Liu D, Pavlovic D, Chen M C, Flodström M, Sandler S, Eizirik D L
Gene Transcription Unit, Diabetes Research Center, Vrije Universiteit Brussels, Belgium.
Diabetes. 2000 Jul;49(7):1116-22. doi: 10.2337/diabetes.49.7.1116.
Prolonged exposure of rodent beta-cells to combinations of cytokines induces the inducible form of nitric oxide synthase (iNOS) expression and Fas expression, nitric oxide (NO) production, and cell death. It also induces the expression of potential "defense" genes, such as manganese superoxide dismutase (MnSOD) and heat shock protein (hsp) 70. NO is a radical with multifaceted actions. Recent studies have shown that NO, in addition to having cytotoxic actions, may regulate gene transcription. It remains unclear whether NO mediates cytokine-induced gene expression and subsequent beta-cell death. Previous studies using NO synthase blockers yielded conflicting results, which may be due to nonspecific effects of these agents. In this study, we examined the effects of cytokines on gene expression, determined by reverse transcriptase-polymerase chain reaction (RT-PCR), and viability, determined by nuclear dyes, of pancreatic islets or fluorescence-activated cell sorter (FACS)-purified beta-cells isolated from iNOS knockout mice (iNOS-/-, background C57BL/6x129SvEv) or their respective controls (C57BL/6x129SvEv). The combination of cytokines used was interleukin-1beta (50 U/ml) plus gamma-interferon (1,000 U/ml) plus tumor necrosis factor-alpha (1,000 U/ml). The lack of cytokine-induced iNOS activity in the iNOS-/- islet cells was confirmed by RT-PCR and nitrite determination. Cytokines induced a >3-fold increase in Fas and MnSOD mRNA expression in wild-type (WT) and iNOS-/- islets. On the other hand, hsp 70 was induced in WT but not in iNOS-/- islets. Prolonged (6-9 days) exposure of WT islets to cytokines leads to an 80-90% decrease in islet cell viability, whereas viability decreased by only 10-30% in iNOS-/- islet cells. To determine the mode of cytokine-induced cell death, FACS-purified beta-cells were exposed to the same cytokines. After 9 days, the apoptosis index was similarly increased in WT (39 +/- 3%) and iNOS4-/- (33 +/- 4%) beta-cells. On the other hand, cytokines increased necrosis in WT (20 +/- 4%) but not in iNOS-/- (7 +/- 3%) beta-cells. From these data, we concluded that 1) NO is required for cytokine-induced hsp 70 mRNA expression but not for Fas and MnSOD expression, 2) cytokines induce both apoptosis and necrosis in mouse beta-cells, and 3) cytokine-induced apoptosis is mostly NO-independent, whereas necrosis requires NO formation.
将啮齿动物的β细胞长时间暴露于细胞因子组合中，会诱导诱导型一氧化氮合酶（iNOS）表达、Fas表达、一氧化氮（NO）生成以及细胞死亡。它还会诱导潜在“防御”基因的表达，如锰超氧化物歧化酶（MnSOD）和热休克蛋白（hsp）70。NO是一种具有多方面作用的自由基。最近的研究表明，NO除了具有细胞毒性作用外，还可能调节基因转录。目前尚不清楚NO是否介导细胞因子诱导的基因表达以及随后的β细胞死亡。先前使用NO合酶阻滞剂的研究得出了相互矛盾的结果，这可能是由于这些药物的非特异性作用。在本研究中，我们检测了细胞因子对从iNOS基因敲除小鼠（iNOS-/-，背景为C57BL/6x129SvEv）或其各自对照（C57BL/6x129SvEv）分离的胰岛或荧光激活细胞分选仪（FACS）纯化的β细胞的基因表达（通过逆转录聚合酶链反应（RT-PCR）测定）和活力（通过核染料测定）的影响。所使用的细胞因子组合为白细胞介素-1β（50 U/ml）加γ干扰素（1000 U/ml）加肿瘤坏死因子-α（1000 U/ml）。通过RT-PCR和亚硝酸盐测定证实了iNOS-/-胰岛细胞中缺乏细胞因子诱导的iNOS活性。细胞因子使野生型（WT）和iNOS-/-胰岛中Fas和MnSOD mRNA表达增加了3倍以上。另一方面，WT胰岛中诱导了hsp 70，而iNOS-/-胰岛中未诱导。将WT胰岛长时间（6 - 9天）暴露于细胞因子会导致胰岛细胞活力降低80 - 90%，而iNOS-/-胰岛细胞活力仅降低10 - 30%。为了确定细胞因子诱导细胞死亡的方式，将FACS纯化的β细胞暴露于相同的细胞因子。9天后，WT（39±3%）和iNOS4-/-（33±4%）β细胞中的凋亡指数同样增加。另一方面，细胞因子增加了WT（20±4%）β细胞中的坏死，但未增加iNOS-/-（7±3%）β细胞中的坏死。根据这些数据，我们得出结论：1）NO是细胞因子诱导hsp 70 mRNA表达所必需的，但不是Fas和MnSOD表达所必需的；2）细胞因子在小鼠β细胞中诱导凋亡和坏死；3）细胞因子诱导的凋亡大多不依赖于NO，而坏死需要NO的形成。
