Effect of RNA Interference of BID and BAX mRNAs on Apoptosis in Granulosa Cell-derived KGN Cells

Abstract

Abstract. In mitochondrion-dependent type II apoptosis, BH3-interacting domain death agonist (BID) and BCL-2-associated X protein (BAX) promote death ligand and receptor-mediated cell death. In porcine ovaries, the levels of BID and BAX increase in follicular granulosa cells during atresia. In the present study, to confirm the pro-apoptotic activity of BID and BAX in granulosa cells, we examined the effect of RNA interference of BID or BAX on apoptosis using a human ovarian granulosa tumor cell line, KGN. By reverse transcription polymerase chain reaction (RT-PCR) and Western blotting, expression of BID and BAX was detected in KGN cells. Then, we suppressed BID and BAX mRNA expression in KGN cells using small interfering RNA (siRNA). When BID or BAX was suppressed, a significant decrease in the apoptotic cell rate was noted. In granulosa-derived cells, BID and BAX showed pro-apoptotic activity. These results suggest that BID and BAX act as signal-transducing factors in mitochondrion-dependent type II apoptosis. Key words: BCL-2-associated X protein (BAX), BH3-interacting domain death agonist (BID), Mitochondrion-dependent type II apoptosis, RNA interference (RNAi) (J. Reprod. Dev. 58: [112][113][114][115][116] 2012) I n mammalian ovaries, more than 99% of follicles degenerate at various stages of follicular growth and development [1]. The degeneration is explained, at least in part, by the apoptosis of granulosa cells [2][3][4][5][6]. In porcine ovarian follicles in the early stages of atresia, characteristics typical of apoptosis are observed in scattered granulosa cells, but not in cumulus cells, oocytes or the cells of internal or external thecal layers [7]. As previously reported [8], it is considered that selective granulosa cell apoptosis is induced by death ligand and receptor systems as follows: When trimerized death ligands bind with trimerized death receptors located on the cell membrane, the receptors are activated. An adaptor protein (Fasassociated death domain protein) binds with activated receptors to construct death-inducing signal complex (DISC), which binds procaspase-8. Then, procaspase-8 is truncated and activated in DISC. Two types of cell death ligand and receptor-dependent signaling pathways, type I and II, have been found. Granulosa cells undergo mitochondrion-dependent type II apoptosis. Activated caspase-8 shortens BH3-interacting domain death agonist (BID) protein [9,10], and the truncated-BID (tBID) stimulates BCL-2-associated X protein (BAX). Then, BAX is transferred to the outer membrane of the mitochondrion and releases cytochrome c [11,12]. Cytochrome c binds with apoptotic protease-activating factor 1 and procaspase-9, and this complex is called an apoptosome [13]. The procaspase-9 is activated in the apoptosome and shortens procaspase-3, and the activated caspase-3 truncates caspase-3-activated DNase (CAD) [14,15]. The shortened CAD moves into the nucleus and cuts the genomic DNA leading to apoptosis.A large amount of caspase-8 is activated in type I apoptosis, but a small amo...

DRAM1 regulates apoptosis through increasing protein levels and lysosomal localization of BAX

“…Moreover, this study showed that 3NP induced the release of cytochrome c , and the activation of caspase-3 was significantly attenuated after knockdown of BAX with siRNA ( Figure 5c and Figure 6c ), suggesting that DRAM1-dependent BAX upregulation is involved in 3NP-induced mitochondrial apoptotic pathway. To confirm if BID cleavage contributes to 3NP-induced apoptosis, BID siRNAs, which have been previously validated, 31 were used to silence BID ( Figure 6d ). The data showed that 3NP-induced caspase-3 activation was partially inhibited by BID siRNA ( Figure 6e ).…”

Effects of an Inhibitor of the Gamma-Secretase Complex on Proliferation and Apoptotic Parameters in a FOXL2-Mutated Granulosa Tumor Cell Line (KGN)1

“…The BAX/BCL2 and BCLXs/BCLXl proapoptotic ratios were increased when Notch activation was inhibited, demonstrating an impaired function of mitochondria in these cells and the participation of the intrinsic apoptotic pathway in DAPT-induced apoptosis in KGN cells. The proapoptotic activity of BH3-interacting domain death agonist (BID) and BAX proteins has been recently confirmed in KGN cells by using the siRNA technique [47]. BAX expression has also been associated with apoptosis in ovarian cancer tissue, and patients expressing high levels of BAX in tumors have a significantly longer median survival than patients with low BAX expression [48,49].…”

The role of BH3-only proteins in apoptosis within the ovary

“…More than 99% of the growing follicles in the ovary are destined for atresia, which is usually first evident by apoptosis of the granulosa cells. Notably, BID expression increases during follicular atresia in the porcine ovary and knockdown of BID expression in human granulosa cell-derived KGN cells can suppress apoptosis (Sai et al 2011(Sai et al , 2012, suggesting a role for BID in regulating the normal process of follicular atresia. In addition to this natural process, BID may also be responsible for triggering follicular atresia following exposure to exogenous environmental toxicants such as di-n-butyl phthalate and the organochlorine pesticide methoxychlor, which are known to increase follicular atresia (Paulose et al 2012, Craig et al 2013.…”