Matsuda Fuko, Inoue Naoko, Goto Yasufumi, Maeda Akihisa, Cheng Yuan, Sakamaki Kazuhiro, Manabe Noboru
Research Unit for Animal Life Sciences, Animal Resource Science Center, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Ibaraki-Kasama, Japan.
J Reprod Dev. 2008 Oct;54(5):314-20. doi: 10.1262/jrd.20051. Epub 2008 Jul 7.
More than 99% of follicles in mammalian ovaries undergo atresia, but the mechanisms regulating the strict selection process are still unclear. Granulosa cell apoptosis is considered the trigger of follicular atresia, which occurs in advance of the death of an oocyte. Cellular FLICE-like inhibitory protein (cFLIP), a homologue of procaspase-8 (also called FLICE), is an intracellular anti-apoptotic protein. It is expressed in granulosa cells of porcine ovaries, where its levels decreases during follicular atresia. We hypothesized that cFLIP regulates granulosa cell apoptosis by acting as a pro-survival factor. In the present study, to further reveal the function of cFLIP in granulosa cells, we examined the anti-apoptotic mechanism of cFLIP using KGN, a human granulosa tumor cell line. Fas-mediated apoptosis was induced by co-treatment with anti-Fas antibody (CH-11), which acts as an agonist of Fas-ligand, and cycloheximide (CHX). When cFLIP was stably expressed in KGN cells following transfection of an expression vector, the Fas-mediated apoptosis was inhibited. Suppression of cFLIP by small interfering RNA (siRNA) spontaneously induced cell death. Silencing of cFLIP promoted cleavage of procaspase-8, and the cell death caused by cFLIP siRNA was completely blocked by a caspase-8 inhibitor (Z-IETD-FMK), indicating that cFLIP regulates apoptosis in KGN cells by inhibiting cleavage of procaspase-8. In conclusion, cFLIP is an essential pro-survival factor for granulosa cells, and it prevents granulosa cell apoptosis by inhibiting procaspase-8 activation.
哺乳动物卵巢中超过99%的卵泡会发生闭锁，但其调控严格选择过程的机制仍不清楚。颗粒细胞凋亡被认为是卵泡闭锁的触发因素，它发生在卵母细胞死亡之前。细胞FLICE样抑制蛋白（cFLIP）是procaspase-8（也称为FLICE）的同源物，是一种细胞内抗凋亡蛋白。它在猪卵巢的颗粒细胞中表达，在卵泡闭锁过程中其水平会降低。我们推测cFLIP通过作为一种促生存因子来调节颗粒细胞凋亡。在本研究中，为了进一步揭示cFLIP在颗粒细胞中的功能，我们使用人颗粒细胞瘤细胞系KGN研究了cFLIP的抗凋亡机制。通过与作为Fas配体激动剂的抗Fas抗体（CH-11）和环己酰亚胺（CHX）共同处理来诱导Fas介导的凋亡。当表达载体转染后cFLIP在KGN细胞中稳定表达时，Fas介导的凋亡受到抑制。小干扰RNA（siRNA）抑制cFLIP会自发诱导细胞死亡。cFLIP沉默促进了procaspase-8的裂解，并且cFLIP siRNA引起的细胞死亡被caspase-8抑制剂（Z-IETD-FMK）完全阻断，这表明cFLIP通过抑制procaspase-8的裂解来调节KGN细胞的凋亡。总之，cFLIP是颗粒细胞必不可少的促生存因子，它通过抑制procaspase-8的激活来防止颗粒细胞凋亡。
