Activin-binding protein follistatin messenger ribonucleic acid and secreted protein levels are induced by chorionic gonadotropin in cultured human granulosa-luteal cells.
We studied the effect of hCG on follistatin (FS) messenger RNA (mRNA) steady state levels and protein secretion in cultures of human granulosa-luteal (GL) cells obtained at oocyte harvest for in vitro fertilization. Three different overlapping FS complementary DNA (cDNA) fragments were generated by reverse transcription-polymerase chain reaction from human GL cell RNA. Together, these fragments covered the open reading frame, which appeared to be identical in sequence to previously isolated human testis-derived cDNAs. An alternative splicing event at the 3'-end of the FS transcript previously shown to give rise to transcripts encoding 344 amino acid (aa) and 317-aa proteins was also observed. In Northern analysis of human GL cell RNA, a major 2.5-kilobase transcript and a minor 1.5-kilobase FS transcript were detected, and the steady state levels of both mRNAs were induced by an 8-h stimulation with hCG (30 ng/ml). Time and concentration dependence studies on the effect of hCG were performed with cells cultured for 6-8 days before hormone treatment. Time-course experiments indicated that hCG (30 ng/ml) markedly induces FS mRNA levels as early as 2 h after stimulation. The maximal response to hCG stimulation, about 9-fold (mean of five experiments) above basal levels, was observed at 6-8 h, and thereafter, only moderate or no induction of FS mRNA levels could be detected at 24 or 48 h. Concentration dependence studies performed 8 h after stimulation indicated that the maximal induction occurred with 30-100 ng/ml hCG, with an ED50 of about 3-10 ng/ml. When the cells were treated with the protein synthesis inhibitor cycloheximide (20 micrograms/ml) 20 min before stimulation of the cells with hCG, both basal and hCG-stimulated FS mRNA levels increased at 24 h, indicating stabilization of the transcripts. However, it did not affect the rapid induction of FS mRNA levels by hCG at 2 h. The decline in FS transcript levels in untreated and hCG-treated cells was studied by blocking the transcription with 5 microM actinomycin-D. The degradation rate of FS mRNA was increased in hCG-treated compared to control cells. To study whether the transiently induced FS mRNAs are translated to proteins in hCG-treated and untreated human GL cells, metabolic labeling and immunoprecipitation experiments were performed to detect secreted [35S]FS proteins with the specific anti-FS antiserum Rb 32.(ABSTRACT TRUNCATED AT 400 WORDS)
Department of Bacteriology and Immunology, University of Helsinki, Finland.
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Tuuri
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Erämaa
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Hildén
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Ritvos
D001483:Base Sequence; D002478:Cells, Cultured; D006063:Chorionic Gonadotropin / Q000494:pharmacology*; D018076:DNA, Complementary / Q000032:analysis / Q000235:genetics; D004305:Dose-Response Relationship, Drug; D005260:Female; D038681:Follistatin; D006023:Glycoproteins / Q000032:analysis* / Q000235:genetics*; D006107:Granulosa Cells / Q000737:chemistry* / Q000166:cytology* / Q000187:drug effects; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011233:Precipitin Tests; D012333:RNA, Messenger / Q000032:analysis* / Q000235:genetics; D013997:Time Factors
D006063:Chorionic Gonadotropin; D018076:DNA, Complementary; D038681:Follistatin; D006023:Glycoproteins; D012333:RNA, Messenger