Nuclear factor-κB-mediated X-linked inhibitor of apoptosis protein expression prevents rat granulosa cells from tumor necrosis factor α-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-alpha (TNF alpha) 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 TNF alpha (20 ng/ml), the protein synthesis inhibitor cycloheximide (10 muM), 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 las assessed by in situ terminal deoxynucleotidyl transferase-mediated deox-UTP-biotin 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-kappaB 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 TNF alpha via a nuclear factor-kappaB-mediated mechanism.