Northern Ireland Centre for Stratified Medicine, School of Biomedical Sciences, Ulster University, C-TRIC Building, Altnagelvin Hospital Campus, Derry/Londonderry, Northern Ireland, UK.
J Mol Endocrinol. 2021 Sep 28;67(4):189-201. doi: 10.1530/JME-21-0076.
TNFAIP3 encodes a zinc finger protein called A20, which has potent anti-inflammatory and anti-apoptotic properties. A20 promotes beta-cell survival and protects against islet graft rejection in experimental models. The current study sought to investigate the mechanisms underlying the protective role of A20 in the pancreatic beta-cell. Two islet cell types were used for experiments: the insulin-secreting BRIN-BD11 cell line and human islet cells. A20 was silenced using siRNA against TNFAIP3, and knockdown was confirmed by qPCR and immunostaining of cells. Cell viability, cytotoxicity and apoptosis were assessed using the ApotoxGlo assay. Glucose-stimulated insulin secretion and production of inflammatory cytokines (TNFa, IL1b and IFNg) were measured by ELISA. Expression of beta-cell regulatory genes (Abcc8, Kcnj11, Kcnq1, Gck, Scl2a2) and transcription factors (Hnf1a, Pdx1, Nkx6.1, Ngn3) was determined by qPCR. A20 deficiency increased apoptosis, impaired glucose-induced insulin secretion, and reduced expression of beta-cell regulatory genes and transcription factors. Addition of recombinant A20 normalized gene expression profiles. TNFa, IL1b and IFNg were elevated in A20 deficient cells and found to independently elicit changes in gene expression. Analysis of PCR array data suggests that A20 action in the beta cell is largely, although not exclusively, driven by the P65 subunit of NF-kB. The current report demonstrates a role for A20 in controlling beta-cell integrity and survival, which likely results from the regulation of inflammatory signalling. Of particular note is the impact that A20 deficiency has on the expression of transcription factors regulating the maturation and normal function of beta cells.
TNFAIP3 编码一种锌指蛋白，称为 A20，具有强大的抗炎和抗凋亡特性。A20 促进β细胞存活，并在实验模型中保护胰岛移植物免受排斥。本研究旨在探讨 A20 在胰岛β细胞中发挥保护作用的机制。实验中使用了两种胰岛细胞类型：胰岛素分泌的 BRIN-BD11 细胞系和人胰岛细胞。使用针对 TNFAIP3 的 siRNA 沉默 A20，并通过 qPCR 和细胞免疫染色确认敲低。使用 ApotoxGlo 测定法评估细胞活力、细胞毒性和细胞凋亡。通过 ELISA 测量葡萄糖刺激的胰岛素分泌和炎症细胞因子（TNFa、IL1b 和 IFNg）的产生。通过 qPCR 测定β细胞调节基因（Abcc8、Kcnj11、Kcnq1、Gck、Scl2a2）和转录因子（Hnf1a、Pdx1、Nkx6.1、Ngn3）的表达。A20 缺乏增加细胞凋亡，损害葡萄糖诱导的胰岛素分泌，并降低β细胞调节基因和转录因子的表达。添加重组 A20 可使基因表达谱正常化。A20 缺乏细胞中 TNFa、IL1b 和 IFNg 升高，发现它们独立地引起基因表达的变化。PCR 阵列数据分析表明，A20 在β细胞中的作用主要（尽管不是完全）由 NF-kB 的 P65 亚基驱动。本报告表明 A20 在控制β细胞完整性和存活方面发挥作用，这可能是由于炎症信号的调节。值得注意的是，A20 缺乏对调节β细胞成熟和正常功能的转录因子的表达产生影响。
