Submission Number: CHA-4-1-21

Abstract Number: 239

NFB-MEDIATED INDUCTION OF FLICE-LIKE INHIBITORY PROTEIN (FLIP) BY TUMOR NECROSIS FACTOR- IN RAT GRANULOSA CELLS.

Chao Wu Xiao ^1,2 and Benjamin K Tsang* ^1,2

Reproductive Biology Unit, Department of Obstetrics & Gynecology and Cellular & Molecular Medicine, University of Ottawa,Ottawa, Ontario, Canada K1Y 4E9 ¹
Loeb Health Research Institute, Ottawa, Ontario, Canada K1Y 4E9 ²

Abstract:
The role of tumor necrosis factor- (TNF) in the regulation of granulosa cell (GC)(pro- or anti-apoptosis) and follicle (atresia or growth) fate is unclear. While its action is generally accepted to be mediated through death and survival pathways, precisely how TNF functions as an anti-apoptotic regulator of GC survival remains to be determined. The purpose of the present studies was to examine the role and regulation of the anti-apoptotic protein FLIP in rat GCs in vitro by TNF. GCs from 24 to 25-day old immature rats 24 h post-eCG were plated for 24 h in RPMI 1640 medium supplemented with 10% fetal bovine serum and subsequently cultured in serum-free RPMI TNF (20 ng/ml), cycloheximide (CHX, a protein synthesis inhibitor; 10 g/ml) and/or SN50 (a specific inhibitor of NFB translocation, 200 g/ml; SM50, a mutated inactive peptide as control). Total and phosphorylated IB as well as NFB binding ability was measured by Western blot and electrophoresis mobility shift assay (EMSA), respectively. Apoptosis was assessed by Hoechst nuclear staining, in situ TUNEL and DNA fragmentation analysis while FLIP steady state levels were determined by semi-quantitative RT-PCR. TNF alone failed to induce GC death but remarkably increased the apoptotic cell number in the presence of CHX, suggesting that an intracellular apoptosis-preventing factor might have been induced by TNF. To determine if FLIP are possible candidates, both FLIP_L (long form) and FLIP_S (short form) mRNA abundance was measured by RT-PCR. TNF significantly up-regulated FLIP_S, but not FLIP_L expression. TNF induced IB phosphorylation and NFB activation. SN50, but not SM50, attenuated TNF-induced FLIP_S expression and enhanced TNF-induced apoptosis. A 485 bp fragment of rat FLIP_S had been cloned and sequenced, showing its high homology to mouse FLIP_S (87%). These findings demonstrate that, in addition to its reported pro-apoptotic function, TNF is survival factor in the control of follicular development and atresia and that TNF-induced, NFB-mediated FLIP_S expression is a determinant of GC fate. The physiological modulator(s) of GC FLIP_S expression remains to be determined (supported by MRC of Canada). .

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This abstract is being presented at: 2:15 PM in session:
SESSION 11: APOPTOSIS IN REPRODUCTIVE BIOLOGY