Anti-apoptotic activity of porcine cFLIP in ovarian granulosa cell lines
Anti-apoptotic activity of porcine cFLIP in ovarian granulosa cell lines
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DOI：
10.1002/mrd.20602
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发表时间：
2007-09-01
影响因子：
2.5
通讯作者：
Manabe, Noboru
中科院分区：
文献类型：
--
作者：
Matsuda-Minehata, Fuko;Goto, Yasufumi;Manabe, Noboru
在哺乳动物的卵巢中，99%以上的卵泡在生长和发育过程中经历闭锁。最近，我们在猪卵巢颗粒细胞中发现，在卵泡闭锁过程中，细胞FLICE样抑制蛋白长形蛋白(cFIP(L))的表达减少。在人类和其他物种中，cFlIP的短形式(S)和长形式(L)都被认为是细胞生存因子，抑制死亡配体受体介导的细胞凋亡。由于猪cFLIP(PcFLIP)在颗粒细胞中的抗凋亡活性尚未确定，我们使用颗粒细胞系检测了pcFLIP对存活的影响。选用人宫颈癌细胞系HeLa、人卵巢颗粒瘤细胞系KGN和猪颗粒细胞系JC-410。经Western blotting检测，所有细胞系均可检测到L的内源性cFlIP，而HeLa和KGN细胞中仅检测到微量的cFlIP(S)。在HeLa和KGN细胞中过表达pcFLIP(S)或pcFLIP(L)，以检测其抗凋亡活性。高表达的细胞在Fas诱导的细胞凋亡中存活，而空载体转染组几乎全部死亡。然后，我们利用小干扰RNA(SiRNA)抑制了猪cFlIP(S)和/或cFlIP(L)在JC-410细胞中的表达。当cFlIP(S)和cFlIP(L)同时抑制或仅cFlIP(L)被抑制时，细胞存活率显著下降。结果表明，猪CFIP及其类似物L具有抗颗粒细胞凋亡的作用。有人强烈建议，pcFLIP在颗粒细胞中作为一种生存促进因子，并决定了猪卵巢卵泡是存活还是闭锁。
In mammalian ovaries, more than 99% of follicles undergo atresia during growth and development. Recently, we found that the expression of cellular FLICE-like inhibitory protein long form (cFLIP(L)) decreased during follicular atresia in granulosa cells of porcine ovaries. In humans and other species, both the short (cFLIP(S)) and long (cFLIP(L)) forms of cFLIP are considered to function as cell survival factors that inhibit death ligand receptor-mediated apoptosis. Since the anti-apoptotic activity of porcine cFLIP (pcFLIP) in granulosa cells had not been determined, we examined the effect of pcFLIP on survival using granulosa-derived cell lines. A human cervix adenocarcinoma cell line, HeLa, human ovarian granulosa tumor cell line, KGN, and porcine granulosa-derived cell line, JC-410, were used. By Western blotting, internal cFLIP(L) was detected in all cell lines, but only trace levels of cFLIP(S) were found in HeLa and KGN cells. To examine the anti-apoptotic activity, pcFLIP(S) or pcFLIP(L) was overexpressed in HeLa and KGN cells. Transfected cells in which pcFLIP(S) or pcFLIP(L) was overexpressed, survived the induction of Fas-mediated apoptosis, while almost all of the cells transfected with empty vector died. Then, we suppressed the expression of porcine cFLIP(S) and/or cFLIP(L) in JC-410 cells using small interfering RNA (siRNA). When both cFLIP(S) and cFLIP(L), or only cFLIP(L) was suppressed, cell viability declined significantly. From the results, we conclude that porcine cFLIPs and cFLIP(L) exhibit anti-apoptotic activity in granulosa-derived cells. It was strongly suggested that pcFLIP acts as a survival-promoting factor in granulosa cells and determines whether porcine ovarian follicles survive or undergo atresia.