Cross-talk between Fas/Fas ligand system and nitric oxide in the pathway subserving granulosa cell apoptosis: A possible regulatory mechanism for ovarian follicle atresia
Cross-talk between Fas/Fas ligand system and nitric oxide in the pathway subserving granulosa cell apoptosis: A possible regulatory mechanism for ovarian follicle atresia
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DOI：
10.1210/en.2004-0579
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发表时间：
2005-02-01
期刊：
影响因子：
4.8
通讯作者：
Taketani, Y
中科院分区：
文献类型：
--
作者：
Chen, QM;Yano, T;Taketani, Y
近年来的研究表明，Fas/Fas配体（FasL）系统和一氧化氮（NO）参与了卵泡闭锁的发生。在这里，我们问是否Fas/Fas配体系统与NO的大鼠颗粒细胞培养相互作用。可溶性重组Fas配体（rFasL），在100 ng/ml，显着降低细胞活力，通过3- 2-（4，5-二甲基噻唑-5-基）（3-羧基甲氧基苯基）2-（4-磺苯基）-2H-四唑，内盐（MTS）测定，而同时加入20 μ M的半胱天冬酶抑制剂Z-VAD-FMK，显著抑制rFasL诱导的细胞毒性。Hoechst 33342染色和流式细胞术分析证实了在干扰素-γ存在下，100 ng/ml rFasL诱导颗粒细胞凋亡，这被同时加入的NO供体S-亚硝基-N-乙酰青霉胺阻断。Western blot分析表明，rFasL显著上调颗粒细胞caspase-3，-8，和-9的活性，这是由S-亚硝基-N-乙酰青霉胺同时处理减弱。实时定量RT-PCR显示rFasL诱导的颗粒细胞凋亡中诱导型NO合酶mRNA水平显著降低。总之，我们证明了Fas/FasL系统参与诱导凋亡，通过激活caspase介导的级联反应在大鼠颗粒细胞，这是再加上诱导型NO合酶表达减少。我们进一步表明，NO抑制Fas/FasL系统诱导的细胞凋亡，通过抑制激活的caspase，指出之间的串扰Fas/FasL系统诱导的细胞凋亡途径和NO介导的抗凋亡途径在卵泡闭锁。
Recent studies have shown the involvement of Fas/Fas ligand ( FasL) system and nitric oxide (NO) in ovarian follicle atresia. Here we asked whether Fas/Fas ligand system interacts with NO using rat granulosa cell culture. Soluble recombinant Fas ligand (rFasL), at 100 ng/ml, significantly decreased cell viability, as measured by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) assay, in the presence of 200 U/ml interferon-gamma, whereas the concurrent addition of a caspase inhibitor, Z-VAD-FMK, at 20 muM, significantly inhibited rFasL-induced cytotoxicity. Hoechst 33342 staining and flow cytometric analysis confirmed the induction of apoptosis in granulosa cells by 100 ng/ml rFasL in the presence of interferon-gamma, which was blocked by the concomitant addition of an NO donor, S-nitroso-N-acetylpenicillamine. Western blot analysis demonstrated that rFasL significantly up-regulated caspase-3, -8, and -9 activities in granulosa cells, which were attenuated by concurrent treatment with S-nitroso-N-acetylpenicillamine. Real-time quantitative RT-PCR revealed a significant decrease in inducible NO synthase mRNA levels in rFasL-induced apoptotic granulosa cells. In conclusion, we demonstrated the involvement of Fas/FasL system in inducing apoptosis through activation of a caspase-mediated cascade in rat granulosa cells, which is coupled with a decrease in inducible NO synthase expression. We further showed that NO inhibited Fas/FasL system-induced apoptosis by suppressing activation of the caspases, pointing to a cross-talk between Fas/FasL system-induced apoptosis pathway and NO-mediated antiapoptotic pathway in ovarian follicle atresia.