Regulation of prostate branching morphogenesis by activin A and follistatin

Ventral prostate development occurs by branching morphogenesis and is an androgen-dependent process modulated by growth factors. Many growth factors have been implicated in branching morphogenesis including activins (dimers of beta(A) and beta(B) subunits); activin A inhibited branching of lung and kidney in vitro. Our aim was to examine the role of activins on prostatic development in vitro and their localization in vivo. Organ culture of day 0 rat ventral prostates for 6 days with activin A (+/- testosterone) inhibited prostatic branching and growth without increasing apoptosis. The activin-binding protein follistatin increased branching in vitro in the absence (but not presence) of testosterone, suggesting endogenous activins may reduce prostatic branching morphogenesis. In vivo, inhibin alpha subunit was not expressed until puberty, therefore inhibins (dimers of alpha and beta subunits) are not involved in prostatic development. Activin beta(A) was immunolocalized to developing prostatic epithelium and mesenchymal aggregates at ductal tips. Activin beta(B) immunoreactivity was weak during development, but was upregulated in prostatic epithelium during puberty. Activin receptors were expressed throughout the prostatic epithelium. Follistatin mRNA and protein were expressed throughout the prostatic epithelium. The in vitro evidence that activin and follistatin have opposing effects on ductal branching suggests a role for activin as a negative regulator of prostatic ductal branching morphogenesis.     

Control of digit formation by activin signalling

Major advances in the genetics of vertebrate limb development have been obtained in recent years. However, the nature of the signals which trigger differentiation of the mesoderm to form the limb skeleton remains elusive. Previously, we have obtained evidence for a role of TGFbeta2 in digit formation. Here, we show that activins A and B and/or AB are also signals involved in digit skeletogenesis. activin betaA gene expression correlates with the initiation of digit chondrogenesis while activin betaB is expressed coincidently with the formation of the last phalanx of each digit. Exogenous administration of activins A, B or AB into the interdigital regions induces the formation of extra digits. follistatin, a natural antagonist of activins, is expressed, under the control of activin, peripherally to the digit chondrogenic aggregates marking the prospective tendinous blastemas. Exogenous application of follistatin blocks physiological and activin-induced digit formation. Evidence for a close interaction between activins and other signalling molecules, such as BMPs and FGFs, operating at the distal tip of the limb at these stages is also provided. Chondrogenesis by activins is mediated by BMPs through the regulation of the BMP receptor bmpR-1b and in turn activin expression is upregulated by BMP signalling. In addition, AER hyperactivity secondary to Wnt3A misexpression or local administration of FGFs, inhibits activin expression. In correlation with the restricted expression of activins in the course of digit formation, neither activin nor follistatin treatment affects the development of the skeletal components of the stylopod or zeugopod indicating that the formation of the limb skeleton is regulated by segment-specific chondrogenic signals.     

Expression of mRNA for activin-binding protein (follistatin) during early embryonic development of Xenopus laevis.

Follistatin is a specific activin-binding protein and is supposed to control activin functions. During Xenopus embryonic development, activin is thought to act as a natural mesoderm-inducing factor. We isolated here the Xenopus follistatin cDNA from Xenopus ovary cDNA library and studied the expression of Xenopus follistatin gene during the course of early embryonic development. The Xenopus follistatin has an 84% homology at the level of deduced amino acid sequence with human and porcine follistatin. Its 3.5 kb mRNA is first expressed at the gastrula stage, when the expression of activin mRNA becomes first detectable, and increased thereafter. Another species of 2 kb mRNA become detectable from early neurula and also increased dramatically in tadpole. These results suggest that the follistatin acts also as a regulator of activin in inductive interactions during amphibian embryonic development.     

Isolation and characterization of native activin B.

To examine whether activin binds to follistatin, an activin-binding protein, to form a complex in vivo, we attempted to purify activin-follistatin complex from porcine follicular fluid. Our results thus obtained indicated that almost equimolar amounts of activins A, AB, and B are present as a complex with follistatin in the follicular fluid. Reverse-phase high performance liquid chromatography of the purified complex yielded follistatin and activins A, AB, and B. The activity of the purified activin B was found to be significantly lower than those of other activins in various assay systems such as stimulation of follicle-stimulating hormone secretion, induction of erythrodifferentiation, and potentiation of expression of gonadotropin receptors on ovarian cells. Moreover, binding of 125I-activin A to erythroleukemic cells which are activin-responsive was competed by activin B with approximately 10-fold lower potency compared with other activins. In contrast to these results, activin B was proved to have a potent Xenopus mesoderm-inducing activity, comparable with that of other activins. This indicates that, unlike activins A and AB, activin B can only elicit mesoderm-inducing activity and cannot function in other biological systems, suggesting a specific role of activin B in early development and unknown biological functions.     

Uterine-embryonic interaction in pig: activin, follistatin, and activin receptor II in uterus and embryo during early gestation

The mRNA expression patterns of activin beta(A) and follistatin in the uterus and embryo, the mRNA expression of the activin receptor II in the embryo, and the localization in the uterus of the immunoreactive activin beta(A) and the receptor II proteins in the uterus were examined at gestation days 7-12 after ovulation in pig. Activin was located predominantly at the mesometrial side of the uterus during all stages of pregnancy studied. Follistatin mRNA was absent in the uterus during these stages, suggesting that activin of uterine origin is not inhibited by intra-uterine follistatin. The receptor was localized throughout the glandular and luminal epithelium of the uterus. In the embryo, activin was expressed predominantly in the epiblast before unfolding, but after unfolding of the epiblast activin expression shifted to the trophoblast. The expression pattern of follistatin mRNA was contrarily to that of activin, i.e., before unfolding predominantly in the trophoblast (days 8-9), and shifted to the epiblast at day 10. During streak stages, follistatin was detected in the node and primitive streak. Activin receptor II mRNA was first detected at day 8 in the embryoblast. At day 11, it was expressed in trophoblast cells near the epiblast, and in the first ingressing mesoderm cells. During the streak stages, it was expressed predominantly in the trophoblast. The presence of activin and its receptor in uterine epithelium and early embryonic tissues indicate that both embryonic and uterine activin are involved in intra-uterine processes, such as attachment and early embryonic development. Mol. Reprod. Dev. 59: 390-399, 2001.     

Regional localization of activin-βA,activin-βC,follistatin, proliferation,and apoptosis in adult and developing mouse prostate ducts

Activins and inhibins, members of the TGF-β superfamily, are growth and differentiation factors involved in the regulation of several biological processes, including reproduction, development, and fertility. Previous studies have shown that the activin-β_A subunit plays a pivotal role in prostate development. Activin-A inhibits branching morphogenesis in the developing prostate, and its expression is associated with increased apoptosis in the adult prostate. Follistatin, a structurally unrelated protein to activins, is an antagonist of activin-A. A balance between endogenous activin-A and follistatin is required to maintain prostatic branching morphogenesis. Deregulation of this balance leads to branching inhibition or excessive branching and increased maturation of the stroma surrounding the differentiating epithelial ducts. Recent work identified another member of the TGF-β superfamily, the activin-β_C subunit, as a novel antagonist of activin-A. Over-expression of activin-C (β_C-β_C) alters prostate homeostasis, by interfering with the activin-A signaling. The current study characterized the spatiotemporal localization of activin-A, activin-C and follistatin in the adult and developing mouse prostate using immunohistochemical analysis. Results showed activin-C and follistatin are differentially expressed during prostate development and suggested that the antagonistic property of follistatin is secondary to the action of activin-C. In conclusion, the present study provides evidence to support a role of activin-C in prostate development and provides new insights in the spatiotemporal localization of activins and their antagonists during mouse prostate development.     

Roles of intracerebral activin,inhibin, and follistatin in the regulation of Kiss-1 gene expression: Studies using primary cultures of fetal rat neuronal cells

Hypothalamic kisspeptin, encoded by the Kiss-1 gene, governs the hypothalamic-pituitary-gonadal axis by directly regulating the release of gonadotropin-releasing hormone. In this study, we examined the roles of activin, inhibin, and follistatin in the regulation of Kiss-1 gene expression using primary cultures of fetal rat neuronal cells, which express the Kiss-1 gene and kisspeptin. Stimulation with activin significantly increased Kiss-1 gene expression in these cultures by 2.02 ± 0.39-fold. In contrast, a significant decrease in Kiss-1 gene expression was observed with inhibin A and follistatin treatment. Inhibin B did not modulate Kiss-1 gene expression. Activin, inhibin, and follistatin were also expressed in fetal rat brain cultures and their expression was controlled by estradiol (E2). The inhibin α, βA, and βB subunits were upregulated by E2. Similarly, follistatin gene expression was significantly increased by E2 in these cells. Our results suggest the possibility that activin, inhibin, and follistatin expressed in the brain participate in the E2-induced feedback control of the hypothalamic-pituitary-gonadal axis.     

A novel BMP expressed in developing mouse limb, spinal cord, and tail bud is a potent mesoderm inducer in Xenopus embryos

The bone morphogenetic proteins (BMPs) play critical roles in patterning the early embryo and in the development of many organs and tissues. We have identified a new member of this multifunctional gene family, BMP-11, which is most closely related to GDF-8/myostatin. During mouse embryogenesis, BMP-11 is first detected at 9.5 dpc in the tail bud with expression becoming stronger as development proceeds. At 10.0 dpc, BMP-11 is expressed in the distal and posterior region of the limb bud and later localizes to the mesenchyme between the skeletal elements. BMP-11 is also expressed in the developing nervous system, in the dorsal root ganglia, and dorsal lateral region of the spinal cord. To assess the biological activity of BMP-11, we tested the protein in the Xenopus ectodermal explant (animal cap) assay. BMP-11 induced axial mesodermal tissue (muscle and notochord) in a dose-dependent fashion. At higher concentrations, BMP-11 also induced neural tissue. Interestingly, the activin antagonist, follistatin, but not noggin, an antagonist of BMPs 2 and 4, inhibited BMP-11 activity on animal caps. Our data suggest that in Xenopus embryos, BMP-11 acts more like activin, inducing dorsal mesoderm and neural tissue, and less like other family members such as BMPs 2, 4, and 7, which are ventralizing and anti-neuralizing signals. Taken together, these data suggest that during vertebrate embryogenesis, BMP-11 plays a unique role in patterning both mesodermal and neural tissues.     

Follistatin attenuates early liver fibrosis: effects on hepatic stellate cell activation and hepatocyte apoptosis

Activin A, a member of the transforming growth factor-beta superfamily, is constitutively expressed in hepatocytes and regulates liver mass through tonic inhibition of hepatocyte DNA synthesis. Follistatin is the main biological inhibitor of activin bioactivity. These molecules may be involved in hepatic fibrogenesis, although defined roles remain unclear. We studied activin and follistatin gene and protein expression in cultured rat hepatic stellate cells (HSCs) and in rats given CCl4 for 8 wk and examined the effect of follistatin administration on the development of hepatic fibrosis. In activated HSCs, activin mRNA was upregulated with high expression levels, whereas follistatin mRNA expression was unchanged from baseline. Activin A expression in normal lobular hepatocytes redistributed to periseptal hepatocytes and smooth muscle actin-positive HSCs in the fibrotic liver. A 32% reduction in fibrosis, maximal at week 4, occurred in CCl4-exposed rats treated with follistatin. Hepatocyte apoptosis decreased by 87% and was maximal at week 4 during follistatin treatment. In conclusion, activin is produced by activated HSCs in vitro and in vivo. Absence of simultaneous upregulation of follistatin gene expression in HSCs suggests that HSC-derived activin is biologically active and unopposed by follistatin. Our in vivo and in vitro results demonstrate that activin-mediated events contribute to hepatic fibrogenesis and that follistatin attenuates early events in fibrogenesis by constraining HSC proliferation and inhibiting hepatocyte apoptosis.     

Site-specific mutagenesis of human follistatin

Follistatin is a monomeric protein originally discovered in ovarian follicular fluid as a suppressor of pituitary follicle-stimulating hormone (FSH) secretion, and later identified as a binding protein for activin. To explore the role of the Asn-linked carbohydrate chains on the follistatin molecule in regard to the inhibition of FSH secretion and activin binding ability, site-specific mutations were introduced at either or both of the two potential Asn-linked glycosylation sites of human follistatin with 315 amino acids (hFS-315). The three types of follistatin mutants were expressed individually in Chinese hamster ovary cells. When tested for their ability to inhibit FSH secretion and to bind activin, each mutant was found to have a similar property as the non-mutated recombinant hFS-315, suggesting that glycosylation of the follistatin molecule has no effect in these functions. However, a two amino acid insertion in between the second and the third amino acid residues in hFS-315 caused the resulting compound to lose completely its inhibitory activity on FSH secretion from the pituitary as well as its binding ability to activin. This finding suggests that the amino-terminal region of the follistatin molecule is critical for both of these functions.     

Inhibition of mesoderm formation by follistatin

 Mesoderm induction requires interaction between cells of the animal and vegetal hemispheres of the embryo. Several molecules have been proposed as candidates for mesoderm-inducing signals, with activin a particularly strong candidate. However, it has not been possible to inhibit mesoderm formation in vivo by specifically blocking activin action. Follistatin is able to inhibit the action of activin but not that of the mature region of Vg1, a member of the transforming growth factor β family. Follistatin therefore provides a useful tool for distinguishing between signalling by these two factors. We have overexpressed Xenopus follistatin mRNA and analysed the expression of several mesodermal markers. Our results show an inhibition of mesodermal formation by follistatin in a concentration-dependent manner, showing the requirement of activin for mesodermal induction. Received: 22 August 1997 / Accepted: 16 January 1998     

Follistatin is a developmentally regulated cytokine in neural differentiation.

Activin acts mitogenically on P19 cells as well as being inhibitory of the differentiation of retinoic acid-treated P19 cells and some neuroblastoma cell lines. Here, we show some lines of evidence that follistatin, an activin-binding protein, is also involved in neural differentiation. Counteracting the activity of activin, addition of follistatin suppresses the anchorage-independent growth of P19 cells in soft agar and stimulates neurite outgrowth of a neuroblastoma cell line, IMR-32 cells. While activin does not seem to be expressed significantly, follistatin is demonstrated in the conditioned medium of these cells. Furthermore, the expression of follistatin in P19 cells is subject to dynamic fluctuations in response to retinoic acid treatment. These neural cells may produce follistatin in a cell stage-specific manner in order to interact with exogenously derived activin.     

FOXL2-induced follistatin attenuates activin A-stimulated cell proliferation in human granulosa cell tumors

Human granulosa cell tumors (GCTs) are rare, and their etiology remains largely unknown. Recently, the FOXL2 402C > G (C134W) mutation was found to be specifically expressed in human adult-type GCTs; however, its function in the development of human GCTs is not fully understood. Activins are members of the transforming growth factor-beta superfamily, which has been shown to stimulate normal granulosa cell proliferation; however, little is known regarding the function of activins in human GCTs. In this study, we examined the effect of activin A on cell proliferation in the human GCT-derived cell line KGN. We show that activin A treatment stimulates KGN cell proliferation. Treatment with the activin type I receptor inhibitor SB431542 blocks activin A-stimulated cell proliferation. In addition, our results show that cyclin D2 is induced by treatment with activin A and is involved in activin A-stimulated cell proliferation. Moreover, the activation of Smad signaling is required for activin A-induced cyclin D2 expression. Finally, we show that the overexpression of the wild-type FOXL2 but not the C134W mutant FOXL2 induced follistatin production. Treatment with exogenous follistatin blocks activin A-stimulated cell proliferation, and the overexpression of wild-type FOXL2 attenuates activin A-stimulated cell proliferation. These results suggest that FOXL2 may act as a tumor suppressor in human adult-type GCTs by inducing follistatin expression, which subsequently inhibits activin-stimulated cell proliferation.     

Developmental profiles of activin betaA, betaB, and follistatin expression in the zebrafish ovary: evidence for their differential roles during sexual maturation and ovulatory cycle

Our recent experiments showed that gonadotropin(s) stimulated activin betaA and follistatin expression through the cAMP-PKA pathway but suppressed betaB via a cAMP-dependent but PKA-independent pathway in cultured zebrafish follicle cells. Given that pituitary gonadotropins are the major hormones controlling the development and function of the ovary, the differential expression of activin betaA and betaB as well as follistatin in response to gonadotropin(s) raises an interesting question about the temporal expression patterns of these molecules in vivo during sexual maturation and ovulatory cycle. Three experiments were performed in the present study. In the first experiment using sexually immature zebrafish, we followed the expression of activin betaA, betaB, and follistatin at the whole ovary level during a 10-day period in which the ovary developed from the primary growth stage to the one with nearly full-grown follicles. Activin betaA expression was very low at the primary growth stage but significantly increased with the growth of the ovary, and its rise was accompanied by an increase in follistatin expression. In contrast, the expression of activin betaB could be easily detected in the ovary of all stages; however, it did not exhibit an obvious trend of variation during the development. The second experiment examined the stage-dependent expression of activin betaA, betaB, and follistatin at the follicle level in the adult mature zebrafish. The expression of activin betaA was again low in the follicles during the primary growth stage, but exhibited a phenomenal increase after the follicles entered vitellogenesis with the peak level reached at midvitellogenic stage; in contrast, activin betaB mRNA could be easily detected at all stages with a slight increase during follicle growth. The expression of follistatin, on the other hand, also increased significantly during vitellogenesis; however, its level dropped sharply after reaching the peak at the midvitellogenic stage. In the third experiment, we investigated the dynamic changes of the ovarian activin betaA, betaB, and follistatin expression during the daily ovulatory cycle. The expression of activin betaA and follistatin gradually increased from 1800 h onward and reached the peak level around 0400 h when the germinal vesicles had migrated to the periphery in the full-grown oocytes. In contrast, activin betaB expression steadily declined, although not statistically significant, during the same period, but increased sharply at 0700 h when mature oocytes started to appear in most of the ovaries collected. In conclusion, activin betaA and betaB exhibit distinct expression patterns during the development of the ovary and the daily ovarian cycle of the zebrafish. It seems that activin betaA is involved in promoting ovary and follicle growth, whereas activin betaB may have a tonic role throughout follicle development but becomes critical at the late stage of oocyte maturation and/or ovulation.     

BMP-2 induces the expression of activin betaA and follistatin in vitro

Activins are members of the transforming growth factor beta (TGF-beta) superfamily and have been shown to be multifunctional regulators of development and cell differentiation. Increasing evidence suggests activin betaA is involved in skeletal development. Using differential display PCR we have identified activin betaA as a gene associated with recombinant human bone morphogenetic protein-2 (rhBMP-2) induced differentiation of a mouse limb bud cell line, MLB13MYC clone 17, from a prechondroblastic to an osteoblastic phenotype. The expression of activin betaA peaks at 24 h of rhBMP-2 treatment, before detection of osteocalcin mRNA expression. Cycloheximide treatment inhibits induction of activin betaA, indicating a requirement for new protein synthesis. The induction of the mRNA encoding follistatin, an activin binding protein, was also examined. Follistatin mRNA increases within 18 h of rhBMP-2 treatment, as activin betaA mRNA increases but before it peaks. Treatment of MLB13MYC clone 17 cells with purified activin betaA concomitant with rhBMP-2 does not affect markers of chondrocyte or osteoblast differentiation, nor does treatment with purified activin betaA alone. This suggests that activin betaA exerts its effect via a paracrine mechanism. In situ hybridization analysis demonstrates that activin betaA expression is localized to cells in the developing interphalangeal joints of embryonic mouse limbs. This is consistent with in vivo induction by BMP-2 which is also expressed in the developing joints. Activin betaA, therefore, is downstream from BMP-2 in the cascade of events that result in skeletal development.