Inhibition of protein kinase CK2 suppresses tumor necrosis factor (TNF)-α-induced leukocyte–endothelial cell interaction

Biochimica et Biophysica Acta 1852 (2015) 2123–2136

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Inhibition of protein kinase CK2 suppresses tumor necrosis factor (TNF)-α-induced leukocyte–endothelial cell interaction Emmanuel Ampofo a,⁎, Jeannette Rudzitis-Auth a, Indra N. Dahmke a, Oliver G. Rössler b, Gerald Thiel b, Mathias Montenarh b, Michael D. Menger a, Matthias W. Laschke a a b

Institute for Clinical & Experimental Surgery, Saarland University, 66421 Homburg/Saar, Germany Medical Biochemistry and Molecular Biology, Saarland University, 66421 Homburg/Saar, Germany

a r t i c l e

i n f o

Article history: Received 9 December 2014 Received in revised form 29 June 2015 Accepted 15 July 2015 Available online 17 July 2015 Keywords: Leukocyte adhesion NF-κB ICAM-1 VCAM-1 Dorsal skinfold chamber Intravital fluorescence microscopy

a b s t r a c t Inflammatory endothelial processes are regulated by the nuclear factor-κB (NF-κB) pathway, which involves phosphorylation of p65. Because p65 is a substrate of CK2, we herein investigated, whether this pleiotropic protein kinase may be a beneficial anti-inflammatory target. For this purpose, we analyzed in human dermal microvascular endothelial cells (HDMEC) the effect of CK2 inhibition by quinalizarin and CX-4945 on cell viability, adhesion molecule expression and NF-κB pathway activation. Leukocyte binding to HDMEC was assessed in an in vitro adhesion assay. Dorsal skinfold chambers in BALB/c mice were used to study leukocyte– endothelial cell interaction and leukocyte transmigration by means of repetitive intravital fluorescence microscopy and immunohistochemistry. We found that quinalizarin and CX-4945 effectively suppressed the activity of CK2 in HDMEC without affecting their viability. This was associated with a significant down-regulation of tumor necrosis factor (TNF)-α-induced E-selectin, intercellular adhesion molecule (ICAM)-1 and vascular cell adhesion molecule (VCAM)-1 expression due to a reduction of shuttling, phosphorylation and transcriptional activity of the NF-κB complex. In consequence, leukocyte binding to quinalizarin- and CX-4945-treated HDMEC was diminished. Finally, CX-4945 treatment significantly decreased the numbers of adherent and transmigrated leukocytes in dorsal skinfold chambers exposed to TNF-α in vivo. These findings indicate that CK2 is a key regulator of leukocyte–endothelial cell interaction in inflammation by regulating the expression of E-selectin, ICAM-1 and VCAM-1 via affecting the transcriptional activity of the NF-κB complex. Accordingly, CK2 represents a promising target for the development of novel anti-inflammatory drugs. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Leukocyte–endothelial cell interaction is a key process in tumor necrosis factor (TNF)-α-induced inflammation and is characterized by a coordinated sequence of adhesive interactions between leukocytes and the microvascular endothelium [1,2]. Upon TNF-α stimulation, activated endothelial cells express different cell adhesion proteins on their surface. These include selectins, such as E-selectin, which promote leukocyte rolling along the endothelial lining [3] and members of the immunoglobulin superfamily, such as intercellular adhesion molecule (ICAM)-1 and vascular cell adhesion molecule (VCAM)-1, which mediate firm leukocyte adhesion to the endothelium [4,5]. Firm adhesion is a major prerequisite for the subsequent transendothelial migration of leukocytes into inflamed tissue [6]. A fundamental TNF-α-induced cellular pathway, which mediates the expression of E-selectin, ICAM-1 and VCAM-1 during inflammatory conditions, involves signaling via nuclear factor-κB (NF-κB) [7]. In non⁎ Corresponding author. E-mail address: [email protected] (E. Ampofo).

http://dx.doi.org/10.1016/j.bbadis.2015.07.013 0925-4439/© 2015 Elsevier B.V. All rights reserved.

stimulated cells, the transcription factor p65, which is a component of the heterodimeric NF-κB complex, is sequestered by IκBα proteins in the cytoplasm [8,9]. Under stimulation with TNF-α, these IκBα proteins are phosphorylated and subsequently degraded. This degradation promotes the translocation of p65 from the cytoplasm into the nucleus, where it acts as a transcriptional activator for E-selectin, ICAM-1 and VCAM-1 [10–13]. The transcriptional activity of p65 is dependent on its phosphorylation, which is regulated by a variety of different kinases, including protein kinase CK2, formerly known as casein kinase II [14,15]. The CK2 holoenzyme is a tetramer consisting of two catalytic subunits CK2α and CK2α′ and two non-catalytic subunits CK2β [16]. The consensus sequence contains serine or threonine residues surrounded by acidic amino acids [17]. This kinase is involved in various cellular processes, including proliferation, differentiation, apoptosis and angiogenesis [18–21]. However, the specific function of CK2 on leukocyte–endothelial cell interaction during TNF-α-induced inflammation remains elusive. Therefore, the aim of the present study was to analyze in vitro the effect of CK2 inhibition on i) endothelial expression of E-selectin, ICAM1 and VCAM-1, ii) subcellular localization and transcriptional activity of

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p65 in endothelial cells and iii) leukocyte binding to endothelial cells. In addition, dorsal skinfold chambers were prepared in BALB/c mice, treated with CX-4945 or vehicle, to study in vivo the effect of CK2 inhibition on TNF-α-up-regulated leukocyte–endothelial cell interaction and leukocyte transmigration by means of repetitive intravital fluorescence microscopy and immunohistochemistry. 2. Materials and methods 2.1. Chemical and biological reagents Endothelial Cell Basal Medium (C-22210) was purchased from PromoCell (Heidelberg, Germany), γ[32P]ATP from Hartmann Analytic (Braunschweig, Germany), human TNF-α from Provitro (Berlin, Germany), murine TNF-α from PeproTech (Hamburg, Germany), calcein-AM from Molecular Probes (Eugene, OR, USA), CX-4945 from AdooQ (Irvine, CA, US), quinalizarin, hexadimethrine bromide, fluorescein isothiocyanate (FITC)-labeled dextran 150,000 and rhodamine 6G from Sigma-Aldrich (München, Germany), a set of three small interfering RNA (siRNA) duplexes directed against CK2α, CK2α′ and non-targeting siRNA control (ON-TARGET plus SMARTpools) from Dharmacon/GE Healthcare (Germany), HiPerFect transfection reagent from Qiagen (Hilden, Germany), 4′,6-diamidino-2′-phenylindole dihydrochloride (DAPI) from Roche (Mannheim, Germany), Mayer's hemalaun solution from Merck (Darmstadt, Germany), ketamine (Ursotamin®) from

Pharmacia GmbH (Erlangen, Germany), and xylazine (Rompun®) from Bayer (Leverkusen, Germany). 2.2. Endothelial cell culture Human dermal microvascular endothelial cells (HDMEC) were purchased from PromoCell and cultivated in Endothelial Cell Basal Medium at 37 °C under a humidified 95% to 5% (v/v) mixture of air and CO2. Cells were passaged at a split ratio of 1:3 after reaching confluence. All experiments were carried out with confluent cells between the third and seventh passage. 2.3. Antibodies Anti-GAPDH antibody (FL335), anti-α-tubulin antibody (sc-53646), anti-p65 antibody (sc-372) and anti-phospho-p65 (Ser529) antibody (sc-101751) were obtained from Santa Cruz Inc. (Heidelberg, Germany). The antibodies anti-CD54 (ICAM-1) (555511), anti-CD106 (VCAM-1) (555647), anti-CD31 (platelet endothelial cell adhesion molecule (PECAM-1)) (555446), anti-CD62E (E-selectin) (551145) and IgG1-κ Isotype Control (555749) were bought from BD Biosciences (Heidelberg, Germany). The antibodies anti-CK2α #26 and anti-CK2α′ #30 were generated as described previously [22]. Peroxidase-labeled anti-rabbit antibody (NIF 824) and peroxidase-labeled anti-mouse antibody (NIF 825) were purchased from GE healthcare (Freiburg, Germany).

Fig. 1. CX-4945 and quinalizarin decreases CK2 kinase activity without affecting cell viability. (A) HDMEC were cultivated with DMSO (vehicle) or the indicated concentrations of CX-4945 or quinalizarin (Q) in the presence of 10 ng/mL TNF-α for 5 h. After cell lysis, CK2 kinase activity was measured by CK2 kinase assay. Vehicle treated cells were used as control and set 100%. Mean ± SEM. *P b 0.05 vs. vehicle. (B) Expression of CK2α and GAPDH in HDMEC, which were cultivated as described above and assessed by Western blot analysis. Western blots are representative of experiments conducted in triplicate. (C) Formazan reduction (% of vehicle) of HDMEC, which were cultivated without TNF-α, with TNF-α (10 ng/mL) and DMSO (vehicle) or the indicated concentrations of CX-4945 or quinalizarin (Q) for 5 h. Viability was determined by WST-1 assay. Mean ± SEM. (D) LDH release (% of vehicle) of HDMEC, which were treated as described in (C) and analyzed by LDH assay. Lysed HDMEC (shaded bar) served as high control. Mean ± SEM. *P b 0.05 vs. vehicle.

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Cy3-conjugated anti-rabbit antibody (111-165-144) was from Jackson ImmunoResearch Laboratories (West Groves, PA, USA). The antimyeloperoxidase (MPO) antibody (ab9535) and the secondary biotinylated goat anti-rabbit antibody (ab64256) were obtained from Abcam (Cambridge, UK). 2.4. CK2 in vitro phosphorylation assay HDMEC were lysed on ice for 10 min in lysis buffer (RIPA buffer: 50 mM Tris–HCl, pH 7.2, 0.15 M NaCl, 1.0 mM EDTA, 0.1% SDS, 1.0% Triton X-100, 1.0% sodium deoxycholate). Then, extracts were used for kinase filter assays. The incorporation rate of [32P] phosphate into the CK2-specific substrate peptide with the sequence RRRDDDSDDD was measured. 20 μL of kinase buffer (50 mM Tris/HCl, pH 7.5, 100 mM NaCl, 10 mM MgCl2, 1 mM DTT) containing 30 μg of proteins was mixed with 30 μL CK2 mix (25 mM Tris-HCl, pH 8.5, 150 mM NaCl, 5 mM MgCl2, 50 μM ATP, 1 mM DTT, 0.19 mM substrate peptide) containing 10 μCi/500 μL γ[32P]ATP. The mixture was spotted onto a P81 ion exchange paper. The paper was washed three times with 85 mM H3PO4. After treatment with ethanol the paper was dried and the Čerenkov-radiation was determined in a scintillation counter.

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2.8. Western blot analysis Nuclear and cytoplasmic extracts were prepared as previously described [25]. Proteins were separated through a 10% SDS polyacrylamide gel and transferred onto a PVDF membrane. The membrane was blocked with 5% dry milk in PBS (0.1% Tween20) for 1 h and then incubated with the specific antibodies, which were diluted in PBS (0.1% Tween20) containing 1% dry milk. The membrane was incubated with a peroxidase-coupled secondary antibody (anti-rabbit 1:30.000 or anti-mouse 1:10.000) for 1 h. Protein expression was visualized by luminol-enhanced chemiluminescence (ECL; GE-Healthcare, Freiburg, Germany) and exposure of the membranes to a blue light sensitive autoradiography film (Hyperfilm ECL, GE-Healthcare). 2.9. siRNA transfection HDMEC were reversely transfected with siRNA duplexes (100 nM) directed against CK2α and CK2α′ or with scramble-siRNA as a control, using HiPerFect transfection reagent for 72 h. The transfection was refreshed every 24 h. The medium was then removed and the cells were stimulated with fresh medium containing 10 ng/mL TNF-α for 5 h. Subsequently, the cells were used for flow cytometry or Western blot analyses.

2.5. Water-soluble tetrazolium (WST)-1 assay 2.10. Isolation of leukocytes A WST-1 assay was used to measure the metabolic activity of treated HDMEC. In this assay, WST salt is reduced to formazan and the absorbance can be measured with a microplate reader at 480 nm. The WST1 assay was carried out according to the manufacturer's instructions. HDMEC were seeded in a 96 well cell culture plate and treated with 10 ng/mL TNF-α in combination with different concentrations of the CK2 inhibitors quinalizarin or CX-4945 or vehicle (DMSO) in a total volume of 100 μL for 5 h. Then, 10 μL WST-1 reagent per well was added and the plate was incubated for 1 h at 37 °C. The absorption was measured at 480 nm with 620 nm as reference using a microplate reader and corrected to blank values (wells without cells).

Venous blood from four human healthy donors was collected into citrate tubes and 1:1 diluted with PBS. Afterwards, mononuclear cells (MNC) were isolated on a Ficoll density gradient by centrifugation for 25 min at 1600 rpm. Interphase cells were collected, washed with PBS by centrifugation for 8 min at 1600 rpm and resuspended in a small volume of 1 × PBS. MNC were counted by a Coulter Counter (AcTdiff, Coulter, Hamburg, Deutschland) and stained with 500 μL of calceinAM (8 μg/mL) for 15 min. Stained cells were washed by centrifugation for 8 min at 1600 rpm and were used for the adhesion assay. 2.11. Leukocyte adhesion assay

2.6. Lactate dehydrogenase (LDH) assay PLUS

, Roche) was used to A LDH assay (Cytotoxicity Detection Kit analyze the cytotoxicity of the test compounds according to the manufacturer's instructions. For this purpose, HDMEC were treated as described above (WST-1 assay). After treatment, 100 μL of LDH-reaction mix per 100 μL medium was added to each well. After 10 min of incubation at room temperature in the dark, absorption was measured at 492 nm with 620 nm as reference after the addition of 50 μL stop solution using a microplate reader and corrected to blank values (wells without cells).

HDMEC were seeded in a 24 well cell culture plate in a total volume of 1 mL/well and allowed to grow to confluence. Then, the medium was removed and cells were stimulated with fresh medium containing 10 ng/mL TNF-α in combination with different concentrations of the CK2 inhibitors or vehicle (DMSO) for 5 h. After that, cells were washed three times and 1 × 105 freshly isolated MNC were added and incubated for 1 h in a total volume of 1 mL medium. After co-cultivation, cells were gently washed three times in PBS and images of labeled cells that adhered to the HDMEC were taken by fluorescence microscopy (BZ8000; Keyence, Osaka, Japan). Stained MNC were counted in two randomly selected microscopic fields in each well by means of the ImageJ 1.47 software.

2.7. Lentiviral gene transfer and luciferase assay 2.12. Immunofluorescence microscopy The lentiviral transfer vector pFWHIVLTRluc that contains the HIV LTR sequence (sequence from −120 to +83) upstream of the luciferase open reading frame, has been described recently [23]. Viral particles were produced as previously described [24] by triple transfection of 293T/17 cells with the gag–pol–rev packaging plasmid, the env plasmid encoding VSV glycoprotein and the transfer vector. Viral particles were added to HDMEC seeded in a 24 well cell culture plate (1 mL/well volume, 90% confluence) for 20 h in the presence of hexadimethrine (8 μg/mL). Following infection, cells were washed and cultivated in 1 mL/well of Endothelial Cell Basal Medium for 48 h before drug treatment. Cell extracts were prepared using reporter lysis buffer (Promega, Mannheim, Germany) and analyzed for luciferase activities. Luciferase activity was normalized to the protein concentration (relative luciferase units (RLU)/mg protein).

HDMEC were seeded on coverslips. After 24 h cells were stimulated with medium containing 10 ng/mL TNF-α in combination with different concentrations of CX-4945, quinalizarin or DMSO (vehicle) for 5 h. Then, cells were fixed in PBS with 3.7% paraformaldehyde for 10 min, permeabilized in PBS with 0.5% Triton X-100 for 10 min and blocked in PBS with 5% bovine serum albumin (BSA) for 30 min at room temperature. Afterwards, cells were incubated with the anti-p65 or anti-phospho-p65 (Ser529) antibodies for 30 min at 37 °C, washed with 1× PBS and incubated with Cy3-conjugated anti-rabbit antibody for 30 min at 37 °C. The coverslips were washed and incubated with DAPI for 30 min at 37 °C. The coverslips were sealed with mounting media and analyzed by fluorescence microscopy (BX60F; Olympus Optical, Tokyo, Japan).

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Fig. 3. Silencing of CK2 by siRNA reduces expression of cell adhesion molecules. (A) HDMEC were treated with siRNA against CK2α and CK2α′ or scrambled siRNA for 72 h. Subsequently, HDMEC were stimulated with 10 ng/mL TNF-α for 5 h and the protein levels of CK2α and CK2α′ were assessed by Western blot analysis. Western blots are representative of experiments conducted in triplicate. (B) CK2/α-tubulin (% of scr-siRNA), as assessed by quantitative analysis of Western blots. Mean ± SEM. *P b 0.05 vs. scr-siRNA. (C–E) HDMEC were treated as described above and the fold increase MFI E-selectin (C), ICAM-1 (D) and VCAM-1-positive cells (E) were analyzed by flow cytometry. Scrambled siRNA-treated cells were used as control and set 100%. Mean ± SEM. *P b 0.05 vs. control.

2.13. Flow cytometry HDMEC were seeded in a 24 well cell culture plate in a total volume of 1 mL/well and allowed to grow to confluence. Then, the medium was removed and cells were stimulated with fresh medium containing 10 ng/mL TNF-α supplemented with different concentrations of the CK2 inhibitors quinalizarin or CX-4945 or DMSO (vehicle) for 5 h. Cells were washed and detached by incubation for 15 min at 37 °C with accutase. Subsequently, cells were fixed in PBS containing 1% formalin for 10 min at 4 °C, washed by centrifugation (1800 ×g for 10 min, RT) in PBS and incubated with the antibodies against E-selectin, ICAM-1, VCAM-1, PECAM-1 or control antibody (isotype control) for 30 min at room temperature. Thereafter, cells were washed in PBS, centrifugated and fixed with PBS containing 1% formalin. The fluorescence per 5000 cells was analyzed in a FACScan flow cytometer (Becton

Dickinson, San Jose, CA, USA) using CellQuest software. Because unstimulated cells exhibited high levels of PECAM-1, E-selectin and ICAM-1, the expression of these adhesion molecules was determined by analyzing the mean fluorescence intensity in the FL2 channel. In contrast, the expression of VCAM-1 was measured by counting numbers of VCAM-1 positive cells. 2.14. Ethics statement All animal care and experimental procedures were approved by the local governmental animal care committee (Landesamt für Verbraucherschutz, Abteilung C Lebensmittel- und Veterinärwesen, Saarbrücken, Germany; Permit Number: 08/2013) and were conducted in accordance with the European legislation on protection of animals (Guideline 2010/63/EU) and the NIH Guidelines for the Care and Use

Fig. 2. Inhibition of CK2 affects TNF-α-induced expression of cell adhesion molecules. (A, C, E, G) Flow cytometry histograms: Open histogram areas represent PECAM-1, E-selectin, ICAM-1 and VCAM-1 expression of HDMEC treated with vehicle (DMSO) and TNF-α (10 ng/mL) for 5 h, filled histogram areas represent HDMEC treated with CX-4945 (50 μM) and TNF-α (10 ng/mL) for 5 h. (B, D, F, H) Quantitative analysis of (A, C, E, G). HDMEC were cultivated without TNF-α, with TNF-α (10 ng/mL) and DMSO (vehicle) or the indicated concentrations of CX-4945 or quinalizarin (Q) for 5 h. Fold increase MFI PECAM-1 (B), E-selectin (D), ICAM-1 (F) and VCAM-1-positive cells (H) were analyzed by flow cytometry. Vehicle treated cells were used as control and set 100%. Mean ± SEM. *P b 0.05 vs. vehicle.

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of Laboratory Animals (http://oacu.od.nih.gov/regs/index.htm. 8th Edition; 2011). 2.15. Dorsal skinfold chamber model BALB/c mice with a body weight of 20–25 g were used for the experiments. They were housed in a temperature-controlled environment under a 12 h/12 h light–dark cycle and received standard pellet food (Altromin, Lage, Germany) and water ad libitum. The dorsal skinfold chamber model was used to investigate the effect of CK2 inhibition on leukocyte–endothelial cell interaction in vivo. The chamber was prepared as described previously in detail [26]. Briefly, mice were anesthetized by an intraperitoneal (i.p.) mixture of ketamine (75 mg/kg) and xylazine (15 mg/kg). Two symmetrical titanium frames were implanted on the extended dorsal skinfold of the animals, so that they sandwiched the double layer of the skin. Next, one layer of the skin was completely removed in a circular area of 15 mm in diameter. The remaining layers consisting of the epidermis, subcutaneous tissue and striated skin muscle were covered with a glass coverslip. To avoid alterations of the microcirculation due to anesthesia or surgical trauma, the mice were allowed to recover for 72 h before the in vivo analyses. Subsequently, the chamber tissue was topically exposed to 200 ng murine TNF-α (dissolved in 100 μL saline) to induce a local inflammation [27,28]. To analyze the effect of CK2 inhibition on leukocyte–endothelial cell interaction, a group of 10 animals was treated with 75 mg/kg CX4945 i.p. (dissolved in 50 μL DMSO) 19 h and 1 h before exposure to TNF-α, whereas 10 vehicle-treated animals served as controls. In additional experiments, a group of 4 animals was treated again with CX4945 or vehicle, as described above. However, the chamber tissue was topically exposed to saline instead of TNF-α. 2.16. Intravital fluorescence microscopy For in vivo microscopic observation, mice were immobilized on a Plexiglas stage and the dorsal skinfold chamber was attached to the microscopic stage. After retrobulbary i.v. injection of 0.05 mL 5% FITCdextran 150,000 for contrast enhancement by staining of blood plasma and 0.05 mL 0.1% rhodamine 6G for staining of leukocytes, intravital epiillumination fluorescence microscopy was performed using a Zeiss microscope (Zeiss, Oberkochen, Germany) with a 100 W mercury lamp attached to a blue and green filter block. The microscopic images were recorded by a charge-coupled device video camera (FK6990; Pieper, Schwerte, Germany) and transferred to a monitor (Trinitron; Sony, Tokyo, Japan) and DVD system (DVD-HR775; Samsung, Eschborn, Germany) for off-line evaluation. Quantitative off-line analyses of the microscopic images were performed using the software package CapImage (Zeintl, Heidelberg, Germany). For this purpose, eight postcapillary or collecting venules per chamber were randomly selected for the determination of leukocyte–endothelial cell interaction, microhemodynamic parameters and macromolecular leakage at baseline conditions 19 h before topical exposure of the chamber tissue to TNF-α as well as 0.5 h, 2 h and 4 h after TNF-α treatment. Leukocytes were classified according to their interaction with the microvascular endothelium as rolling or adherent cells [29]. Rolling leukocytes were defined as cells moving with a velocity less than two-fifths of the centerline velocity, and are given as number of cells per minute, passing a reference point within the microvessel. Adherent leukocytes

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were defined as cells that did not move or detach from the endothelial lining within a 20 s observation period, and are given as number of cells per square millimeter of endothelial surface. Endothelial surface was calculated from the diameter and length of the vessel segment studied, assuming a cylindrical vessel geometry. Diameters, centerline red blood cell (RBC) velocity, volumetric blood flow, and wall shear rate were determined in those venules in which leukocyte–endothelial cell interaction was analyzed. Diameters (d) were measured in μm perpendicularly to the vessel path. Centerline RBC velocity (v) was analyzed by the computer-assisted image analysis system using the line shift method [30]. Volumetric blood flow was calculated by Q = π ∗ (d ∕ 2)2 ∗ v ∗ 1.6 [pL/s], where 1.6 represents the Baker– Wayland factor [31] to correct for the parabolic velocity profile in microvessels with diameters N20 μm. Additionally, wall shear rate (y) was calculated based on the Newtonian definition: y = 8 ∗ v ∕ d. Macromolecular leakage as a parameter of microvascular permeability and, thus, endothelial integrity was assessed after intravenous injection of the macromolecular fluorescent dye FITC-labeled dextran 150,000 by determining densitometrically gray levels in the tissue directly adjacent to the venular vessel wall (E1), as well as in the marginal cell free plasma layer within the vessel (E2). Extravasation (E) was then calculated as E = E1 ∕ E2 [32]. At the end of the in vivo experiments, the animals were sacrificed with an overdose of the anesthetic and the dorsal skinfold preparations were excised for further immunohistochemical analyses. 2.17. Immunohistochemistry Formalin-fixed specimens of the dorsal skinfold chamber preparations were embedded in paraffin. For the analysis of transmigrated leukocytes, 2 μm-thick sections were cut and stained with a polyclonal rabbit anti-mouse antibody against the neutrophil granulocyte marker myeloperoxidase (MPO) followed by biotinylated secondary goat antirabbit IgG antibody. Sections solely incubated with the secondary antibody were used as negative controls. The sections were counterstained with Mayer's hemalaun solution and examined by light microscopy (BX60F). MPO-positive cells were assessed in 10 high power fields (HPFs) per section. 2.18. Statistical analysis After testing the data for normal distribution and equal variance, differences between two groups were analyzed by the unpaired Student's t-test and differences between multiple groups were analyzed by ANOVA followed by the appropriate post hoc test, including correction of the alpha error according to Bonferroni's probabilities (SigmaStat; Jandel Corporation, San Rafael, CA, USA). To test for time effects in the individual groups, ANOVA for repeated measures was applied followed by the Bonferroni-t-test. All values are expressed as means ± SEM. Statistical significance was accepted for a value of P b 0.05. 3. Results 3.1. Effect of CX-4945 and quinalizarin on CK2 kinase activity and cell viability In a first set of experiments, we analyzed the effect of the two inhibitors CX-4945 and quinalizarin on the CK2 kinase activity in endothelial

Fig. 4. CK2 inhibition affects subcellular localization of p65. (A) Immunofluorescence images of HDMEC, which were cultivated without TNF-α, with TNF-α (10 ng/mL) and DMSO (vehicle), CX-4945 (50 μM) or (Q) quinalizarin (50 μM) for 1 h or 5 h. P65 was detected with an anti-p65 antibody and labeled by Cy3-conjugated secondary antibody. Cell nuclei were stained with DAPI. Scale bars: 20 μm. Images are representative of experiments conducted in triplicate. (B) HDMEC were treated for 5 h as described above and the subcellular localization of p65 was analyzed by Western blot analysis. Western blots are representative of experiments conducted in triplicate. (C and D) p65/α-tubulin (% of vehicle) (C) and p65/nucleolin (% of vehicle) (D), as assessed by quantitative analysis of Western blots. Mean ± SEM. *P b 0.05 vs. vehicle.

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cells. To mimic inflammatory conditions in cell culture, HDMEC were exposed to the indicated concentrations of CX-4945 (1–50 μM) or quinalizarin (1–50 μM) in the presence of 10 ng/mL TNF-α for 5 h. Both inhibitors decreased the activity of CK2 (Fig. 1A). Notably, the inhibitory effect of CX-4945 was more pronounced when compared to quinalizarin. Western blot analyses of the cells further revealed that the inhibition of CK2 activity by both compounds was not associated with a reduced expression of the catalytic subunit CK2α of the holoenzyme (Fig. 1B). For the determination of cell viability after CK2 inhibition, we performed two different cytotoxicity assays, i.e. LDH and WST-1 assays. For this purpose, HDMEC were identically treated as described above. Under these conditions, inhibition of CK2 with CX4945 or quinalizarin did not affect the viability of the cells (Fig. 1C and D). 3.2. Effect of CK2 inhibition on surface protein expression of HDMEC Fluorescence cytometry was performed to assess the expression of PECAM-1, E-selectin, ICAM-1 and VCAM-1 on TNF-α-stimulated and non-stimulated HDMEC, which were exposed to different concentrations of the CK2 inhibitors CX-4945 or quinalizarin (Fig. 2A, C, E and G). We found that PECAM-1 expression on HDMEC was neither affected by TNF-α nor by TNF-α in combination with the two CK2 inhibitors (Fig. 2B). In contrast, TNF-α significantly increased the endothelial expression of E-selectin, ICAM-1 and VCAM-1 (Fig. 2D, F and H). Of interest, this effect of TNF-α was diminished by CX-4945 and quinalizarin (Fig. 2D, F and H), indicating that CK2 kinase activity is required for TNFα-induced up-regulation of E-selectin, ICAM-1 and VCAM-1 expression. Noteworthy, treatment of HDMEC solely with the CK2 inhibitors CX4945 and quinalizarin did not affect the expression of PECAM-1, Eselectin, ICAM-1 and VCAM-1 (data not shown). To exclude that the observed effects of CX-4945 and quinalizarin on the expression of E-selectin, ICAM-1 and VCAM-1 were primarily caused by off target effects of the inhibitors, we additionally transfected HDMEC with siRNA against CK2α and α′. In this control experiment, silencing of CK2 reduced the levels of CK2α and α′ in the primary cells to 55% and 69% of controls (Fig. 3A and B). The expression of E-selectin, ICAM-1 and VCAM-1 was reduced to a similar extent (Fig. 3C–E). 3.3. Effect of CK2 inhibition on subcellular localization of p65 and phosphorylated p65 and its transcriptional activity The expression of E-selectin, ICAM-1 and VCAM-1 is controlled by the NF-κB pathway. Moreover, the transcriptional activity of p65, a component of this pathway, is strictly regulated by its shuttling from the cytoplasm into the nucleus as well as its posttranscriptional modifications. It has been shown that CK2 phosphorylates p65 on serine 529 and the loss of this phosphorylation decreases the transcriptional activity of p65 [14,33]. To determine whether inhibition of CK2 affects shuttling of p65 between the cytoplasm and nucleus we analyzed its subcellular localization 1 h and 5 h after TNF-α stimulation (Fig. 4A). As expected, p65 was solely localized in the cytoplasm of unstimulated HDMEC. Treatment of HDMEC for 1 h with TNF-α showed a translocation of p65 into the nucleus. After 5 h treatment we detected a partial redistribution of p65 from the nucleus to the cytoplasm. Interestingly, treatment of HDMEC with TNF-α and CX-4945 or quinalizarin elevated

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the nuclear localization of p65 after 1 h and 5 h. This indicates that inhibition of CK2 led to an accumulation of p65 in the nucleus. These findings were confirmed by additional Western blot analyses, quantifying the cytoplasmatic and nuclear levels of p65 in quinalizarin- and CX-4945treated HDMEC after 5 h (Fig. 4B–D). To exclude that the inhibitors solely affect the subcellular localization of p65, HDMEC were treated for 5 h with CX-4945 or quinalizarin. Under these conditions, p65 was mainly detected in the cytoplasm (data not shown). To further clarify whether the inhibition of CK2 affects the phosphorylation of p65, HDMEC were treated as described above and phosphorylated p65 was detected with phospho-specific antibodies against the CK2 phosphorylation site serine 529 in the polypeptide chain of p65. We found that quinalizarin and CX-4945 markedly decreased the nuclear signal of phosphorylated p65 in TNF-α-stimulated HDMEC when compared to vehicle-treated controls (Fig. 5A–C). Finally, we analyzed the transcription activity of the NF-κB complex in HDMEC. We used lentiviral gene transfer to integrate a luciferase reporter gene under the control of the HIV LTR into the chromatin of HDMEC to ensure that the reporter gene is embedded into a nucleosomal structure. The LTR contains two binding sites for NF-κB. A schematic depiction of the integrated provirus is shown in Fig. 5D. In line with previous results [33], the transcriptional activity of NF-κB was increased in TNF-α-stimulated HDMEC when compared to untreated cells (Fig. 5E). Moreover, NF-κB reporter activity was lower in HDMEC, which were treated with TNF-α and CX-4945 or quinalizarin (Fig. 5E). Taken together, these results indicate that inhibition of CK2 markedly decreased the NF-κB gene expression in TNF-α-stimulated endothelial cells. In control experiments, we analyzed the subcellular localization and phosphorylation of p65 in siRNA-treated HDMEC. In line with our experiments using the CK2 inhibitors, we found that CK2 silencing also significantly decreased the nuclear signal of phosphorylated p65 (Fig. 6A–E). 3.4. Effect of CK2 inhibition on leukocyte binding to endothelial cells in vitro Because ICAM-1 and VCAM-1 mediate firm leukocyte adhesion to the inflamed endothelium, we next examined the binding capacity of leukocytes to quinalizarin- or CX-4945-treated HDMEC under TNF-αstimulation. For this purpose, HDMEC were treated without, with TNFα and vehicle or the indicated concentration of CX-4945 or quinalizarin for 5 h and subsequently co-cultivated with MNC from whole blood for 1 h. As shown in Fig. 7A treatment of HDMEC with TNF-α increased binding of MNC whereas CX-4945 (50 μM) or quinalizarin (50 μM) decreased the binding capacity. Quantification of bound MNC revealed the inhibitory effect of the CK2 inhibitors on MNC binding to HDMEC (Fig. 7B). 3.5. Effect of CK2 inhibition on leukocyte–endothelial cell interaction in vivo The effect of CK2 inhibition by CX-4945 on leukocyte–endothelial cell interaction was analyzed in postcapillary and collecting venules of TNF-α-exposed dorsal skinfold chambers. For this purpose, we first assessed important microhemodynamic parameters of the analyzed vessels. In line with former studies [34,35], we found that TNF-α stimulation increased the venular diameter and volumetric blood flow when

Fig. 5. CK2 inhibition affects phosphorylation of p65. (A) Immunofluorescence images of HDMEC, which were cultivated without TNF-α, with TNF-α (10 ng/mL) and DMSO (vehicle), CX-4945 (50 μM) or (Q) quinalizarin (50 μM) for 1 h or 5 h. Phopho-p65 was detected with an anti-phospho-p65 antibody and labeled by Cy3-conjugated secondary antibody. Cell nuclei were stained with DAPI. Scale bars: 20 μm. Images are representative of experiments conducted in triplicate. (B) HDMEC were treated for 5 h as described above and the nuclear localization of phospho-p65 was analyzed by Western blot analysis. Western blots are representative of experiments conducted in triplicate. (C) Phospho-p65/nucleolin (% of vehicle), as assessed by quantitative analysis of Western blots. Mean ± SEM. *P b 0.05 vs. vehicle. (D) Schematic representation of the integrated provirus encoding an HIVLTR/luciferase reporter gene. The LTR sequence is upstream of the luciferase open reading frame. The location of the NF-κB and Sp1 binding sites within the LTR is depicted. Additionally the nucleotide sequences of the NF-κB binding sites are shown. The U3 region of the 5′ LTR of the transfer vector is deleted. The locations of the woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) and the HIV flap element are indicated. (E) RLU (% of vehicle) of HDMEC, which infected with a recombinant lentivirus encoding the NF-κB-responsive HIVLTR/luciferase reporter gene for 20 h. HDMEC were serum-starved for 48 h and afterward cultivated without TNF-α, with TNF-α (10 ng/mL) and DMSO (vehicle) or the indicated concentrations of CX-4945 or quinalizarin (Q) for 5 h. Cell extracts were prepared and analyzed for luciferase activities. Luciferase activity was normalized to the protein concentration. Vehicle treated cells were used as control and set 100%. Mean ± SEM. *P b 0.05 vs. vehicle.

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Fig. 7. CK2 inhibition reduces leukocyte–endothelial cell interaction in vitro. (A) Immunofluorescence images of leukocyte bound to HDMEC. HDMEC were cultivated without TNF-α, with TNF-α (10 ng/mL) and DMSO (vehicle) or the indicated concentrations of CX-4945 or quinalizarin (Q) for 5 h. Then, isolated MNC from whole blood were stained with calcein-AM and co-cultured for 1 h with the treated HDMEC. After washing, bound MNC were analyzed by immunofluorescence. Scale bars: 400 μm. (B) Quantitative analysis of (A). Vehicle-treated cells were used as control and set 100%. Mean ± SEM. *P b 0.05 vs. vehicle.

Fig. 6. Inhibition of CK2 by siRNA affects subcellular localization and phosphorylation of p65. (A) HDMEC were treated with siRNA against CK2α and CK2α′ or scrambled siRNA (control) for 72 h. After HDMEC were stimulated with 10 ng/mL TNF-α for 5 h, the subcellular localization of p65 was analyzed by Western blot. Western blots are representative of experiments conducted in triplicate. (B and C) p65/nucleolin (% of scr-siRNA) (B) and p65/α-tubulin (% of scr-siRNA) (C), as assessed by quantitative analysis of Western blots. Mean ± SEM. (D) HDMEC were treated as described above and the phosphorylation of p65 was assessed by Western blot analysis. Western blots are representative of experiments conducted in triplicate. (E) Phospho-p65/nucleolin (% of scr-siRNA), as assessed by quantitative analysis of Western blots. Mean ± SEM. *P b 0.05 vs. scr-siRNA.

compared to baseline conditions (Table 1). Throughout the entire observation period, however, we did not detect any significant differences in diameter, centerline RBC velocity, wall shear rate and volumetric blood

flow of venules in CX-4945-treated and vehicle-treated control animals (Table 1). This indicates that the leukocyte–endothelial cell interaction could be analyzed in both groups under comparable microhemodynamic conditions. TNF-α stimulation of the chamber tissue resulted in an elevated number of rolling and adherent leukocytes and a reduced functional capillary density in CX-4945-treated and vehicle-treated control animals (Fig. 8A– D). However, in line with our in vitro results, the numbers of adherent leukocytes were significantly lower in CX-4945-treated mice 0.5 h and 4 h after TNF-α treatment (Fig. 8C). This was associated with a markedly decreased macromolecular leakage (Fig. 8E), indicating an improved endothelial integrity. In an additional set of control experiments, animals were solely treated with CX-4945 or vehicle without exposure to TNF-α. Under these non-inflamed conditions, CX-4945 did not affect leukocyte– endothelial cell interaction, functional capillary density and macromolecular leakage within the chamber tissue (Fig. S1).

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Table 1 Diameter (μm), centerline RBC velocity (μm/s), wall shear rate (s−1) and volumetric blood flow (pL/s) of postcapillary and collecting venules in dorsal skinfold chambers of CX-4945-treated and vehicle-treated BALB/c mice 19 h before as well as 0.5 h, 2 h and 4 h after topical TNF-α application. −19 h Diameter (μm) Vehicle CX-4945 Centerline RBC velocity (μm/s) Vehicle CX-4945 Wall shear rate (s−1) Vehicle CX-4945 Volumetric blood flow (pL/s) Vehicle CX-4945

+0.5 h

+2 h

+4 h

32.3 ± 2.4 36.1 ± 2.3

37.2 ± 3.1 39.3 ± 2.7

46.4 ± 3.8a 51.2 ± 3.9a

45.5 ± 3.3a 50.7 ± 4.2a

407.8 ± 38.8 455.5 ± 50.6

539.9 ± 110.8 500.1 ± 62.8

451.3 ± 77.3 425.2 ± 65.3

496.9 ± 72.1 441.7 ± 74.2

103.84 ± 10.8 104.9 ± 14.5

115.3 ± 20.4 104.4 ± 14.3

76.3 ± 12.4 65.8 ± 6.4a

87.7 ± 12.4 75.1 ± 16.3a

227.1 ± 45.2 302.2 ± 58.8

444.5 ± 149.8 420.3 ± 105.1

581.3 ± 173.8b 645.1 ± 204.4b

573.7 ± 145.2b 638.7 ± 196.6b

Mean ± SEM. a P b 0.05 vs. −19 h and +0.5 h in each individual group. b P b 0.05 vs −19 h in each individual group.

Additional immunohistochemical analyses of dorsal skinfold chamber preparations 4 h after TNF-α exposure further exhibited a significantly reduced number of transmigrated MPO-positive leukocytes in CX-4945treated animals (5.3 ± 0.5/HPF) when compared to vehicle-treated controls (10.5 ± 0.3/HPF). 4. Discussion The interaction of leukocytes with the microvascular endothelium of inflamed tissue is a highly dynamic process, which is tightly regulated by a complex network of intracellular signaling pathways mediating the expression of adhesion proteins on the surface of endothelial cells and leukocytes [36]. In the present study, protein kinase CK2 was identified as a novel key regulator of this process. In fact, we could demonstrate that inhibition of CK2 suppresses the transcriptional activity of the NF-κB complex and down-regulates the expression of E-selectin, ICAM-1 and VCAM-1. This is associated with a reduced leukocyte adhesion to endothelial cells under TNF-α-induced inflammatory conditions. During the last years, several studies reported that inhibition of CK2 selectively induces apoptosis in tumor cells relative to normal cells [37]. Accordingly, CK2 has been suggested as a potential therapeutic target for the development of novel cancer drugs [38–42]. For this purpose, a broad spectrum of CK2 inhibitors has been developed, including quinalizarin and more recently CX-4945. By now, the latter one is considered to be the most specific CK2 inhibitor with a binding affinity of Ki = 0.38 nM [43]. In contrast, quinalizarin exhibits a binding affinity of Ki = 0.052 μM [38], which is comparable to that of other commonly used CK2 inhibitors, such as 2-dimethylamino-4,5,6,7-tetrabromo-1Hbenzimidazole (DMAT), tetrabromocinnamic acid (TBCA) and 4,5,6,7tetrabromo-1H-benzotriazole (TBB) [44–46]. Quinalizarin and CX-4945 have previously been shown to effectively reduce the activity of CK2 in animal studies focusing on cancer [40,42] and endometriosis [19]. Moreover, CX-4945 has even successfully entered phase II clinical trials [47]. Therefore, we decided to use quinalizarin and CX-4945 to study the effect of CK2 inhibition on the expression of adhesion molecules and on leukocyte binding to endothelial cells in vitro. A reduced CK2 activity in TNF-α-stimulated HDMEC resulted in a decreased expression of the adhesion proteins E-selectin, ICAM-1 and VCAM-1. The expression of these proteins is regulated by a combination of several transcription factors, including NF-κB [13,48–52]. The NF-κB pathway is a fundamental pathway in inflammatory processes. Downregulation of any member of this pathway results in a decreased inflammatory response. P65 is a key factor of the NF-κB pathway, whose transcriptional activity or subcellular localization is crucially determined by phosphorylation events [53–55]. In former studies, it has been shown that CK2 phosphorylates p65 on serine 529 and that the loss of this

phosphorylation decreased the transcriptional activity of p65 [14,33]. In the present study, we found that inhibition of CK2 by CX-4945 or quinalizarin decreased the level of phosphorylated p65 as well as the expression of a luciferase reporter gene controlled by NF-κB binding sites. In addition, we detected an accumulation of nuclear p65 in TNF-αstimulated HDMEC after CK2 inhibition. The inhibitor protein IκBα, which is part of a negative feedback loop for terminating the activity of NF-κB, is necessary for the rapid degradation of p65 after stimulation [56]. Hochreiner et al. reported that hypo-phosphorylation leads to accumulation of p65 in the nucleus due to a defective IκBα synthesis [57]. Thus, it is tempting to speculate that the loss of p65 phosphorylation by CK2 prevents its shuttling from the nucleus into the cytoplasm by down-regulation of this negative feedback loop resulting in nuclear p65 accumulation. PECAM-1 is constitutively expressed on the surface of endothelial cells [58,59]. On the other hand, NF-κB binding sites have been identified in the promoter region of PECAM-1 [60], indicating that this protein may be further up-regulated under inflammatory conditions. However, we did not detect a marked effect on the expression of PECAM-1 in TNFα-stimulated HDMEC. These results are in line with a previous study of Romer et al. [61], demonstrating that the expression of PECAM-1 is not altered in human umbilical venular endothelial cells (HUVEC) in the presence of TNF-α. In addition, we found that PECAM-1 expression on HDMEC is not affected by the treatment with quinalizarin or CX-4945. This shows that CK2 is not generally involved in the regulation of all endothelial adhesion proteins, and, thus, is a promising candidate for the selective targeting of specific cell–cell interactions. ICAM-1 is a ligand for leukocyte adhesion protein (LFA)-1 [62], whereas VCAM-1 interacts with integrin α4β1 on leukocytes [63]. Both proteins specifically mediate firm leukocyte adhesion to the microvascular endothelium. Accordingly, inhibition of CK2 by quinalizarin or CX-4945 markedly decreased the number of bound MNC in our leukocyte adhesion assay. To confirm these in vitro results in vivo, we used the dorsal skinfold chamber, which has been proven to be a suitable model for the analysis of leukocyte–endothelial cell interaction under physiological and inflammatory conditions by means of intravital fluorescence microscopy [27,28,64]. For this purpose, we repetitively assessed the number of rolling and adherent leukocytes in postcapillary and collecting venules of TNF-α-treated chambers. The endothelium of these vessels typically acts as a gatekeeper to leukocyte adhesion and transmigration into the surrounding tissue [65]. Because the interaction of leukocytes and endothelial cells is dependent on intravascular blood flow and wall shear rates [66], we first analyzed microhemodynamic parameters of the vessels, which did not differ between CX-4945treated and vehicle-treated control animals. Under these standardized microhemodynamic conditions, inhibition of CK2 by CX-4945

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Fig. 8. Inhibition of CK2 reduces leukocyte adhesion in vivo. (A) Intravital fluorescence microscopic images of postcapillary venules in dorsal skinfold chambers of CX-4945-treated and vehicle-treated BALB/c mice 19 h before as well as 4 h after topical TNF-α application. Blue-light epi-illumination with contrast enhancement by staining of plasma with FITC-dextran or green-light epi-illumination with staining of leukocytes (arrows) with rhodamine 6G. Scale bars: 50 μm. (B–E) Rolling leukocytes (min−1) (B), adherent leukocytes (mm−2) (C), functional capillary density (cm−1) (D) and macromolecular leakage (E1/E2) (E) in dorsal skinfold chambers of CX-4945-treated and vehicle-treated BALB/c mice 19 h before as well as 0.5 h, 2 h and 4 h after topical TNF-α application, as assessed by intravital fluorescence microscopy and computer-assisted image analysis. Mean ± SEM. *P b 0.05 vs. vehicle. aP b 0.05 vs −19 h in each individual group; bP b 0.05 vs. −19 h and + 0.5 h in each individual group.

resulted in a significantly reduced number of adherent leukocytes to the microvascular endothelium. However, we did not detect a difference in numbers of rolling leukocytes despite our in vitro finding that CX-4945 suppresses the expression of E-selectin. This unexpected observation may be due to the fact that other members of the selectin family, such as Pselectin, are coexpressed in inflamed tissue [67]. Studies indicate that there is an overlapping function of these selectins in leukocyte rolling [68]. Accordingly, they may have compensated the downregulation of E-selectin in the present experimental setting. We additionally detected a markedly decreased number of transmigrated leukocytes in CX-4945-treated chambers 4 h after stimulation with TNF-α. This observation may be primarily due to the

reduced expression of VCAM-1 after CK2 inhibition, because VCAM-1 does not only mediate firm leukocyte adhesion to endothelial cells, but is also crucially involved in diapedesis [5]. Besides, there are some evidences that CK2 phosphorylates proteins of the extracellular matrix, such as vitronectin, which interacts with integrin αvβ3 on leukocytes [69,70]. Integrin αvβ3, in turn, has also been shown to be involved in the process of transmigration [71]. Phosphorylation of tight junction components is critically involved in the regulation of tight junction assembly, maintenance and function. Previous studies indicate that some of these components are substrates of CK2 [72,73]. Thus, it may be speculated that inhibition of CK2 disrupts endothelial integrity. However, in the present study we observed a

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reduced macromolecular leakage in chambers of CX-4945-treated mice when compared to vehicle-treated controls. A possible explanation for this finding may be the beneficial effect of CK2 inhibition on leukocyte-mediated endothelial injury in our experimental setting of TNF-α-induced tissue inflammation. In conclusion, we have demonstrated that protein kinase CK2 regulates TNF-α-stimulated leukocyte–endothelial cell interaction. In fact, inhibition of CK2 suppresses NF-κB gene expression by affecting the phosphorylation state of p65 and its subcellular localization. This down-regulates the expression of E-selectin, ICAM-1 and VCAM-1, which is associated with a reduced leukocyte adhesion to endothelial cells. Accordingly, CK2 represents a promising target for the development of novel anti-inflammatory drugs. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.bbadis.2015.07.013. Transparency document The Transparency document associated with this article can be found, in the online version. Acknowledgments We are grateful for the excellent technical assistance of Ruth M. Nickels. References [1] E.C. Butcher, Leukocyte–endothelial cell adhesion as an active, multi-step process: a combinatorial mechanism for specificity and diversity in leukocyte targeting, Adv. Exp. Med. Biol. 323 (1992) 181–194. [2] E.C. Butcher, Leukocyte–endothelial cell recognition: three (or more) steps to specificity and diversity, Cell 67 (1991) 1033–1036. [3] A. Zarbock, K. Ley, R.P. McEver, A. Hidalgo, Leukocyte ligands for endothelial selectins: specialized glycoconjugates that mediate rolling and signaling under flow, Blood 118 (2011) 6743–6751. [4] C. Lawson, S. Wolf, ICAM-1 signaling in endothelial cells, Pharmacol. Rep. 61 (2009) 22–32. [5] H.E. Matheny, T.L. Deem, J.M. Cook-Mills, Lymphocyte migration through monolayers of endothelial cell lines involves VCAM-1 signaling via endothelial cell NADPH oxidase, J. Immunol. 164 (2000) 6550–6559. [6] L. Lindbom, X. Xie, J. Raud, P. Hedqvist, Chemoattractant-induced firm adhesion of leukocytes to vascular endothelium in vivo is critically dependent on initial leukocyte rolling, Acta Physiol. Scand. 146 (1992) 415–421. [7] P. Viatour, M.P. Merville, V. Bours, A. Chariot, Phosphorylation of NF-kappaB and IkappaB proteins: implications in cancer and inflammation, Trends Biochem. Sci. 30 (2005) 43–52. [8] T. Guan, Q. Liu, Y. Qian, H. Yang, J. Kong, J. Kou, B. Yu, Ruscogenin reduces cerebral ischemic injury via NF-kappaB-mediated inflammatory pathway in the mouse model of experimental stroke, Eur. J. Pharmacol. 714 (2013) 303–311. [9] F. Mercurio, H. Zhu, B.W. Murray, A. Shevchenko, B.L. Bennett, J. Li, D.B. Young, M. Barbosa, M. Mann, A. Manning, A. Rao, IKK-1 and IKK-2: cytokine-activated IkappaB kinases essential for NF-kappaB activation, Science 278 (1997) 860–866. [10] F. Aoudjit, N. Brochu, B. Belanger, C. Stratowa, J. Hiscott, M. Audette, Regulation of intercellular adhesion molecule-1 gene by tumor necrosis factor-alpha is mediated by the nuclear factor-kappaB heterodimers p65/p65 and p65/c-Rel in the absence of p50, Cell Growth Differ. 8 (1997) 335–342. [11] M.S. Hayden, S. Ghosh, Shared principles in NF-kappaB signaling, Cell 132 (2008) 344–362. [12] L.L. Paxton, L.J. Li, V. Secor, J.L. Duff, S.M. Naik, N. Shibagaki, S.W. Caughman, Flanking sequences for the human intercellular adhesion molecule-1 NF-kappaB response element are necessary for tumor necrosis factor alpha-induced gene expression, J. Biol. Chem. 272 (1997) 15928–15935. [13] U. Schindler, V.R. Baichwal, Three NF-kappa B binding sites in the human E-selectin gene required for maximal tumor necrosis factor alpha-induced expression, Mol. Cell. Biol. 14 (1994) 5820–5831. [14] T.A. Bird, K. Schooley, S.K. Dower, H. Hagen, G.D. Virca, Activation of nuclear transcription factor NF-kappaB by interleukin-1 is accompanied by casein kinase IImediated phosphorylation of the p65 subunit, J. Biol. Chem. 272 (1997) 32606–32612. [15] M.S. Brown, O.T. Diallo, M. Hu, R. Ehsanian, X. Yang, P. Arun, H. Lu, V. Korman, G. Unger, K. Ahmed, C. Van Waes, Z. Chen, CK2 modulation of NF-kappaB, TP53, and the malignant phenotype in head and neck cancer by anti-CK2 oligonucleotides in vitro or in vivo via sub-50-nm nanocapsules, Clin. Cancer Res. 16 (2010) 2295–2307. [16] M. Salvi, S. Sarno, O. Marin, F. Meggio, E. Itarte, L.A. Pinna, Discrimination between the activity of protein kinase CK2 holoenzyme and its catalytic subunits, FEBS Lett. 580 (2006) 3948–3952.

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