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 α (TNFα) in vitro. Granulosa cells from immature rats primed with eCG were cultured in serum-free RPMI in the absence or presence of TNFα (20 ng/ml), cycloheximide (CHX, 10 μg/ml), SN50 (a specific inhibitor of nuclear factor κB [NFκB] translocation, 100 or 200 μg/ml), or a combination of these. (SM50, a mutated inactive peptide of SN50, was used as control.) Inhibitor κB (IκB; total and phosphorylated forms) and NFκB 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. TNFα alone failed to induce granulosa cell death but significantly increased the apoptotic cell number in the presence of cycloheximide. TNFα significantly up-regulated the expression of the short form of FLIP (FLIPS) but not the long form (FLIPL). TNFα induced IκB phosphorylation and NFκB activation. SN50, but not SM50, attenuated TNFα-induced FLIPS expression and enhanced TNFα-induced apoptosis. Down-regulation of TNFα-induced FLIPS by FLIPS antisense expression enhanced TNFα-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, TNFα can induce an intracellular survival factor for the maintenance of follicular development. TNFα-induced, NFκB-mediated FLIPS expression is a determinant of granulosa cell fate.
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