title: Cross-Talk between Fas/Fas ligand system and nitric oxide in the pathway subserving granulosa cell apoptosis: a possible regulatory mechanism for ovarian follicle atresia.
abstract: Recent studies have shown the involvement of Fas/Fas ligand (FasL) system and nitric oxide (NO) in ovarian follicle atresia. Here we asked whether Fas/Fas ligand system interacts with NO using rat granulosa cell culture. Soluble recombinant Fas ligand (rFasL), at 100 ng/ml, significantly decreased cell viability, as measured by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) assay, in the presence of 200 U/ml interferon-gamma, whereas the concurrent addition of a caspase inhibitor, Z-VAD-FMK, at 20 microm, significantly inhibited rFasL-induced cytotoxicity. Hoechst 33342 staining and flow cytometric analysis confirmed the induction of apoptosis in granulosa cells by 100 ng/ml rFasL in the presence of interferon-gamma, which was blocked by the concomitant addition of an NO donor, S-nitroso-N-acetylpenicillamine. Western blot analysis demonstrated that rFasL significantly up-regulated caspase-3, -8, and -9 activities in granulosa cells, which were attenuated by concurrent treatment with S-nitroso-N-acetylpenicillamine. Real-time quantitative RT-PCR revealed a significant decrease in inducible NO synthase mRNA levels in rFasL-induced apoptotic granulosa cells. In conclusion, we demonstrated the involvement of Fas/FasL system in inducing apoptosis through activation of a caspase-mediated cascade in rat granulosa cells, which is coupled with a decrease in inducible NO synthase expression. We further showed that NO inhibited Fas/FasL system-induced apoptosis by suppressing activation of the caspases, pointing to a cross-talk between Fas/FasL system-induced apoptosis pathway and NO-mediated antiapoptotic pathway in ovarian follicle atresia.
authors.affiliation: Associate Professor, Department of Obstetrics and Gynecology, Faculty of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan.
authors.forename: Qiumei
authors.initials: Q
authors.lastname: Chen
authors.forename: Tetsu
authors.initials: T
authors.lastname: Yano
authors.forename: Hirotaka
authors.initials: H
authors.lastname: Matsumi
authors.forename: Yutaka
authors.initials: Y
authors.lastname: Osuga
authors.forename: Naomi
authors.initials: N
authors.lastname: Yano
authors.forename: Jiping
authors.initials: J
authors.lastname: Xu
authors.forename: Osamu
authors.initials: O
authors.lastname: Wada
authors.forename: Kaori
authors.initials: K
authors.lastname: Koga
authors.forename: Toshihiro
authors.initials: T
authors.lastname: Fujiwara
authors.forename: Koji
authors.initials: K
authors.lastname: Kugu
authors.forename: Yuji
authors.initials: Y
authors.lastname: Taketani
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D020169:Caspases / Q000378:metabolism; D002470:Cell Survival / Q000502:physiology; D002478:Cells, Cultured; D053222:Fas Ligand Protein; D005260:Female; D005496:Follicular Atresia / Q000378:metabolism*; D006107:Granulosa Cells / Q000166:cytology* / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000378:metabolism*; D009569:Nitric Oxide / Q000378:metabolism*; D020030:Nitric Oxide Donors / Q000494:pharmacology; D019001:Nitric Oxide Synthase / Q000378:metabolism; D052247:Nitric Oxide Synthase Type II; D010396:Penicillamine / Q000031:analogs & derivatives* / Q000494:pharmacology; D051381:Rats; D017208:Rats, Wistar; D020239:Receptor Cross-Talk / Q000502:physiology; D019014:fas Receptor / Q000378:metabolism*
chemical_list: D053222:Fas Ligand Protein; C505662:Faslg protein, rat; D008562:Membrane Glycoproteins; D020030:Nitric Oxide Donors; C110881:S-nitro-N-acetylpenicillamine; D019014:fas Receptor; D009569:Nitric Oxide; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; C496318:Nos2 protein, rat; D020169:Caspases; D010396:Penicillamine