Service of Medical Genetics, CHUV Hospital, Chemin des Falaises 1, 1011, Lausanne, Switzerland.
Diabetologia. 2009 Sep;52(9):1871-80. doi: 10.1007/s00125-009-1431-7. Epub 2009 Jul 16.
AIMS/HYPOTHESIS: In insulin-secreting cells, activation of the c-Jun NH(2)-terminal kinase (JNK) pathway triggers apoptosis. Whereas JNK1 and JNK2 are ubiquitously produced, JNK3 has been described exclusively in neurons. This report aims to characterise the expression and role in apoptosis of the three JNK isoforms in insulin-secreting cells exposed to cytokines.
Sections of human and mouse pancreases were used for immunohistochemistry studies with isoform-specific anti-JNK antibodies. Human, pig, mouse and rat pancreatic islets were isolated by enzymatic digestion and RNA or protein extracts were prepared. RNA and protein levels were determined by quantitative RT-PCR and western blotting respectively, using JNK-isoform-specific primers and isoform-specific antibodies; activities of the three JNK isoforms were determined by kinase assays following quantitative immunoprecipitation/depletion of JNK3. JNK silencing was performed with small interfering RNAs and apoptotic rates were determined in INS-1E cells by scoring cells displaying pycnotic nuclei.
JNK3 and JNK2 mRNAs are the predominant isoforms expressed in human pancreatic islets. JNK3 is nuclear while JNK2 is also cytoplasmic. In INS-1E cells, JNK3 knockdown increases c-Jun levels and caspase-3 cleavage and sensitises cells to cytokine-induced apoptosis; in contrast, JNK1 or JNK2 knockdown is protective.
CONCLUSIONS/INTERPRETATION: In insulin-secreting cells, JNK3 plays an active role in preserving pancreatic beta cell mass from cytokine attacks. The specific localisation of JNK3 in the nucleus, its recruitment by cytokines, and its effects on key transcription factors such as c-Jun, indicate that JNK3 is certainly an important player in the transcriptional control of genes expressed in insulin-secreting cells.
目的/假设：在胰岛素分泌细胞中，c-Jun NH（2）-末端激酶（JNK）途径的激活会引发细胞凋亡。虽然 JNK1 和 JNK2 广泛产生，但 JNK3 仅在神经元中描述过。本报告旨在描述在细胞因子暴露下胰岛素分泌细胞中三种 JNK 同工型的表达和在细胞凋亡中的作用。
使用同工型特异性抗 JNK 抗体对人胰腺和小鼠胰腺的组织切片进行免疫组织化学研究。通过酶消化分离人、猪、鼠和大鼠胰岛，并制备 RNA 或蛋白质提取物。使用 JNK-同工型特异性引物和同工型特异性抗体，通过定量 RT-PCR 和 Western 印迹分别测定 RNA 和蛋白质水平；通过定量免疫沉淀/耗尽 JNK3 后测定三种 JNK 同工型的活性。用小干扰 RNA 进行 JNK 沉默，并通过计数显示核固缩的细胞来确定 INS-1E 细胞中的凋亡率。
JNK3 和 JNK2 mRNA 是人类胰岛中表达的主要同工型。JNK3 是核内的，而 JNK2 也是细胞质的。在 INS-1E 细胞中，JNK3 敲低增加了 c-Jun 水平和半胱天冬酶-3 切割，并使细胞对细胞因子诱导的凋亡敏感；相比之下，JNK1 或 JNK2 敲低是保护的。
结论/解释：在胰岛素分泌细胞中，JNK3 在保护胰岛β细胞免受细胞因子攻击方面发挥积极作用。JNK3 在核内的特定定位、细胞因子的募集及其对关键转录因子（如 c-Jun）的影响，表明 JNK3 肯定是胰岛素分泌细胞中表达基因转录控制的重要参与者。
