The immunoproteasome is induced by cytokines and regulates apoptosis in human islets

1. View larger version:

   • In this window
   • In a new window

   • Download as PowerPoint Slide

   Figure 1

   Gene set enrichment analysis (GSEA) and validation of selected genes in INS-1E cells exposed to cytokines and HDAC inhibitors. (A) INS-1E cells were exposed to cytokines (IL-1β + TNFα + IFNγ) ± the HDAC1–3 selective inhibitor MS-275 or the HDAC1–2 selective inhibitor ‘3’ for 6 h. RNA was purified, and a microarray followed by GSEA was performed. The top scoring gene set is shown here. Selected gene expressions are highlighted in yellow, n = 1. (B, C, D and E) INS-1E cells were exposed to vehicle (white bars), cytokines (black bars) ± MS-275 (light grey bars) or BRD0476 (dark grey bars) for 3, 6, 9, 12 or 24 h. Expression of (B) Psma2, (C) Psme1, (D) Psma7, (E) Psmb10 was analyzed by qPCR. Results are shown as means + s.e.m., n = 4. *P < 0.05, **P < 0.01 and ***P < 0.001 vs vehicle (CTRL), ^#P < 0.05, ^###P < 0.001 vs cytokines.
2. View larger version:

   • In this window
   • In a new window

   • Download as PowerPoint Slide

   Figure 2

   Expression of selected proteasomal subunit genes in human islets exposed to cytokines. The expression of PSMB5, PSMB6, PSMB7, PSMB8, PSMB9 and PSMB10 was examined in human islets exposed to (A) a combination of IL-1β and IFNγ (black bars) or (B) only IL-1β (grey bars) or IFNγ (black bars). Results are shown as means + s.e.m., n = 4–8. *P < 0.05 and **P < 0.01 vs other groups.
3. View larger version:

   • In this window
   • In a new window

   • Download as PowerPoint Slide

   Figure 3

   Proteasomal activities in human islets exposed to cytokines. The trypsin-like, chymotrypsin-like and caspase-like proteasomal activities were analyzed in human islets exposed to (A) a combination of IL-1β and IFNγ (black bars) or (B) only IL-1β (grey bars) or IFNγ (black bars). Results are shown as means + s.e.m., n = 4–7. *P < 0.05 and **P < 0.01 vs other groups.
4. View larger version:

   • In this window
   • In a new window

   • Download as PowerPoint Slide

   Figure 4

   Cytokine-induced apoptosis is exacerbated by inhibition of the proteasome in human islets. Quantification of apoptotic human islet beta cells (A), identified by chromatin condensation and/or blebs in cells containing typical β granules by transmission electron microscopy analysis of human islets (B). The quantified analysis in panel A depicts data as means + s.e.m. for islets exposed to vehicle (white bar), ONX-914 (light grey bar), cytokines (black bar) or cytokines + ONX-914 (dark grey bar); n = 7, *P < 0.05 vs other groups.
5. View larger version:

   • In this window
   • In a new window

   • Download as PowerPoint Slide

   Figure 5

   Proteasomal activity of INS-1E cells exposed to cytokines and small molecule inhibitors of cytokine-induced apoptosis. Chymotrypsin-like proteasomal activity was measured in (A) a time-dependent manner and (B) in INS-1E cells exposed for cytokines ± MS-275/BRD0476 for 12 h. The broad proteasome inhibitor MG-132 was used as a positive control. (A) Eleven independent wells, (B) twenty-two independent wells, results are shown as means + STD. *P < 0.01 and **P < 0.001 vs CTRL, NS, no significance.
6. View larger version:

   • In this window
   • In a new window

   • Download as PowerPoint Slide

   Figure 6

   I-proteasome inhibition does not cause accumulation of oxidized proteins in cytokine-exposed INS-1 cells. (A) Thiol concentration in INS-1 cell lysates incubated with cytokines (Cyt), 200 nM Psmb8 inhibitor ONX-914 (Ctl/ONX) or both (Cyt/ONX) for 6, 12, 18 or 24 h. Data are presented as mean ± s.d., n = 6. (B) Carbonyl concentration in INS-1 cell lysates incubated with cytokines (Cyt), Psmb8 inhibitor (Ctl/ONX) or both (Cyt/ONX) for 6, 12, 18 or 24 h. Data are presented as mean ± s.d., n = 5. Two-way ANOVA: P = 0.0275; One-way ANOVAs: 6 h P = 0.9786; 12 h P = 0.0421; 18 h P = 0.4129; 24 h P = 0.0835. Ad hoc Student’s t-tests for 12 h: *P < 0.05, **P < 0.01.
7. View larger version:

   • In this window
   • In a new window

   • Download as PowerPoint Slide

   Figure 7

   (A, B, C and D) INS-1E cells were exposed to 1 ng/mL IL-1β plus 50 ng/mL IFNγ for 6 h without or with ONX-914 (200 nM). Western blots were performed using anti-p50, p52, IκB alpha or p65 as well as anti-beta actin and anti-tubulin antibodies. For p65 analysis (C), cytoplasmic fractions were analyzed. (E) INS-1 cells were exposed to 1 ng/mL IL-1β plus 50 ng/mL IFNγ for 30 min without or with ONX-914 (200 nM). Western blots were performed using IκB alpha and anti-tubulin antibodies. Data are expressed as ratios to house-keeping protein in percentage of control, with n = 3–5. *P < 0.05 vs CTRL and ONX-0914 (ANOVA followed by Student’s t-test with Bonferroni correction). Results are shown as means + s.e.m.

• © 2017 Society for Endocrinology