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Regular Article |
B-Mediated Induction of Flice-Like Inhibitory Protein Prevents Tumor Necrosis Factor 
-Induced Apoptosis in Rat Granulosa Cells1
a Reproductive Biology Unit and Division of Reproductive Medicine, Department of Obstetrics & Gynecology and Cellular & Molecular Medicine, University of Ottawa, Ottawa Health Research Institute, The Ottawa Hospital (Civic Campus), Ottawa, Ontario, Canada K1Y 4E9
b Département de Chimie-Biologie, Université du Québec à Trois-Rivières, C.P. 500, Trois-Rivières, Québec, Canada G9A 5H7
The purpose of the present studies was to examine the role and regulation of the antiapoptotic Flice-like inhibitory protein (FLIP) in rat granulosa cells by tumor necrosis factor 
(TNF) in vitro. Granulosa cells from immature rats primed with eCG were cultured in serum-free RPMI in the absence or presence of TNF (20 ng/ml), cycloheximide (CHX, 10 µg/ml), SN50 (a specific inhibitor of nuclear factor 
B [NFB] translocation, 100 or 200 µg/ml), or a combination of these. (SM50, a mutated inactive peptide of SN50, was used as control.) Inhibitor B (IB; total and phosphorylated forms) and NFB binding abilities were measured by Western blot and electrophoretic mobility shift assay, respectively. Apoptosis was assessed by in situ TUNEL assay, whereas FLIP mRNA levels were determined by semiquantitative reverse transcriptase-polymerase chain reaction. TNF alone failed to induce granulosa cell death but significantly increased the apoptotic cell number in the presence of cycloheximide. TNF significantly up-regulated the expression of the short form of FLIP (FLIPS) but not the long form (FLIPL). TNF induced IB phosphorylation and NFB activation. SN50, but not SM50, attenuated TNF-induced FLIPS expression and enhanced TNF-induced apoptosis. Down-regulation of TNF-induced FLIPS by FLIPS antisense expression enhanced TNF-induced apoptosis. A full length of rat FLIPS, with high homology to mouse FLIPS (85%), had been cloned and sequenced. These findings suggest that, in addition to its proapoptotic function, TNF can induce an intracellular survival factor for the maintenance of follicular development. TNF-induced, NFB-mediated FLIPS expression is a determinant of granulosa cell fate.
1 This work was supported by grant MOP-15691 from the Canadian Institutes of Health Research. C.W.X. and E.A. were recipients of Canadian Institute of Health Research Postdoctoral Fellowships.
2 Correspondence: Benjamin K. Tsang, Ottawa Health Research Institute, The Ottawa Hospital (Civic Campus), 725 Parkdale Avenue, Ottawa, ON, Canada K1Y 4E9. FAX: 613 761 4403; btsang{at}ohri.ca
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