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B-Mediated X-Linked Inhibitor of Apoptosis Protein Expression Prevents Rat Granulosa Cells from Tumor Necrosis Factor 
-Induced Apoptosis
Reproductive Biology Unit and Division of Reproductive Medicine, Departments of Obstetrics and Gynecology and Cellular and Molecular Medicine, University of Ottawa, Loeb Health Research Institute, The Ottawa Hospital, Ottawa, Ontario, Canada K1Y 4E9
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: btsang{at}lri.ca
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-
(TNF) 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 (20 ng/ml), the protein synthesis inhibitor
cycloheximide (10 µM), and/or adenoviral Xiap sense or
antisense complementary DNA. TNF 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 TNF 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-
B
activation by N-acetyl-cysteine and SN50 suppressed Xiap
protein expression and enhanced apoptosis induced by TNF. 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 TNF death signaling pathway in
granulosa cells. Its expression is regulated by the TNF via a
nuclear factor-B-mediated mechanism.
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