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

Abstract

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.

Fig. 1

Effects of DRB and DMAT on β cell survival. a, b INS-1E cells and c, d mouse islets were cultured for 48 h in the absence or presence of cytokines (IL-1β, TNF-α, and IFN-γ) (3mix) and with DRB (5–75 μM) (DRB_5–75) or DMAT (5–10 μM) (DMAT_5–10) as indicated before determination of cell viability. a, b Results are mean ± SEM (n = 4–12); a *P < 0.001 versus 3mix; b *P < 0.001 versus 3mix. c, d Results are mean ± SEM (n = 4); c *P < 0.001 versus control, **P < 0.001 versus 3mix; d *P < 0.001 versus control, **P < 0.001 versus 3mix

Fig. 2

Role of CK2 in cytokine-induced β cell apoptosis. Mouse islets were cultured for 48 h in the absence or presence of cytokines (IL-1β, TNF-α, and IFN-γ) (3mix) and with DRB (50 μM) (DRB₅₀) as indicated before determination of apoptosis. Results are mean ± SEM (n = 4), *P < 0.001 versus control, **P < 0.001 versus 3mix

Fig. 3

Role of CK2 in H₂O₂- and palmitate-induced β cell death. INS-1E cell were cultured for 24 h with a H₂O₂ (25–50 μM) [(H₂O₂)_25–50] or b palmitate (125–250 μM) (Palm_125–250) plus bovine serum albumin (2.75 mg/ml) (BSA_2.75) and with DRB (50 μM) (DRB₅₀) as indicated before determination of cell viability. Results are mean ± SEM (n = 4–6)

Fig. 4

Role of NFκB in cytokine-induced β cell death. a INS-1E cells and b mouse islets were cultured for 48 h in the absence or presence of cytokines (IL-1β, TNF-α, and IFN-γ) (3mix) and with andrographolide (5–50 μM) (Andro_5–50) as indicated before determination of cell viability. a Results are mean ± SEM (n = 8), *P < 0.001 versus 3mix, b Results are mean ± SEM (n = 4), *P < 0.05 versus control, **P < 0.05 versus 3mix

Fig. 5

Western blots of IκBα and NFκB p65 proteins. INS-1E cells were cultured for 30 min or 2 h in the absence or presence of cytokines (IL-1β, TNF-α, and IFN-γ) (3mix) and DRB (50 μM) (DRB₅₀) or DMAT (5 μM) (DMAT₅) as indicated before determination of a, b IκBα and c, d NFκB p65 protein levels. Results are mean ± SEM (n = 3–6) with a representative experiment shown below bars. a *P < 0.001 versus 3mix; b *P < 0.001 versus 3mix

Fig. 6

Effects of cytokines on CK2 activity in INS-1E cells. INS-1E cells were cultured for 30 min or 2 h in the absence or presence of cytokines (IL-1β, TNF-α, and IFN-γ) (3mix). Results are mean ± SEM (n = 6)

Fig. 7

Effects of DRB and DMAT on NFκB p65 phosphorylations. INS-1E cells were cultured for 30 min or 2 h in the absence or presence of cytokines (IL-1β, TNF-α, and IFN-γ (3mix) and DRB (50 μM) (DRB₅₀) or DMAT (5 μM) (DMAT₅) as indicated. a NFκB p65 phosphoserine 536 (pNFκB_ser536), b NFκB p65 phosphoserine 276 (pNFκB_ser276), c, d NFκB p65 phosphoserine 529 (pNFκB_ser529). Results are mean ± SEM (n = 3–7) with a representative experiment shown below bars. c *P < 0.001 versus control; d *P < 0.01 versus control, **P < 0.01 versus 3mix, ***P < 0.001 versus control, ****P < 0.001 versus 3mix

Fig. 8

Effects of DRB on HDACs 1–3 phosphorylations. INS-1E cells were cultured for 30 min or 2 h in the absence or presence of cytokines (IL-1β, TNF-α, and IFN-γ) (3mix) and DRB (50 μM) (DRB₅₀) as indicated. a HDAC1 phosphoserine 421/423 (pHDAC1_ser421/423), b HDAC2 phosphoserine 394 (pHDAC2_ser394), c HDAC3 phosphoserine 424 (pHDAC3_ser424). Results are mean ± SEM (n = 3–5) with a representative experiment shown below bars

Fig. 9

Effects of DRB and DMAT on STAT1 serine 727 phosphorylation. INS-1E cells were cultured for 30 min or 2 h in the absence or presence of cytokines (IL-1β, TNF-α, and IFN-γ) (3mix) and DRB (50 μM) (DRB₅₀) or DMAT (5 μM) (DMAT₅) as indicated before determination of STAT1 phosphoserine 727 (pSTAT1_ser727). Results are mean ± SEM (n = 3–5) with a representative experiment shown below bars. a *P < 0.01 versus 3mix; b *P < 0.001 versus 3mix

Fig. 10

Role of IFN-γ in STAT1 phosphorylations. INS-1E cells were cultured for 30 min or 2 h in the absence or presence of IL-1β and TNF-α with IFN-γ (3mix) or without IFN-γ (2mix) and with DRB (50 μM) (DRB₅₀) as indicated before determination of a, b STAT1 phosphotyrosine 701 (pSTAT_tyr701) and c, d STAT1 phosphoserine 727 (pSTAT1_ser727). Results are mean ± SEM (n = 3) with a representative experiment shown below bars. d *P < 0.01 versus 3 mix

Fig. 11

Role of STAT1 in CK2-dependent NFκB activation and β cell death. a, b INS-1E cells were cultured for 30 min or 2 h in the absence or presence of IL-1β and TNF-α with IFN-γ (3mix) or without IFN-γ (2mix) and with DRB (50 μM) (DRB₅₀) as indicated before determination of IκBα. Results are mean ± SEM (n = 3) with a representative experiment shown below bars. b *P < 0.001 versus 3mix, **P < 0.001 versus 2 mix. c INS-1E cells were cultured for 48 h in the absence or presence of IL-1β, TNF-α with IFN-γ (3mix) or without IFN-γ (2mix) and with DRB (50 μM) (DRB₅₀) as indicated before determination of cell viability. Results are mean ± SEM (n = 7). *P < 0.001 versus 3mix, **P < 0.001 versus 2mix

Fig. 12

Role of CK2 and NFκB in glucose-induced insulin secretion. Mouse islets were cultured for 24 h in the absence or presence of a DRB (50 μM) (DRB₅₀), b DMAT (5 μM) (DMAT₅) or c andrographolide (50 μM) (Andro₅₀) before determination of islet insulin secretion and insulin content as indicated. Islets were perifused for 45 min with 3.3 mM glucose (Gl_3.3) and then stimulated for 60 min with 16.7 mM glucose (Gl_16.7). Results are mean ± SEM (n = 3–4)