Follistatin-like 3 Expression Research Articles

Growth Differentiation Factor 9 (GDF9) Suppresses Follistatin and Follistatin-Like 3 Production in Human Granulosa-Lutein Cells

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.

Brief Communication: Sexual dimorphic expression of myostatin and follistatin like-3 in a rat trans-generational under-nutrition model

The detrimental effects of maternal under-nutrition during gestation on fetal development are well known with an increased propensity of metabolic disorders identified in the adult offspring. Understanding exactly how and by which molecular pathways inadequate nutrition can impact upon offspring phenotype is critical and necessary for the development of treatment methods and ultimately prevention of any negative health effects. Myostatin, a negative regulator of muscle development, has recently been shown to effect glucose homeostasis and fat deposition. The involvement of myostatin in glucose metabolism and adipogenesis thus supports its ability to act in the continued alterations to the postnatal phenotype of the offspring. This hypothesis was examined in the current study using a trans-generational gestationally under-nourished rat model exposed to a high-fat (HF) diet post-weaning. The body weight, body fat, plasma glucose and insulin concentrations of the offspring, both male and female, were investigated in relation to the protein expression of myostatin and its main inhibitor; follistatin like-3 (FSTL-3), in skeletal muscle of mature offspring. Sexual dimorphism was clearly evident in the majority of these measures, including myostatin and FSTL-3 expression. Generally males displayed higher (P < 0.05) myostatin precursor and dimer expression than females, which was especially apparent (P < 0.01) in both chow and HF trans-generationally undernourished (UNAD) groups. In females only, myostatin precursor and dimer expression was altered by both trans-generational under-nutrition and postnatal diet. Overall FSTL-3 expression did not differ between sexes, although difference between sexes within certain treatments and diets were evident. Most notably, HF fed UNAD females had higher (P < 0.05) FSTL-3 expression than HF fed UNAD males. The former group also displayed higher (P < 0.01) FSTL-3 expression compared to all other female groups. In summary, myostatin may prove to be a key mediator of the effects of inadequate prenatal nutrition, independently or in combination with a high-fat postnatal diet on offspring phenotype. Consequently, further study of myostatin may provide a novel therapeutic pathway for the treatment of metabolic disorders; however, it is vital that the influence of nutrition and gender should be taken into consideration.

Placental expression of myostatin and follistatin-like-3 protein in a model of developmental programming

Maternal undernutrition during gestation is known to be detrimental to fetal development, leading to a propensity for metabolic disorders later in the adult lives of the offspring. Identifying possible mediators and physiological processes involved in modulating nutrient transport within the placenta is essential to prevent and/or develop treatments for the effects of aberrant nutrition, nutrient transfer, and detrimental changes to fetal development. A potential role for myostatin as a mediator of nutrient uptake and transport from the mother to the fetus was shown through the recent finding that myostatin acts within the human placenta to modulate glucose uptake and therefore homeostasis. The mRNA and protein expression of myostatin and its inhibitor, follistatin-like-3 (FSTL3), was studied in the placenta and skeletal muscle of a transgenerational Wistar rat model of gestational maternal undernutrition in which the F2 offspring postweaning consumed a high-fat (HF) diet. Alterations in placental characteristics and offspring phenotype, specifically glucose homeostasis, were evident in the transgenerationally undernourished (UNAD) group. Myostatin and FSTL3 protein expression were also higher (P < 0.05) in the placentae of the UNAD compared with the control group. At maturity, UNAD HF-fed animals had higher (P < 0.05) skeletal muscle expression of FSTL3 than control animals. In summary, maternal undernutrition during gestation results in the aberrant regulation of myostatin and FSTL3 in the placenta and skeletal muscle of subsequent generations. Myostatin, through the disruption of maternal nutrient supply to the fetus, may thus be a potential mediator of offspring phenotype.

Activin βA subunit, follistatin and follistatin-like 3 are expressed in the endometrium of ovariectomized rats and regulated by estrogen replacement

Activin A is a growth factor expressed in the endometrium, where it modulates tissue remodeling and enhances decidualization. The effects of activin A are counteracted by two binding proteins, namely follistatin and follistatin-like 3 (FSTL3). We have evaluated the effects of estrogen and progestin on the endometrial expression of activin betaA subunit, follistatin and FSTL3 in ovariectomized rats. Adult female Wistar rats (n = 21) were ovariectomized and received one week later a single dose of estradiol benzoate (1.5 mg/kg body weight, i.m. injection), either alone (n = 7) or associated with depot medroxyprogesterone acetate (3 mg/kg body weight, i.m. injection, n = 7), or oil vehicle (control group, n = 7). One week later, activin betaA subunit mRNA levels had increased significantly in the uteri of rats treated with estradiol alone (7.4 fold increase over controls, P < 0.05) and to the same extent in rats receiving estradiol plus medroxyprogesterone (6.1 fold increase over controls, P < 0.05). This was accompanied by increase of betaA subunit immunostaining in estradiol and estroprogestin treated rats, which was noted only in the surface endometrial epithelium. Follistatin mRNA expression, conversely, showed a significant decrease in the groups treated with estrogen alone and estrogen plus progestin (P < 0.05), and follistatin immunostaining in the glandular epithelium was weaker in estradiol and estroprogestin-treated rats compared to controls. FSTL3 expression was similar in the 3 groups. In conclusion, the expression of activin betaA subunit increases and that of follistatin decreases following estrogen replacement in the endometrium of ovariectomized rats, and these effects are not further altered by the addition of progestin.

Hypoxia Enhances the Expression of Follistatin-like 3 in Term Human Trophoblasts

Hypoxic injury hinders placental differentiation and alters trophoblast gene expression. We tested the hypothesis that the expression of follistatin-like 3 (FSTL3), a member of the follistatin family of proteins, is modulated by hypoxia in primary human trophoblast (PHT). Using immunofluorescence of human term placental villi we detected the expression of FSTL3 protein in placental villi, primarily in trophoblasts. We verified this finding in cultured term PHT cells. Basal expression of FSTL3 transcript in cultured PHT cells, determined using quantitative PCR, was stable over the culture period. Importantly, when compared to culture in FiO 2 = 20% or FiO 2 = 8%, PHT cells cultured in FiO 2 <1% exhibited a 4–6 fold increase in FSTL3 mRNA expression as early as 4 h in hypoxia. Whereas cellular FSTL3 protein was unchanged in hypoxia, we found that hypoxia increased the level of FSTL3 in the medium. Lastly, the exposure of PHT cells to either the hypoxia-mimetic cobalt chloride or the proline hydroxylase inhibitor dimethyloxaloylglycine upregulated the expression of FSTL3 transcript. Our data indicate that hypoxia enhances the expression of FSTL3 and its release from PHT cells. Our finding that hypoxia-mimetic agents enhance FSTL3 expression implicates HIF1α in this process.