ACTIVIN-BINDING PROTEIN FOLLISTATIN MESSENGER-RIBONUCLEIC-ACID AND SECRETED PROTEIN-LEVELS ARE INDUCED BY CHORIONIC-GONADOTROPIN IN CULTURED HUMAN GRANULOSA-LUTEAL CELLS

Citation
T. Tuuri et al., ACTIVIN-BINDING PROTEIN FOLLISTATIN MESSENGER-RIBONUCLEIC-ACID AND SECRETED PROTEIN-LEVELS ARE INDUCED BY CHORIONIC-GONADOTROPIN IN CULTURED HUMAN GRANULOSA-LUTEAL CELLS, Endocrinology, 135(5), 1994, pp. 2196-2203
Citations number
42
Categorie Soggetti
Endocrynology & Metabolism
Journal title
ISSN journal
00137227
Volume
135
Issue
5
Year of publication
1994
Pages
2196 - 2203
Database
ISI
SICI code
0013-7227(1994)135:5<2196:APFMAS>2.0.ZU;2-0
Abstract
We studied the effect of hCG on follistatin (FS) messenger RNA (mRNA) steady state levels and protein secretion in cultures of human granulo sa-luteal (GL) cells obtained at oocyte harvest for in vitro fertiliza tion. Three different overlapping FS complementary DNA (cDNA) fragment s were generated by reverse transcription-polymerase chain reaction fr om human GL cell RNA. Together, these fragments covered the open readi ng frame, which appeared to be identical in sequence to previously iso lated human testis-derived cDNAs. An alternative splicing event at the 3'-end of the FS transcript previously shown to give rise to transcri pts encoding 344 amino acid (as) and 317- as proteins was also observe d. In Northern analysis of human GL cell RNA, a major 2.5-kilobase tra nscript 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 w ith hCG (30 ng/ml). Time and concentration dependence studies on the e ffect of hCG were performed with cells cultured for 6-8 days before ho rmone treatment. Time-course experiments indicated that hCG (30 ng/ml) markedly induces FS mRNA levels as early as 2 h after stimulation. Th e maximal response to hCG stimulation, about 9-fold (mean of five expe riments) above basal levels, was observed at 6-8 h, and thereafter, on ly moderate or no induction of FS mRNA levels could be detected at 24 or 48 h. Concentration dependence studies performed 8 h after stimulat ion indicated that the maximal induction occurred with 30-100 ng/ml hC G, with an ED(50) of about 3-10 ng/ml. When the cells were treated wit h the protein synthesis inhibitor cycloheximide (20 mu g/ml) 20 min be fore stimulation of the cells with hCG, both basal and hCG-stimulated FS mRNA levels increased at 24 h, indicating stabilization of the tran scripts. However, it did not affect the rapid induction of FS mRNA lev els by hCG at 2 h. The decline in FS transcript levels in untreated an d hCG-treated cells was studied by blocking the transcription with 5 m u M actinomycin-D. The degradation rate of FS mRNA was increased in hC G-treated compared to control cells. To study whether the transiently induced FS mRNAs are translated to proteins in hCG-treated and untreat ed human GL cells, metabolic labeling and immunoprecipitation experime nts were performed to detect secreted [S-35]FS proteins with the speci fic anti-FS antiserum Rb 32. Several immunoreactive proteins in the ra nge of 32-39 kilodaltons, corresponding to the expected mol wt of vari ably processed forms of FS, were detected by polyacrylamide gel electr ophoresis. A clear increase in their levels was observed in medium of hCG-treated cultures. The relatedness of the immunoprecipitated protei ns to FS was verified by the ability of the recombinant human FS to di splace their binding to anti-FS antiserum. We conclude that 1) the hum an GL cell-derived FS cDNAs are indistinguishable from previously repo rted cDNA clones derived from human testis; 2) the alternative splicin g variant of FS transcript that encodes the 344-aa protein is more abu ndantly expressed in human GL cells than that giving rise to the 317-a a FS protein; 3) FS transcript levels are rapidly and transiently indu ced by hCG in a time- and concentration-dependent manner; 4) the rapid decline in FS mRNA levels after their initial induction by hCG is pro bably explained by increased degradation of the transcripts in hCG-tre ated cells compared to the controls; and 5) the induced mRNAs are tran slated to secreted immunoreactive FS proteins. These results support a physiological role for hCG (or LH) as a regulator of FS mRNA and prot ein expression in the human ovary.