Abstract
BackgroundWe have demonstrated that growth differentiation factor 9 (GDF9) enhances activin A-induced inhibin β B-subunit mRNA levels in human granulosa-lutein (hGL) cells by regulating receptors and key intracellular components of the activin signaling pathway. However, we could not exclude its effects on follistatin (FST) and follistatin-like 3 (FSTL3), well recognized extracellular inhibitors of activin A.MethodologyhGL cells from women undergoing in vitro fertilization (IVF) treatment were cultured with and without siRNA transfection of FST, FSTL3 or GDF9 and then treated with GDF9, activin A, FST, FSTL3 or combinations. FST, FSTL3 and inhibin β B-subunit mRNA, and FST, FSTL3 and inhibin B protein levels were assessed with real-time RT-PCR and ELISA, respectively. Data were log transformed before ANOVA followed by Tukey's test.Principal FindingsGDF9 suppressed basal FST and FSTL3 mRNA and protein levels in a time- and dose-dependent manner and inhibited activin A-induced FST and FSTL3 mRNA and protein expression, effects attenuated by BMPR2 extracellular domain (BMPR2 ECD), a GDF9 antagonist. After GDF9 siRNA transfection, basal and activin A-induced FST and FSTL3 mRNA and protein levels increased, but changes were reversed by adding GDF9. Reduced endogenous FST or FSTL3 expression with corresponding siRNA transfection augmented activin A-induced inhibin β B-subunit mRNA levels as well as inhibin B levels (P values all <0.05). Furthermore, the enhancing effects of GDF9 in activin A-induced inhibin β B-subunit mRNA and inhibin B production were attenuated by adding FST.ConclusionGDF9 decreases basal and activin A-induced FST and FSTL3 expression, and this explains, in part, its enhancing effects on activin A-induced inhibin β B-subunit mRNA expression and inhibin B production in hGL cells.
Highlights
Inhibin and activin are structurally related dimeric glycoprotein hormones that inhibit and stimulate, respectively, the secretion of FSH by pituitary gonadotrophs
To further evaluate if the enhancing effect of growth differentiation factor 9 (GDF9) on activin A-induced inhibin bB-subunit mRNA is related to FST or follistatin-like 3 (FSTL3) expression, we measured these changes in activin Atreated human granulosa-lutein (hGL) cells at different doses of FST or FSTL3
When 100 ng/ml of GDF9 was preincubated with BMPR2 extracellular domain (ECD) for 30 min before adding to the cell culture, the inhibitory effects of GDF9 on FST and FSTL3 mRNA levels were attenuated (Figure 1, C and D)
Summary
Inhibin and activin are structurally related dimeric glycoprotein hormones that inhibit and stimulate, respectively, the secretion of FSH by pituitary gonadotrophs. Among the extracellular regulators are some members of the follistatin (FST)-related protein family. This family consists of a large group of proteins which contain a highly conserved module of cysteine-rich sequence termed the follistatin domain and includes follistatin [4], follistatin-like (FSTL) proteins such as FSTL3 [5], and agrin [6]. Apart from follistatin and FSTL3 {formerly known as FST-related protein (FSRP) and FST-related gene (FLRG)} which are major regulators of activin action, most proteins in this family do not have this follistatin-like activity. We have demonstrated that growth differentiation factor 9 (GDF9) enhances activin A-induced inhibin bBsubunit mRNA levels in human granulosa-lutein (hGL) cells by regulating receptors and key intracellular components of the activin signaling pathway. We could not exclude its effects on follistatin (FST) and follistatin-like 3 (FSTL3), well recognized extracellular inhibitors of activin A
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