A critical role for CK2 in cytokine-induced activation of NFκB in pancreatic β cell death

This study aimed to assess the role of constitutive protein kinase CK2 in cytokine-induced activation of NFκB in pancreatic β cell death. The CK2 inhibitors DRB (5,6-dichloro-1-β-d-ribofuranosylbenzimidazole) (50 μM) and DMAT (2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole) (5 μM), which decreased CK2 activity by approx. 65 %, rescued INS-1E β cells and mouse islets from cytokine (IL-1β, TNF-α plus IFN-γ)-induced β cell death without affecting H2O2- or palmitate-induced β cell death. Western blot analysis revealed that while DRB or DMAT did not influence cytokine-induced IκBα degradation, they inhibited NFκB-dependent IκBα resynthesis, demonstrating that cytokine-induced NFκB activity is dependent on CK2. Both DRB and DMAT inhibited the constitutive phosphorylation of NFκB p65 at serine 529, while leaving cytokine-induced phosphorylations of NFκB p65 at serines 276 and 536 unaltered. In comparison, putative phosphorylation sites for CK2 on HDACs 1, 2, and 3 at serines 421/423, 394, and 424, respectively, which may stimulate NFκB transcriptional activity, were unchanged by cytokines and CK2 inhibitors. Whereas IL-1β and TNF-α stimulate IκBα degradation and NFκB activation, IFN-γ potentiates cytokine-induced β cell death through activation of STAT1. DRB and DMAT inhibited IFN-γ-stimulated phosphorylation of STAT1 at serine 727, while leaving IFN-γ-induced phosphorylation of STAT1 at tyrosine 701 unaffected. Inhibition of cytokine-induced β cell death by CK2 inhibitors was, however, not dependent on IFN-γ, and IFN-γ did not affect CK2-dependent IκBα turnover. In conclusion, it is suggested that cytokine-induced activation of NFκB in β cells is dependent on CK2 activity, which phosphorylates NFκB p65 at serine 529.


Introduction
Diabetes mellitus is characterized by a decline in b cell mass and function leading to insufficient insulin secretion and hyperglycemia [1]. In type 1 diabetes mellitus, pancreatic islet inflammation contributes to the progressive loss of insulin producing b cells and inflammatory mediators, e.g., IL-1b, TNF-a, and IFN-c contribute to the suppression of b cell function and subsequent apoptosis [2,3]. In addition, recent evidence suggests that islet inflammation may contribute to pancreatic b cell death in type 2 diabetes mellitus [4].
CK2 (formerly casein kinase 2) is a highly conserved serine/threonine kinase [5], which also has been identified in b cells [6]. This constitutively active and ubiquitously expressed enzyme is known to phosphorylate more than 300 substrates and controls a wide range of processes, including the regulation of cell cycle, apoptosis, and inflammation. Although CK2 is a constitutive active enzyme, its activity has been described to be modulated by several factors, including IL-1b, TNF-a, and IFN-c, which have been shown to stimulate CK2 activity in different cell types [5]. Moreover, CK2 phosphorylates and stimulates several cytokine-stimulated transcription factors, including nuclear factor-kappa B (NFjB) and signal transducer and activator of transcription (STAT1), key regulators of the inflammatory response [5].
NFjB is activated by a range of stimuli, including inflammatory cytokines, like IL-1b and TNF-a, and is generally regarded as one of the most important master regulators of the inflammatory response [7]. NFjB is a dimer composed of the five mammalian Rel proteins, p65-RelA, c-Rel, RelB, NFjB1-p50, and NFjB2-p52 where p65-p50 is the predominant form in b cells [2]. In unstimulated cells, NFjB dimer is complexed with proteins called inhibitor of jB (IjBs), which sequester the transcription factor in the cytoplasm by masking their nuclear localization sequence. Activators of NFjB initiate multiple intracellular signaling pathways that converge at IjB kinase (IKK) complex, which phosphorylates IjBs. This phosphorylation is a prerequisite for the subsequent ubiquitinylation and degradation of IjBs via the proteasomal pathway. NFjB is now free to migrate into the nucleus and activate gene transcription by binding to its recognition sequence in the promoter and enhancer regions of target genes [7].
CK2 has been found to phosphorylate IjBa at a number of sites, including serines 283, 289, 293, and threonine 291, and this has been found, at least in certain context, to trigger the degradation of the protein and thereby activation of NFjB [8,9]. In addition to IjB, the NFjB p65 subunit has also been found to be subject to CK2-mediated phosphorylation with implications to inflammation [10]. NFjB p65 is phosphorylated at serines 276, 529, and 536, and whereas CK2 may increase phosphorylation of serine 276 and 536, serine 529 has been described to be a specific substrate for CK2 [11][12][13][14].
In addition, NFjB activity in b cells is dependent on histone deacetylases (HDACs), which through deacetylation of histones promote NFjB binding to DNA and gene expression [15,16]. HDAC1, HDAC2, and HDAC3, which are present in b cells, have been shown to be phosphorylated by CK2 at serines 421/423, serine 394, and serine 424, respectively [17][18][19]. Overall, therefore, these findings suggest a potentially important role for CK2 in the regulation and expression of genes implicated in inflammation through the control of NFjB action.
In most tissues, NFjB activation inhibits cell death. However, under certain circumstances, or in certain cell types as in b cells, activation of NFjB may promote cell death [2]. IL-1b mediates activation of the transcription factor NFjB pathway, a pathway partly shared by TNF-a. IFN-c signaling involves induction of JAKs 1 and 2, leading to activation of STAT1 through phosphorylation of STAT1 at tyrosine 701 [2]. In addition, IFN-c action may be fortified by a CK2-dependent phosphorylation of STAT1 at serine 727 [20]. The concerted action of activated transcription factors initiates a cascade of events leading to the deleterious effects on b cells and to apoptosis in type 1 diabetes mellitus. In this process, NFjB appears to play a central role. Thus, whereas IL-1b and TNF-a trigger NFjB activation, induction of iNOS and apoptosis, IFN-c fortifies b cell death by mechanisms which may involve potentiation of NFjB activity, and iNOS expression [3,21]. Overall, therefore, CK2 may be speculated to promote cytokine-induced NFjB activity and b cell death through phosphorylations of IjBa, NFjB p65, STAT1, and HDACs 1, 2, and 3.
While the concept of insulitis in type 1 diabetes mellitus is well-established, its role in type 2 diabetes mellitus is disputed [3]. Elevation of circulating nutrients such as glucose and free fatty acids (FFAs) induces an inflammatory response within numerous tissues in the body and has been shown to induce IL-1b release in human and rodent islets [4]. Glucolipotoxicity in type 2 diabetes mellitus leading to b cell death has, however, been described to proceed independent of NFjB activation [3,22,23] and may rely on endoplasmic reticulum stress in response to prolonged elevation in FFAs [3,22]. Furthermore, prolonged elevations in glucose and FFAs may lead to oxidative stress with formation of reactive oxygen species (ROS) like O 2 •-, OH • , and H 2 O 2 , which may stimulate b cell death through NFjB-independent pathways [3,23].
The role of CK2 in b cell inflammation and apoptosis has not been investigated. This study, therefore, aimed at investigating the role of CK2 in cytokine-, FFA-, and ROSinduced b cell death. It is demonstrated that constitutive active CK2 does not affect palmitate-or H 2 O 2 -induced b cell death. CK2, however, stimulates cytokine-induced NFjB activity, most likely through phosphorylation of NFjB p65 at serine 529, and furthermore, phosphorylation of STAT1 at serine 727 appears to be dependent on CK2. Thus, in comparison with numerous other cell types, CK2 seems to have a selective pro-apoptotic function in pancreatic b cells.
Isolation and culture of islets Islets were prepared by collagenase digestion of the pancreas of male albino mice (NMRI) (approx. 18-22 g body weight) (Charles River Laboratories Sulzfeld, Germany) fed ad libitum on a standard laboratory diet. Principles of laboratory animal care as approved by The Danish Animal Experiments Inspectorate were followed. Islets were kept in tissue culture for 48-72 h in a 5 % CO 2 incubator in RPMI 1640 medium (11 mM glucose) supplemented with glutamax (2 mM), 10 % (v/v) newborn calf serum (Gibco, Paisley, Strathclyde, UK), 100 units penicillin/ml, and 100 lg streptomycin/ml. Cytokines [IL-1b (160 pg/ml), TNF-a (2 ng/ml), and IFN-c (200 pg/ml)] and other test substances as specified in the text were present during the last 48 h of culture.

MTT assay
The proportion of viable cells in treated versus control cells was determined using the MTT [3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide] assay, which measures succinate dehydrogenase activity [25]. Briefly, INS-1E cells or groups of 50 islets were incubated with 500 lg MTT/ml RPMI 1640 medium for 3 h at 37°C. At the end of the incubation, the medium containing MTT was removed, and islets or cells were dissolved in DMSO. Absorbance was then measured at 540 and 690 nm [24].

Apoptosis assay
The proportion of apoptotic cells in treated versus control islets was determined by the Cell Death Detection ELI-SA PLUS kit (Roche Diagnostics, Mannheim, Germany). Briefly, groups of 25 islets were lysed in 200 ll lysis buffer (Roche Diagnostics, Mannheim, Germany) followed by measurement of histone-associated DNA fragments as outlined by the manufacturer.

Western blot analysis
After culture, INS-1E cells were washed twice in PBS??, lysed in 100 ll radioimmunoprecipitation assay (RIPA) buffer (1 % Triton X-100, 0.5 % sodium deoxycholate, 0.1 % sodium dodecyl sulfate, 1 mM 4-(2-aminoethyl)benzene sulfonyl fluoride, 1 mM orthovanadate, 2 lM okadaic acid, 10 mM b-glycerophosphate, 10 mM NaF, 1 lg/ml aprotinin, and 1 lg/ml leupeptin in PBS), and centrifuged at 11,0009g for 3 min. Protein concentration was then determined in supernatants using the BCA protein assay (Sigma Chemical Co., St. Louis, MO). 50 lg of protein was loaded and separated by electrophoresis on 7 % NuPage Novex Tris-Acetate gels in Tris-Acetate running buffer or 12 % NuPage Novex Bis-Tris gels in Mops running buffer as appropriate (Invitrogen, Taastrup, Denmark) and transferred to nitrocellulose membranes by electroblotting. After incubation of the membrane in blocking buffer (Tris-buffered saline containing 5 % nonfat dry milk), the membrane was exposed overnight at 4°C to primary antibody diluted 1:1,000 in blocking buffer. The membrane was subsequently washed in Tris-buffered saline containing 0.1 % Tween 20, incubated with peroxidaseconjugated goat antirabbit IgG (Dako, Glostrup, Denmark), and proteins detected by chemiluminescence using ECL plus Western blotting detection reagent (GE Healthcare, Chalfont St. Giles, UK). Proteins were then quantified relative to controls on the same gel with ImageJ software as described [26]. b-actin served as a loading control.

Insulin secretion
Insulin release from islets was determined by perifusion in a noncirculating system with beads of 0.25 ml Bio-Gel P2 (Bio-Rad Laboratories, Rockville Center, NY, USA) as a supporting medium. 25 islets per chamber were perifused at 37°C at a flow rate of 0.26 ml/min. The perifusion Endocrine (2014) 47:117-128 119 medium was Krebs-Ringer medium supplemented with 20 mM HEPES, 5 mM NaHCO 3 , 2 mg/ml of bovine serum albumin/ml, and 3.3 mM glucose. Islets were initially perifused for 45 min to obtain a basal release rate and then stimulated with 16.7 mM glucose for 60 min. The effluent medium was collected for periods of 5 or 10 min. Insulin was determined by radioimmunoassay [27].

Islet insulin content
Groups of ten islets were collected in 1 ml of acidified ethanol (0.7 M HCl/ethanol, 1:3 v/v) and incubated for approx. 20 h at 4°C for extraction of insulin [28].
Miscellaneous DRB, DMAT, and andrographolide were added in a small volume of DMSO, final conc. 0.01-0.1 %. Results are given as mean ± SEM for n experiments. Statistical evaluation of the data was made by t test or ANOVA, followed by the Newman-Keuls test for multiple comparisons; not significant, P [ 0.05.

DRB and DMAT inhibit cytokine-induced b cell death
To evaluate the role of CK2 in b cell survival, INS-1E cells were exposed to increasing concentrations of the CK2 inhibitor DRB (Fig. 1a). While DRB in concentrations above 50 lM appeared detrimental to INS-1E cells, DRB at 50 lM and below did not affect b-cell survival. 50 lM of DRB, however, led to a significant protection against b cell death induced by a cytokine mixture of IL-1b, TNF-a, and IFN-c. In addition, DMAT, another CK2 inhibitor, at a concentration of 5 lM, with minimal effect on b cell survival per se, also increased b-cell survival during a 48 h exposure to cytokines (Fig. 1b). Furthermore, both DRB (50 lM) and DMAT (5 lM), which per se did not affect the survival of mouse islets, improved survival of mouse islets during a 48 h exposure to cytokines (Fig. 1c, d). In accordance, DRB (50 lM) also prevented cytokine-induced apoptosis in mouse islets  (Fig. 3), suggesting that CK2 may have a specific role in promoting cytokine-induced b cell death.
DRB (50 lM) and DMAT (5 lM) caused an approximately 65 % inhibition of CK2 activity, measured as phosphorylation of the CK2 substrate RRRDDDSDDD, in cell extracts of both INS-1E cells and mouse islets ( Table 1).

Role of NFjB in CK2-stimulated b cell death
NFjB is a master regulator of cytokine-induced b-cell death and as demonstrated in Fig. 4 inhibition of NFjB by the NFjB inhibitor andrographolide protects both INS-1E cells and mouse islets from cytokine-induced b cell death. To investigate the potential role of NFjB in CK2-mediated b cell death, we, therefore, aimed at investigating the effect of CK2 inhibitors on NFjB activation and activity.
CK2 has been described to phosphorylate, and at least in certain context, to trigger the degradation of IjBa and  Results are mean ± SEM (n) thereby the activation of NFjB [8,9]. However, neither DRB (50 lM) nor DMAT (5 lM) affected cytokineinduced degradation of IjBa as determined after 30 min of incubation (Fig. 5a, b). IjBa contains a jB site in its promoter. To investigate whether CK2 affects NFjB transcriptional activity, the effects of DRB (50 lM) and DMAT (5 lM) on IjBa resynthesis were evaluated. As demonstrated in Fig. 5a, b, extended exposure of b cells to cytokines for 2 h led to a rapid resynthesis of IjBa, which was abrogated by both DRB (50 lM) and DMAT (5 lM), suggesting a critical role for CK2 in NFjB transcriptional activity.
In comparison, NFjB p65 expression as determined after 30 min and 2 h was not affected by CK2 inhibitors and cytokines (Fig. 5c, d).
In addition to IjBa, the NFjB p65 subunit has been found to be subject to CK2-mediated phosphorylation with implications to inflammation. Thus, while serine 529 has been identified as a specific phosphorylation site for CK2, other studies also point at serine 276 and serine 536 as possible targets for CK2 [11][12][13][14].
Culture of INS-1E cells for 30 min or 2 h with cytokines did not affect CK2 activity in cell extracts (Fig. 6), suggesting that cytokines may not increase CK2 activity in b cells. In accordance, exposure of INS-1E cells to cytokines led to phosphorylation of NFjB p65 at serine 536 and to a lesser extent at serine 276, which was insensitive to DRB (50 lM) (Fig. 7a, b). In comparison, however, serine 529 appeared to be constitutively phosphorylated, and sensitive to dephosphorylation by both DRB (50 lM) (Fig. 7c) and DMAT (5 lM) (Fig. 7d), suggesting that constitutive CK2 activity may be necessary for NFjB activity.
Role of STAT1 in CK2-stimulated b cell death Inhibition of CK2 has been described to attenuate the IFNc-regulated expression of several key genes in other tissues, and this is mediated, at least in part, via modulation of STAT1 phosphorylation on serine 727, necessary for maximal activity [20].

Discussion
According to the present experiments, CK2 may have an indispensable and specific function in cytokine-induced b cell death. Hence CK2 inhibitors failed to affect both H 2 O 2 -and palmitate-induced b cell death, but appeared to prevent IL-1b plus TNF-a-induced activation of NFjB, which is considered to be a master regulator of cytokineinduced b cell death. Indeed, while cytokines apparently failed to stimulate CK2 activity, constitutive active CK2 phosphorylated NFjB p65 at serine 529, which is a specific phosphorylation site for CK2.
Phosphorylation of NFjB p65 at serine 529 has previously been demonstrated to be indispensable in intestinal epithelial inflammation [14]. The relative importance of NFjB serine 276, 536, and 529 phosphorylations in cytokine signaling in pancreatic b cells is, however, not known, and since in one study only phosphorylation of serine 276 seemed to be of significance in cytokine signaling in b cells [31], it remains to be fully established that the sole dephosphorylation of serine 529 explains the inhibition of NFjB activity and cell death by CK2 inhibitors.
Cytokines have been described to activate p38 MAP kinase in pancreatic islets [32,33], and p38 MAP kinase has been described to activate CK2 in other tissues [34,35]. The role of p38 MAP kinase in cytokine-induced b-cell death is, however, disputed, and some data suggest that cytokines may not activate p38 MAP kinase in pure b-cell populations [36]. Accordingly, the p38 MAP kinase inhibitor SB203580 did not prevent cytokine-induced b cell death in INS-1E b cells in this (results not given) and a previous study [24], suggesting that CK2 does not promote b cell death through cytokine activation of p38 MAP kinase. Cytokines have also been described to activate ERK 1/2 MAP kinase, which may fortify NFjB activation of iNOS expression [31]. Furthermore, ERK 1/2 may be activated by CK2 through phosphorylation of serines 244 and 246 [37]. In our hands, however, the ERK 1/2 MAP kinase inhibitor PD98059 did not inhibit cytokine-induced death of INS-1E b cells [24], suggesting that CK2 does not promote b cell death through cytokine activation of ERK 1/2 MAP kinase.
A few studies have suggested a possible inhibitory role for CK2 in b cell function [38,39]. Recently, CK2 has been described to inhibit PDX-1 activity and glucose-induced insulin biosynthesis in MIN6 b cells [40,41]. Thus, CK2 may phosphorylate PDX-1 at threonine 231 and serine 232, and culture of MIN6 b cells with CK2 inhibitors, including DMAT, has been described to increase insulin content and glucose-induced insulin secretion [40,41]. Since PDX-1 may inhibit apoptosis in b cells [42], it, therefore, remains to be established whether CK2 inhibition of PDX-1 may promote cytokine-induced b cell death.
In similar experiments in mouse islets, however, culture with DRB (50 lM) or DMAT (5 lM) per se did not affect islet insulin content, but caused an inhibition of glucoseinduced insulin secretion. The reason for this discrepancy from MIN6 b cells [40,41] is not known, and, in fact it remains to be established whether this inhibition of glucose-induced insulin secretion also may rely on an inhibition of NFjB activity, which, as confirmed in the present study, is required for glucose-stimulated insulin secretion [29,30]. Thus, although DRB and DMAT also may inhibit some other protein kinases apart from CK2 [43], DRB ,and DMAT, as described herein, appear to be quite specific CK2 inhibitors in pancreatic b cells.
While IL-1b and TNF-a induce NFjB activity, IFN-c may potentiate NFjB nuclear translocation and activity through activation of STAT1 [3,21]. Thus, IFN-c activates JAKs 1 and 2 which stimulates STAT1 through phosphorylation of tyrosine 701 [2]. In addition, studies in other tissues have shown that phosphorylation of serine 727 by a CK2-dependent mechanism may be needed for full activation of STAT1 [20]. Although this phosphorylation is dependent on CK2, it may be exerted by a yet unknown IFN-c-stimulated downstream protein kinase, dependent on constitutive CK2 activity [20].
According to the present experiments, IFN-c stimulated STAT1 phosphorylation at both tyrosine 701 and serine 727 and potentiated IL-1b plus TNF-a-induced b cell death. Indeed phosphorylation of serine 727 appeared to be dependent on CK2 activity. The significance of this phosphorylation of serine 727 for STAT1 activity is, however, at present not known, and although IFN-c potentiated cytokine-induced cell death, STAT1 was not the primary target for CK2 in cytokine-induced b cell death.
In conclusion, the present study has demonstrated that CK2 may have an indispensable function in cytokineinduced b cell death, which primarily may be exerted by NFjB activation and phosphorylation of NFjB p65 at serine 529.