Nuclear factor kappaB-mediated induction of Flice-like inhibitory protein prevents tumor necrosis factor alpha-induced apoptosis in rat granulosa cells.
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 alpha (TNFalpha) in vitro. Granulosa cells from immature rats primed with eCG were cultured in serum-free RPMI in the absence or presence of TNFalpha (20 ng/ml), cycloheximide (CHX, 10 microg/ml), SN50 (a specific inhibitor of nuclear factor kappaB [NFkappaB] translocation, 100 or 200 microg/ml), or a combination of these. (SM50, a mutated inactive peptide of SN50, was used as control.) Inhibitor kappaB (IkappaB; total and phosphorylated forms) and NFkappaB 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. TNFalpha alone failed to induce granulosa cell death but significantly increased the apoptotic cell number in the presence of cycloheximide. TNFalpha significantly up-regulated the expression of the short form of FLIP (FLIP(S)) but not the long form (FLIP(L)). TNFalpha induced IkappaB phosphorylation and NFkappaB activation. SN50, but not SM50, attenuated TNFalpha-induced FLIP(S) expression and enhanced TNFalpha-induced apoptosis. Down-regulation of TNFalpha-induced FLIP(S) by FLIP(S) antisense expression enhanced TNFalpha-induced apoptosis. A full length of rat FLIP(S), with high homology to mouse FLIP(S) (85%), had been cloned and sequenced. These findings suggest that, in addition to its proapoptotic function, TNFalpha can induce an intracellular survival factor for the maintenance of follicular development. TNFalpha-induced, NFkappaB-mediated FLIP(S) expression is a determinant of granulosa cell fate.
Reproductive Biology Unit and Division of Reproductive Medicine, University of Ottawa, Ottawa Health Research Institute, The Ottawa Hospital (Civic Campus), 725 Parkdale Avenue, Ottawa, Ontario, Canada K1Y 4E9.
Chao Wu
CW
Xiao
Eric
E
Asselin
Benjamin K
BK
Tsang
D000595:Amino Acid Sequence; D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D015153:Blotting, Western; D053446:CASP8 and FADD-Like Apoptosis Regulating Protein; D002352:Carrier Proteins / Q000096:biosynthesis*; D002478:Cells, Cultured; D003001:Cloning, Molecular; D003513:Cycloheximide / Q000494:pharmacology; D016373:DNA, Antisense / Q000494:pharmacology; D018076:DNA, Complementary / Q000032:analysis / Q000235:genetics; D005260:Female; D006107:Granulosa Cells / Q000187:drug effects*; D006801:Humans; D020287:In Situ Nick-End Labeling; D047908:Intracellular Signaling Peptides and Proteins; D051379:Mice; D008969:Molecular Sequence Data; D016328:NF-kappa B / Q000494:pharmacology*; D010455:Peptides / Q000494:pharmacology; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D012333:RNA, Messenger / Q000096:biosynthesis; D051381:Rats; D017207:Rats, Sprague-Dawley; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000037:antagonists & inhibitors* / Q000494:pharmacology*
D053446:CASP8 and FADD-Like Apoptosis Regulating Protein; C506386:CFLAR protein, human; D002352:Carrier Proteins; C506387:Cflar protein, mouse; D016373:DNA, Antisense; D018076:DNA, Complementary; D047908:Intracellular Signaling Peptides and Proteins; D016328:NF-kappa B; D010455:Peptides; D011500:Protein Synthesis Inhibitors; D012333:RNA, Messenger; C118258:SN50 peptide; D014409:Tumor Necrosis Factor-alpha; D003513:Cycloheximide