Mechanism of nuclear factor of activated T-cells mediated FasL expression in corticosterone -treated mouse Leydig tumor cells.
Mechanism of nuclear factor of activated T-cells mediated FasL expression in corticosterone -treated mouse Leydig tumor cells.
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DOI：
10.1186/1471-2121-9-31
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发表时间：
2008-06-12
期刊：
影响因子：
--
通讯作者：
Gao, Hui-Bao
中科院分区：
文献类型：
--
作者：
Chai, Wei-Ran;Chen, Yong;Wang, Qian;Gao, Hui-Bao
Fas和FasL是细胞凋亡的重要介质。我们曾报道应激水平的皮质酮（corticosterone，CORT，glucocorticoid in rat）可增加大鼠睾丸间质细胞Fas/FasL的表达，激活Fas/FasL信号通路，从而导致细胞凋亡。此外，我们的另一项研究表明，活化T细胞核因子（NFAT）可能在皮质醇处理大鼠Leydig细胞上调FasL中发挥潜在作用。目前尚不清楚NFAT是如何参与皮质醇诱导的FasL上调。本研究的目的是探讨NFAT介导的FasL在皮质醇处理的Leydig细胞中表达的分子机制。Western blot分析显示NFAT 2在小鼠Leydig肿瘤细胞（mLTC-1）中表达。通过Western Blot分析确定CORT诱导的mLTC-1中FasL表达的增加，并通过FACS结合annexin-V标记检测CORT诱导的mLTC-1细胞凋亡频率的增加。mLTC-1中NFAT 2-GFP的共聚焦成像显示，高水平的CORT刺激NFAT从细胞质易位到细胞核。RNA干扰介导的NFAT 2敲低显著减弱了CORT诱导的mLTC中FasL表达的上调。这些结果证实了我们以前的发现，NFAT 2参与皮质激素诱导的大鼠Leydig细胞的FasL表达，并表明mLTC-1是一个合适的模型，用于研究皮质激素诱导的FasL表达的机制。对报告基因的分析表明，小鼠FasL基因的-201 ~+71之间的序列是CORT诱导FasL表达所必需的。突变分析表明，CORT诱导的FasL表达是通过位于-201至+71区域的NFAT结合元件介导的。NFAT 2表达载体与FasL基因-201 ~+71区报告基因共转染研究表明，NFAT 2在FasL启动子调控区具有较强的诱导活性。此外，染色质免疫沉淀试验进一步证实了报告基因研究的结果，显示NFAT 2与-201至+71区域的特异性结合。在本研究中，我们证明了NFAT 2直接刺激高水平CORT处理的mLTC-1中FasL的转录。总之，本研究为我们的发现提供了进一步的证据，皮质激素诱导的FasL在Leydig细胞的表达是由NFAT介导的。
Fas and FasL is important mediators of apoptosis. We have previously reported that the stress levels of corticosterone (CORT, glucocorticoid in rat) increase expression of Fas/FasL and activate Fas/FasL signal pathway in rat Leydig cells, which consequently leads to apoptosis. Moreover, our another study showed that nuclear factor of activated T-cells (NFAT) may play a potential role in up-regulation of FasL during CORT-treated rat Leydig cell. It is not clear yet how NFAT is involved in CORT-induced up-regulation of FasL. The aim of the present study is to investigate the molecular mechanisms of NFAT-mediated FasL expression in CORT-treated Leydig cells. Western blot analysis showed that NFAT2 expression is present in mouse Leydig tumor cell (mLTC-1). CORT-induced increase in FasL expression in mLTC-1 was ascertained by Western Blot analysis and CORT-induced increase in apoptotic frequency of mLTC-1 cells was detected by FACS with annexin-V labeling. Confocal imaging of NFAT2-GFP in mLTC-1 showed that high level of CORT stimulated NFAT translocation from the cytoplasm to the nucleus. RNA interference-mediated knockdown of NFAT2 significantly attenuated CORT-induced up-regulation of FasL expression in mLTC. These results corroborated our previous finding that NFAT2 is involved in CORT-induced FasL expression in rat Leydig cells and showed that mLTC-1 is a suitable model for investigating the mechanism of CORT-induced FasL expression. The analysis of reporter constructs revealed that the sequence between -201 and +71 of mouse FasL gene is essential for CORT-induced FasL expression. The mutation analysis demonstrated that CORT-induced FasL expression is mediated via an NFAT binding element located in the -201 to +71 region. Co-transfection studies with an NFAT2 expression vector and reporter construct containing -201 to +71 region of FasL gene showed that NFAT2 confer a strong inducible activity to the FasL promoter at its regulatory region. In addition, chromatin immunoprecipitation assay further confirmed the results of reporter gene studies by showing the specific binding of NFAT2 to the -201 to +71 region. In the present study, we demonstrated that NFAT2 directly stimulates transcription of FasL in high level CORT-treated mLTC-1. In conclusion, the present study provides further evidence for our finding that CORT-induced FasL expression in Leydig cells is mediated by NFAT.
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DOI：
10.1084/jem.181.3.1235
发表时间：
1995-03-01
期刊：
The Journal of experimental medicine
影响因子：
--
作者：
Arase H;Arase N;Saito T
通讯作者：
Saito T
影响因子：
64.8
作者：
BELLGRAU, D;GOLD, D;DUKE, RC
通讯作者：
DUKE, RC
NFAT2 参与皮质酮诱导的大鼠 Leydig 细胞凋亡
影响因子：
2.9
作者：
Chai, Wei-Ran;Wang, Qian;Gao, Hui-Bao
通讯作者：
Gao, Hui-Bao
影响因子：
64.5
作者：
Graef, IA;Wang, F;Crabtree, GR
通讯作者：
Crabtree, GR
影响因子：
64.5
作者：
Graef, IA;Chen, F;Crabtree, GR
通讯作者：
Crabtree, GR