone. This observation was confirmed by the detection of phospho-histone

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H3, which is phosphorylated at serine 10 during mitosis [14] and thus can serve as a marker for mitotic cells. The cells treated with quinalizarin contained considerably

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smaller amounts of phosphorylated histone H3 indicating the presence of less mitotic cells (Fig. 1 C). As expected, control experiments with an antibody for total histone H3 revealed more protein at day 3 compared to day 2 but no differences between quinalizarin treated and untreated cells. To exclude that the reduced cell numbers were caused by apoptosis, we performed an MTT assay that detects cell viability by measuring the metabolic activity. As shown in Fig. 1 D the viability of the cells was elevated at day 2, dropped until day 4 and then increased again until the end of differentiation. After quinalizarin treatment viability was slightly reduced compared to

ACCEPTED MANUSCRIPT 14 the control. However, taking account of the cell number reveals that the cells treated with quinalizarin are even more vital than the DMSO control (Fig. 1 E). Thus, we conclude that the CK2 kinase activity is necessary for the proliferation early after induction of differentiation and that there is no apoptosis caused by quinalizarin

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treatment.

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It is well known that a cascade of different transcription factors is implicated in the

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differentiation of pre-adipocytes into adipocytes. The interaction of the most important ones are shown in Fig. 2 A. In order to detect targets of CK2, the expression of the

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transcription factors in the signalling cascade was compared in DMSO- and quinalizarin-treated cells. Cells were harvested at different time points after the start

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of differentiation and proteins in the cell extract were analysed by SDSpolyacrylamide gel electrophoresis followed by Western blot with specific antibodies.

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Chemically induced adipogenesis begins with the phosphorylation of CREB-1 at

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Ser133 [21], which was examined in Fig. 2 B. Total CREB-1 (left panel) showed a slight increase during the first hour of differentiation with a maximum after 30 minutes

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in the DMSO-treated and after 15 minutes in the quinalizarin-treated cell extracts. Phosphorylated CREB-1S133 (right panel) was detected within 15 minutes after the

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start of the differentiation. This increase was transient, decreasing after 2 h. Furthermore, inhibition of CK2 with quinalizarin did not prevent the phosphorylation of CREB-1 at serine 133 indicating the activation of the signalling cascade leading to adipocytes. Next, we analysed the expression of the C/EBP family members C/EBP, C/EBPand CHOP. As shown in Fig. 2 C there were no gross differences in the expression of C/EBP and C/EBP between DMSO- and quinalizarin-treated cell extracts. The overall levels of C/EBP and C/EBP seemed to oscillate during differentiation. The phosphorylation of C/EBP at threonine 235 (T188 in mouse),

ACCEPTED MANUSCRIPT 15 which is necessary for its DNA-binding ability [42], was also abundant under both conditions. The level of CHOP constantly decreased from day 2 to day 6 after the induction of differentiation (Fig. 2 D) without difference between the presence or absence of quinalizarin. In contrast to these observations the expression of PPAR1 and of the transcription factors responsible for the adipogenic phenotype – C/EBP

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and PPAR2 – was strongly inhibited when differentiation was started in the presence

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of quinalizarin (Fig. 2 E). These results indicate that the mode of action of CK2 is

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located upstream of C/EBP and PPAR on the level of their activation, probably by

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targeting the transcription factors C/EBP or C/EBP.

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Transcription factors have to be localised to the nucleus for a proper functioning. Thus, one idea was, that C/EBP and C/EBP might not translocate into the nucleus,

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so that C/EBP and PPAR2 are not activated. Therefore, cells were harvested at

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day 0, day 2 and day 3 in the presence of quinalizarin and fractionated into a cytoplasmic and nuclear fraction. Proteins were analysed by SDS-polyacrylamide gel electrophoresis followed by Western blot with C/EBP and C/EBP specific

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antibodies. In order to prove the purity of the fraction we used an antibody against

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nucleolin for the nuclear fraction and an antibody against HSP70 for the cytoplasmic fraction. Fig. 3 A shows that both, C/EBP and C/EBP were localised in the nuclear fraction with no difference regarding the presence or absence of quinalizarin. In order to verify this result we repeated the experiment, but instead of a cellular subfractionation we analysed the subcellular localisation of C/EBP and C/EBP by immunofluorescence. As shown in Fig. 3 B and C, we found both proteins exclusively in the nucleus, confirming the result obtained by subfractionation. Thus, an altered

ACCEPTED MANUSCRIPT 16 localisation of the transcription factors C/EBP and C/EBPdoes not seem to be the reason for the reduced expression of C/EBP and PPAR2.

As neither an altered protein level nor an altered subcellular localisation of C/EBP and C/EBP seemed to be responsible for the reduced expression of C/EBP and

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PPAR2, we next asked whether there are differences in the amount of mRNA for

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these two transcription factors. Therefore, cells were differentiated in the presence of

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DMSO or quinalizarin, harvested at given time points and the amount of mRNA was analysed by quantitative RT-PCR. As a control we also analysed the level of mRNA

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for C/EBP and C/EBP. As shown in Fig. 4 A, regardless whether the cells were

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treated with quinalizarin or not, there was no gross difference in the amounts of mRNA for C/EBP and C/EBP. However, a drastic reduction of the amount of

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mRNAs for C/EBP and PPAR2 (Fig. 4 B) could be detected in the presence of

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quinalizarin. These results are in perfect agreement with the results presented for the protein levels of all four transcription factors C/EBP, C/EBP C/EBP and PPAR2

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after treatment of the cells with quinalizarin.

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One reason for the decreased level of C/EBP and PPAR2 mRNAs might be a reduction in the DNA binding activity of C/EBP and C/EBP to their promoters and hence a reduced activation of their transcription. To analyse this, we performed a pull-down assay to directly analyse the amount of C/EBP and C/EBP bound to the PPAR2 promoter. C/EBP exists in three isoforms, as liver-enriched activator proteins LAP* (38 kDa) and LAP (35 kDa) and as liver-enriched inhibitory protein LIP (20 kDa). For adipogenic differentiation LAP is the most important form [24], and therefore we quantified this isoform. As shown in Fig. 5 A and quantified in Fig. 5 B,

ACCEPTED MANUSCRIPT 17 there were no gross differences in the DNA binding of C/EBP at day 2 and day 3 of differentiation in the presence of the CK2 inhibitor quinalizarin. There is a slight reduction in the DNA binding of C/EBP in the presence of quinalizarin compared to the DMSO control. This decrease, however, seems not to be significant. Subsequently, we performed an analysis of the transactivation of the PPAR2

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promoter in 3T3-L1 cells induced to differentiate in the presence or absence of

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quinalizarin using a luciferase reporter assay. Already at day 2 and even stronger at

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day 3 of differentiation, we found an elevated transcriptional activation of the PPAR2 promoter in cells treated with quinalizarin (Fig. 5 C). Thus, the results shown in Fig. 5

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A-C revealed that neither the DNA-binding nor the transcription of the PPAR2 promoter seems to be responsible for the down-regulation of the mRNA as shown in

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Fig. 4. As a consequence, it is unlikely that the role of protein kinase CK2 for the

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regulation of the differentiation is implemented by targeting C/EBP and CEBP.

Since the PPAR2 promoter is even activated after CK2 inhibition, the regulation of the reduced expression might be a posttranscriptional event. Over the last ten years

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it turned out that microRNAs play an important role in differentiation in general [18]

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and in adipogenesis in particular [33]. According to our results described above, it was tempting to speculate that microRNAs might be responsible for the degradation of the PPAR2 mRNA after CK2 inhibition. The four microRNAs miR-27a, miR-27b, miR-130a and miR-130b were already described to have an inhibitory effect on the PPAR2 mRNA [19,20,25,26,33]. Therefore, in the present study we focussed on the analysis of the contribution of these microRNAs on adipogenic differentiation under the inhibition of CK2. Thus, at days 0, 2, 4, 8 and 12 of differentiation, cells were harvested, RNA was extracted by phenol/chloroform extraction and analysed for the

ACCEPTED MANUSCRIPT 18 level of the microRNAs in the presence or absence of quinalizarin using Northern hybridization (left panel) and qRT-PCR (right panel). As shown in Fig. 6 the level of all investigated miRNAs was down-regulated in the DMSO control, which is in agreement with published data [19,20,25,26,33]. However, in the presence of quinalizarin the down-regulation of the microRNAs miR-27a and miR-27b was

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significantly attenuated or completely abrogated. In the case of miR-130a and miR-

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130b the difference between the treatments was detectable, but less distinct. Since it

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was already shown that these microRNAs strongly inhibited PPAR and indirectly C/EBP expression [26], these results may explain the low level of PPAR2 and

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C/EBP mRNA.

4. Discussion

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Obesity is a leading cause for death worldwide [16]. It is characterised by the

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expansion of adipose tissue leading to a dys-regulation of glucose and lipid metabolism, which enhances the risk for diabetes, hypertension and dys-lipidemia.

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Therefore, understanding the adipocyte differentiation and the prevention of excessive adipogenesis is urgent. Here, we have used a well-established cellular

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model system to study adipogenesis, namely mouse 3T3-L1 pre-adipocytes, which can be induced to differentiate by the addition of a mix of insulin, dexamethasone and IBMX. It was previously shown that the activity and the amount of protein kinase CK2 increased at early stages of the differentiation of 3T3-L1 cells and declined with proceeding differentiation [40]. Differentiation of 3T3-L1 cells is efficiently inhibited by the addition of CK2 kinase inhibitors [44]. According to the inhibitor experiments, the kinase activity but not the level of CK2 subunits was required for the function of CK2 during differentiation.

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In order to efficiently and specifically inhibit the kinase activity of CK2 we have chosen quinalizarin, which, according to a recent re-evaluation, was found to be one of the most specific CK2 kinase inhibitors [6]. In order to narrow down the optimal time for the inhibition of differentiation, we added either quinalizarin immediately with

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the differentiation mix or at several time points after induction of differentiation. It

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turned out, that quinalizarin can only inhibit differentiation when it is added within the

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first 72 h of differentiation, indicating that CK2 activity is only needed in early stages of differentiation. This finding is supported by our observation, that MCE is retarded

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when CK2 activity is inhibited.

It is known that adipogenic differentiation is accomplished by an activation cascade of

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transcription factors. C/EBP and C/EBP represent early progressive transcription factors, whereas another member of this transcription factor family, CHOP, seems to

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be a repressor of adipogenesis. In many cases up-regulation of CHOP was reported

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to be responsible for the inhibition of adipogenesis [15,45]. As shown here, there is no increase in the level of CHOP after inhibition of CK2. Two other transcription

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factors, PPAR2 and C/EBP are the key regulators of adipogenesis. Whereas the expression and subcellular localisation of C/EBP and C/EBP is not influenced by

reduced. One

of

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quinalizarin treatment, the expression of their targets C/EBP and PPAR2 is mostly

the

mechanisms

that

regulate

transcription

factors

is

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phosphorylation [17]. Likewise, there have already been several transcription factors described that are regulated by phosphorylation conducted by protein kinase CK2 [35]. This result prompted us to focus on the early activation cascade of transcription factors. This idea was supported by the recent observation that C/EBP had been identified as a substrate for CK2 [39]. The phosphorylation site was mapped to serine

ACCEPTED MANUSCRIPT 20 57 within the transactivation domain of C/EBP. Furthermore, it was shown that its transcription factor activity was elevated by CK2 phosphorylation. Our findings, however, show that the reduced expression of C/EBP and PPAR2 is neither regulated by a reduced DNA binding ability of C/EBP and C/EBP nor by a reduced activation of their transcription. Hence, we conclude that protein kinase CK2 does not

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exert its function on adipogenesis by targeting C/EBP and C/EBP in this context.

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Another possible explanation for this observation is an increased expression of

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microRNAs after the inhibition of CK2. MicroRNAs are non-coding RNAs, which function as post-transcriptional gene regulators either by translational repression or

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by mRNA destabilization. MiR-27a and miR-27b have already been identified as new regulators of adipogenesis [26]. MiR-27a has the potential to target over 3000 genes

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indicating that it can regulate a number of different biological processes. Other roles for miR-27a are the cell-cycle regulation in breast cancer cells [30] and the facilitation

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of the growth of gastric cancer cells [28]. In pre-adipocytes miR-27a and miR-27b are

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predominantly expressed and they decrease during adipogenesis while PPAR expression increases. Time course experiments have shown that miR-27a and miR-

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27b block adipogenic differentiation when introduced before or at the start, but not 24

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h after the start of differentiation. MiR-27a and miR-27b bind to a sequence within the PPAR 3’-UTR, which is highly conserved in mammalian cells. MiR-130 likewise potently represses PPAR expression by targeting both the PPAR mRNA coding region and 3’-UTR [25]. Here, we have shown that the expression of miR-27a and miR-27b significantly increased when CK2 was inhibited with quinalizarin. The expression of miR-130a and miR-130b was elevated as well, but to a lesser extent. Our results have further shown that the reduction of mRNA was specific for PPAR2 and C/EBP because both, C/EBP and C/EBP mRNAs were unaffected after

ACCEPTED MANUSCRIPT 21 inhibition of CK2. Thus, we could show an influence on microRNA expression, which is implicated in the regulation of the PPAR and probably indirectly C/EBP mRNA level. These results indicate for the first time an influence of CK2 on the level of microRNAs.

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Funding: This research did not receive any specific grant from funding agencies in

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the public, commercial, or not-for-profit sectors.

5. References

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ACCEPTED MANUSCRIPT 22 Moro, G. Zagotto, L.A. Pinna, Quinalizarin as a potent, selective and cellpermeable inhibitor of protein kinase CK2, Biochem. J. 421 (2009) pp. 387-395. [6] G. Cozza, A. Venerando, S. Sarno, L.A. Pinna, The selectivity of CK2 inhibotor quinalizarin: A reevaluation, BioMed Research International 2015 (2015) p.

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ACCEPTED MANUSCRIPT 24 [20] S.Y. Kim, A.Y. Kim, H.W. Lee, Y.H. Son, G.Y. Lee, J.W. Lee, Y.S. Lee, J.B. Kim, miR-27a is a negative regulator of adipocyte differentiation via suppressing PPARgamma expression, Biochem. Biophys. Res. Commun. 392 (2010) pp. 323-328.

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serum factor, Proc. Natl. Acad. Sci. USA 75 (1978) pp. 6107-6109. [23] U.K. Laemmli, Cleavage of structural proteins during the assembly of the head

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Differentiation 85 (2013) pp. 20-31. [25] E.K. Lee, M.J. Lee, K. Abdelmohsen, W. Kim, M.M. Kim, S. Srikantan, J.L.

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Martindale, E.R. Hutchison, H.H. Kim, B.S. Marasa, R. Selimyan, J.M. Egan, S.R. Smith, S.K. Fried, M. Gorospe, miR-130 suppresses adipogenesis by inhibiting peroxisome proliferator-activated receptor gamma expression, Mol. Cell Biol. 31 (2011) pp. 626-638. [26] Q. Lin, Z. Gao, R.M. Alarcon, J. Ye, Z. Yun, A role of miR-27 in the regulation of adipogenesis, FEBS J. 276 (2009) pp. 2348-2358.

ACCEPTED MANUSCRIPT 25 [27] D.W. Litchfield, Protein kinase CK2: structure, regulation and role in cellular decisions of life and death, Biochem. J. 369 (2003) pp. 1-15. [28] T. Liu, H. Tang, Y. Lang, M. Liu, X. Li, MicroRNA-27a functions as an oncogene in gastric adenocarcinoma by targeting prohibitin, Cancer Lett. 273 (2009) pp.

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[29] 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

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embryonic development, Mol. Cell Biol. 28 (2008) pp. 131-139.

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[30] S.U. Mertens-Talcott, S. Chintharlapalli, X. Li, S. Safe, The oncogenic microRNA-27a targets genes that regulate specificity protein transcription

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Cancer Research 5 (2016) pp. 49-63. [33] N.L. Price and C. Fernandez-Hernando, miRNA regulation of white and brown adipose tissue differentiation and function, Biochim. Biophys. Acta 1861 (2016) pp. 2104-2110. [34] K. Prowald, H. Fischer, O.G. Issinger, Enhanced casein kinase II activity in human tumour cell cultures., FEBS Letters 176 (1984) pp. 479-483.

ACCEPTED MANUSCRIPT 26 [35] A.J. Rabalski, L. Gyenis, D.W. Litchfield, Molecular Pathways: Emergence of Protein Kinase CK2 (CSNK2) as a Potential Target to Inhibit Survival and DNA Damage Response and Repair Pathways in Cancer Cells, Clin. Cancer Res. 22 (2016) pp. 2840-2847.

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[40] J. Sommercorn and E.G. Krebs, Induction of casein kinase II during differentiation of 3T3-L1 cells, J. Biol. Chem. 262 (1987) pp. 3839-3843. [41] S. Spohrer, E.Y. Dimova, T. Kietzmann, M. Montenarh, C. Götz, The nuclear fraction of protein kinase CK2 binds to the upstream stimulatory factors (USFs) in the absence of DNA, Cell Signal. 28 (2016) pp. 23-31. [42] Q.Q. Tang, M. Gronborg, H. Huang, J.W. Kim, T.C. Otto, A. Pandey, M.D. Lane, Sequential phosphorylation of CCAAT enhancer-binding protein beta by MAPK

ACCEPTED MANUSCRIPT 27 and glycogen synthase kinase 3beta is required for adipogenesis, Proc. Natl. Acad. Sci. U. S. A 102 (2005) pp. 9766-9771. [43] Q.Q. Tang, T.C. Otto, M.D. Lane, Mitotic clonal expansion: a synchronous process required for adipogenesis, Proc. Natl. Acad. Sci. U. S. A 100 (2003) pp.

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implicated in early steps of the differentiation of preadipocytes into adipocytes,

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[45] Q.G. Zhou, X. Peng, L.L. Hu, D. Xie, M. Zhou, F.F. Hou, Advanced oxidation protein products inhibit differentiation and activate inflammation in 3T3-L1

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preadipocytes, J. Cell Physiol 225 (2010) pp. 42-51.

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ACCEPTED MANUSCRIPT 28 Figure legends Fig. 1: 3T3-L1 cells were differentiated in the presence of DMSO or 30 µM quinalizarin (Q). (A) Cells were treated with Q at given time points after induced differentiation. The amount of lipid droplets at day 12 was quantified by the measurement of extracted Oil Red O at 515 nm. (B) The proliferation of cells, treated

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with DMSO or Q, was determined at day 0, 2 and 3 after start of differentiation by

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counting living cells. Cell numbers were normalized to day 0 and plotted in a half-

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logarithmic diagram. (C) Phospho-histone H3 protein and total histone H3 protein expression was detected by Western Blotting using 100 µg of total protein. GAPDH

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was used as loading control. (D) At given time points during differentiation viability was detected using an MTT assay. Results were normalized to day 0. Means and

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standard deviations of three independent experiments are shown. (E) Viability was

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normalized to counted cell numbers (D/B) to exclude cell number differences.

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Fig. 2: Expression of transcription factors after induction of differentiation in the presence or absence of quinalizarin. (A) Schematic overview of the transcription

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factor cascade necessary for adipogenic differentiation. (B-E) 3T3-L1 cells were differentiated in the presence of DMSO (D) or 30 µM quinalizarin (Q) dissolved in

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DMSO and harvested at given time points during differentiation. Protein expression was detected in a Western Blot approach (60 to 100 µg of total protein) using specific antibodies. GAPDH or -tubulin were used as loading controls. One representative result of three experiments is shown.

Fig. 3: Subcellular localization of C/EBP and C/EBP

after induction of

differentiation in the presence or absence of quinalizarin. (A) DMSO (D)- or 30 µM Qtreated 3T3-L1 cells (Q) were harvested at 0, 24 and 48 h after the start of

ACCEPTED MANUSCRIPT 29 differentiation. Cytoplasmic (C, 150 µg) and nuclear (N, 75 µg) extracts were separated on a 12.5% SDS-polyacrylamide gel and transferred to a PVDFmembrane. C/EBP and C/EBPwere detected with specific antibodies. (B, C) DMSO- or 30 µM Q-treated 3T3-L1 cells were fixed 24 h after the start of differentiation, permeabilised and C/EBPβ and C/EBP were detected with specific

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antibodies. The DNA was stained with DAPI. Magnification: 400x, scale bar 50 µm.

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Fig. 4: Expression of mRNA for C/EBPs after induction of differentiation in the presence or absence of quinalizarin. 3T3-L1 cells were differentiated in the presence

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of DMSO or 30 µM quinalizarin (Q) and harvested at given time points during differentiation using the peqGOLD TriFast™ reagent. The total RNA was isolated and

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the mRNA amount of C/EBP and C/EBP (A) as well as of PPAR2 and C/EBP (B) was determined using qRT-PCR. The 18 S rRNA served as endogenous control.

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Means and standard deviations of three independent experiments are shown.

Fig. 5: DNA binding and transactivation of C/EBP and C/EBP after induction of

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differentiation in the presence or absence of quinalizarin. (A) DMSO (D)- or 30 µM Q-

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treated 3T3-L1 cells were differentiated for 24 h (day 2) or 48 h (day 3), harvested and cytoplasmic and nuclear proteins were isolated. The DNA-binding of C/EBP and C/EBP was evaluated by incubating nuclear extracts with a biotinylated PPAR2 DNA probe in a streptavidin-agarose pull-down assay. PD: eluted proteins bound to DNA probe; NE: nuclear extract control, 50 µg. (B) Densitometric quantification of results for C/EBP LAP and C/EBP from at least two experiments. Intensity of DMSO treated cells was set to 1. (C) 3T3-L1 cells were transfected with reporter constructs, differentiation was induced in the presence of DMSO or 30 µM Q and

ACCEPTED MANUSCRIPT 30 PPAR2 promoter activity was determined on day 2 or day 3 of differentiation using the Dual-Luciferase Reporter Assay System. The normalized activity of DMSO was set to 1.

Fig. 6: Expression of microRNAs after induction of differentiation in the presence or

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absence of quinalizarin. (A-H) 3T3-L1 cells were differentiated in the presence of

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DMSO (D) or 30 µM quinalizarin (Q) and harvested at given time points during

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differentiation using the peqGOLD TriFast™ reagent. (A, C, E, G) Equal amounts of total RNA were separated on a 12% urea-polyacrylamide gel and blotted to a nylon-

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membrane. MicroRNAs were detected by hybridization of specific radioactive labelled probes overnight and exposition to a phosphor imaging plate. The separated RNA

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was stained with ethidium bromide and used as loading control. One representative result of three independent experiments is shown. (B, D, F, H) Mature miRNAs were

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reverse transcribed and quantified by qPCR. miRNA levels were normalized against

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SNORD68. The normalized expression of day 0 was set to 1. Means and standard deviations of four independent experiments are shown. *p < 0.05; **p < 0.01; ***p <

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ACCEPTED MANUSCRIPT 38 Highlights:

Inhibition of CK2 by quinalizarin prevents adipogenic differentiation of 3T3-L1

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Inhibition of CK2 is effective within the early phase of differentiation

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Inhibition of CK2 compromises the clonal expansion of 3T3-L1 cells

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mRNA and protein of PPAR and C/EBP are down-regulated in CK2-

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The lack of these key proteins is due to an up-regulation of specific miRNAs

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inhibited cells