Nuclear Factor-kB-Mediated X-Linked Inhibitor of Apoptosis Protein Expression Prevents Rat Granulosa Cells from Tumor Necrosis Factor a-Induced Apoptosis

Although X-linked inhibitor of apoptosis protein (Xiap) is an important intracellular suppressor of apoptosis in a variety of cell types and is present in ovary, its physiological role in follicular development remains unclear. The purpose of the present studies was to examine the modulatory role of Xiap in the proapoptotic action of tumor necrosis factor-a (TNFa) in rat granulosa cells. Granulosa cells from equine CG-primed immature rats were plated in RPMI 1640 medium containing 10% FCS and subsequently cultured in serum-free RPMI in the absence or presence of TNFa (20 ng/ml), the protein synthesis inhibitor cycloheximide (10 mM), and/or adenoviral Xiap sense or antisense complementary DNA. TNFa alone failed to induce granulosa cell death, but in the presence of cycloheximide, it markedly increased the number of apoptotic granulosa cells (as assessed by in situ terminal deoxynucleotidyl transferase-mediated deox-UTPbiotin end labeling and DNA fragmentation analysis). Western analysis indicated that TNFa alone increased the Xiap protein level, a response significantly reduced by adenoviral Xiap antisense expression. Down-regulation of Xiap expression by antisense complementary DNA induced granulosa cell apoptosis, which was potentiated by the cytokine. Inhibition of nuclear factor-kB activation by N-acetylcysteine and SN50 suppressed Xiap protein expression and enhanced apoptosis induced by TNFa. The latter phenomenon was readily attenuated by adenoviral Xiap sense expression. In conclusion, these findings suggest that Xiap is an important intracellular modulator of the TNFa death signaling pathway in granulosa cells. Its expression is regulated by the TNFa via a nuclear factor-kB-mediated mechanism. (Endocrinology 142: 557–563, 2001) I THE MAMMALIAN ovary, only a small proportion of the developing pool of follicles will eventually ovulate, and the majority undergoes atresia involving granulosa cell apoptosis. Granulosa cell fate (survival vs. apoptosis) is determined by a balance between secretion and actions of cell death inducers [e.g. GnRH (1–3) and androgens (4)] and survival factors [e.g. gonadotropin (5, 6), estrogen (4), and epidermal growth factor and transforming growth factor-a (TGFa) (7)]. Tumor necrosis factor-a (TNFa) is a pleiotropic cytokine that can induce differentiation, proliferation, and apoptosis in many cell types (8, 9). It is produced by rat and bovine granulosa cells and oocytes (10–13) and is an important regulator of steroid hormone production and follicular development and atresia (14). In addition, TNFa has been shown to inhibit ovarian cell viability and proliferation (14) and counteract the blocking effect of FSH on spontaneous granulosa cell apoptosis in cultured rat early antral follicles by inducing ceramide production and activating the interleukin-1b-converting enzyme/ced-3-related cysteine protease death pathway (15). Two receptors, TNFR1 and TNFR2, have been identified in a variety of TNFa response cells (16–18). TNFR1 contains an intracellular death domain, which is required for signaling pathways associated with apoptosis and nuclear factor-kB (NFkB) activation. NFkB activation regulates the expression of genes involved in the inflammatory response (19, 20) and in the prevention of TNFa-induced apoptosis, such as zinc finger protein A20 (21–23), members of the Bcl-2 family (24), Bcl-2 homolog Bfl-1/A1 (25), and inhibitor of apoptosis proteins (26, 27). In addition, TNFa activates caspase-8 and -3 and induces apoptosis in TNFa-sensitive cells, such as human U937 tumor cells (28) and bovine endothelial cells (29). In the mouse, TNFa alone does not induce apoptosis in cultured granulosa cells (30). However, treatment of granulosa cells with the protein synthesis inhibitor cycloheximide (CHX) potentiated Fas-mediated cell death, suggesting the presence of endogenous inhibitors of the Fas signaling pathway in granulosa cells (30). Although the rapid activation of NFkB by TNFa has been demonstrated in a number of cell systems (31), whether it is involved in either the proor antiapoptotic actions of TNFa in granulosa cells is unknown. Previous studies from our laboratory have shown that Xlinked inhibitor of apoptosis protein (Xiap) expression is higher in granulosa cells of healthy follicles than in those of atretic ones (5). However, the physiological roles of Xiap in follicular development and atresia remain unclear. In the present studies we used an equine CG (eCG)primed rat granulosa cell culture system to examine whether 1) Xiap plays a role in regulating granulosa cell apoptosis after TNFa challenge, and 2) NFkB is involved in the regulation of Xiap expression by TNFa. Our studies demonstrate that Xiap plays an important regulatory role in granulosa cell apoptosis and that TNFa alone is incapable of inducing this response. The inability of the cell to Received August 9, 2000. Address all correspondence and requests for reprints to: Benjamin K. Tsang, Ph.D., Loeb Health Research Institute, The Ottawa Hospital (Civic Campus), 1053 Carling Avenue, Ottawa, Ontario, Canada K1Y 4E9. E-mail: [email protected]. 0013-7227/01/$03.00/0 Vol. 142, No. 2 Endocrinology Printed in U.S.A. Copyright © 2001 by The Endocrine Society