Lee Hyun Jung, Oh Yeo Kyoung, Rhee Marie, Lim Joong-Yeon, Hwang Ji-Young, Park Yoon Shin, Kwon Yongil, Choi Kyung-Hee, Jo Inho, Park Sang Ick, Gao Bin, Kim Won-Ho
Division of Intractable Diseases, Center for Biomedical Sciences, National Institutes of Health, Eunpyeong-gu, Seoul, South Korea.
J Mol Biol. 2007 Jun 15;369(4):967-84. doi: 10.1016/j.jmb.2007.03.072. Epub 2007 Apr 1.
Previously, we demonstrated that signal transducer and activator of transcription factor 1 (STAT1) plays an essential role in liver injury induced by lipopolysaccharide (LPS)/D-galactosamine (D-GalN); however, the underlying mechanism involved remains unclear. Here, we showed that LPS/D-GalN administration induced secretion of tumor necrosis factor alpha (TNF-alpha) and interferon gamma (IFN-gamma), which mediated apoptosis synergistically. Moreover, LPS/D-GalN-induced apoptosis was associated with increased inducible nitric oxide synthase (iNOS) and nitric oxide (NO) production, as well as elevated reactive oxygen species (ROS) production, which were all strongly inhibited by treatment with the antioxidant N-acetyl-L-cysteine (NAC) and an iNOS/NO inhibitor, L-NMMA. Although STAT1 activation and expression did not change significantly in TNF-alpha/IFN-gamma-cotreated cells compared with cells treated with IFN-gamma alone, the absence of STAT1 or interferon regulatory factor 1 (IRF-1) in genetic knockout mice strongly abrogated the observed effects of TNF-alpha/IFN-gamma on iNOS/NO induction, ROS production, loss of mitochondrial transmembrane potential (DeltaPsim), and apoptosis compared with STAT1(+/+) and IRF-1(+/+) mice. Additionally, the synergistic effects of TNF-alpha/IFN-gamma on iNOS/NO induction, ROS production, and apoptosis were significantly inhibited by overexpression of dominant negative STAT1 in contrast to overexpression of wild-type STAT1. In STAT1-deficient mice, nuclear factor kappaB (NF-kappaB) activation by TNF-alpha/IFN-gamma was attenuated and strongly inhibited by both NAC and L-NMMA. Moreover, the proteasome inhibitor, MG132, inhibited NF-kappaB activation and strongly inhibited iNOS/NO induction, ROS production, and loss of DeltaPsim induced by TNF-alpha/IFN-gamma, thereby inhibiting apoptosis. Interestingly, it appears peroxynitrite, which is produced by TNF-alpha/IFN-gamma, may interfere with STAT1 phosphorylation by inducing STAT1 nitration. Collectively, these findings demonstrate that TNF-alpha/IFN-gamma synergistically potentiates iNOS/NO induction, ROS production, and loss of DeltaPsim via STAT1 overexpression, playing an important role in promoting apoptosis and liver injury induced by LPS/D-GalN.
先前，我们证明信号转导和转录激活因子1（STAT1）在脂多糖（LPS）/D-半乳糖胺（D-GalN）诱导的肝损伤中起关键作用；然而，其潜在机制仍不清楚。在此，我们表明给予LPS/D-GalN可诱导肿瘤坏死因子α（TNF-α）和干扰素γ（IFN-γ）分泌，二者协同介导细胞凋亡。此外，LPS/D-GalN诱导的细胞凋亡与诱导型一氧化氮合酶（iNOS）和一氧化氮（NO）生成增加以及活性氧（ROS）生成升高有关，而抗氧化剂N-乙酰-L-半胱氨酸（NAC）和iNOS/NO抑制剂L-NMMA处理可强烈抑制上述变化。虽然与单独用IFN-γ处理的细胞相比，TNF-α/IFN-γ共处理的细胞中STAT1的激活和表达没有显著变化，但与STAT1(+/+)和IRF-1(+/+)小鼠相比，基因敲除小鼠中STAT1或干扰素调节因子1（IRF-1）的缺失强烈消除了TNF-α/IFN-γ对iNOS/NO诱导、ROS生成、线粒体跨膜电位（ΔΨm）丧失和细胞凋亡的观察到的影响。此外，与野生型STAT1过表达相比，显性负性STAT1过表达显著抑制了TNF-α/IFN-γ对iNOS/NO诱导、ROS生成和细胞凋亡的协同作用。在STAT1缺陷小鼠中，TNF-α/IFN-γ诱导的核因子κB（NF-κB）激活减弱，且NAC和L-NMMA均强烈抑制该激活。此外，蛋白酶体抑制剂MG132抑制NF-κB激活，并强烈抑制TNF-α/IFN-γ诱导的iNOS/NO诱导、ROS生成和ΔΨm丧失，从而抑制细胞凋亡。有趣的是，由TNF-α/IFN-γ产生的过氧亚硝酸盐似乎可能通过诱导STAT1硝化来干扰STAT1磷酸化。总的来说，这些发现表明TNF-α/IFN-γ通过STAT1过表达协同增强iNOS/NO诱导、ROS生成和ΔΨm丧失，在促进LPS/D-GalN诱导的细胞凋亡和肝损伤中起重要作用。
