Nuclear factor-kappaB-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 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 (TNFalpha) 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 TNFalpha (20 ng/ml), the protein synthesis inhibitor cycloheximide (10 microM), and/or adenoviral Xiap sense or antisense complementary DNA. TNFalpha 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 TNFalpha 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-acetyl-cysteine and SN50 suppressed Xiap protein expression and enhanced apoptosis induced by TNFalpha. 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 TNFalpha death signaling pathway in granulosa cells. Its expression is regulated by the TNFalpha via a nuclear factor-kappaB-mediated mechanism.