Nuclear factor-kappa B-mediated X-linked inhibitor of apoptosis protein expression prevents rat granulosa cells from tumor necrosis factor alpha-induced apoptosis

Although X-linked inhibitor of apoptosis protein (Xiap) is an important int racellular suppressor of apoptosis in a variety of cell types and is presen t in ovary, its physiological role in follicular development remains unclea r. The purpose of the present studies was to examine the modulatory role of Xiap in the proapoptotic action of tumor necrosis factor-alpha (TNF alpha) in rat granulosa cells. Granulosa cells from equine CG-primed immature rat s were plated in RPMI 1640 medium containing 10% FCS and subsequently cultu red in serum-free RPMI in the absence or presence of TNF alpha (20 ng/ml), the protein synthesis inhibitor cycloheximide (10 muM), and/or adenoviral X iap sense or antisense complementary DNA. TNFa alone failed to induce granu losa cell death, but in the presence of cycloheximide, it markedly increase d the number of apoptotic granulosa cells las assessed by in situ terminal deoxynucleotidyl transferase-mediated deox-UTP-biotin end labeling and DNA fragmentation analysis). Western analysis indicated that TNFa: alone increa sed 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 b y the cytokine. Inhibition of nuclear factor-kappaB activation by N-acetylc ysteine 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 TNF alpha via a nuclea r factor-kappaB-mediated mechanism.