TGFBR3L is an inhibin B co-receptor that regulates female fertility.

Follicle-stimulating hormone (FSH), a key regulator of ovarian function, is often used in infertility treatment. Gonadal inhibins suppress FSH synthesis by pituitary gonadotrope cells. The TGFß type III receptor, betaglycan, is required for inhibin A suppression of FSH. The inhibin B co-receptor was previously unknown. Here, we report that the gonadotrope-restricted transmembrane protein, TGFBR3L, is the elusive inhibin B co-receptor. TGFBR3L binds inhibin B but not other TGFß family ligands. TGFBR3L knockdown or overexpression abrogates or confers inhibin B activity in cells. Female Tgfbr3l knockout mice exhibit increased FSH levels, ovarian follicle development, and litter sizes. In contrast, female mice lacking both TGFBR3L and betaglycan are infertile. TGFBR3L's function and cell-specific expression make it an attractive new target for the regulation of FSH and fertility.

A Decade Later: Revisiting the TGFß Family's Role in Diabetes.

In 2010, we published a review summarizing the role of the transforming growth factor-beta (TGFß) family of proteins in diabetes. At that time there were still many outstanding questions that needed to be answered. In this updated review, we revisit the topic and provide new evidence that supports findings from previous studies included in the 2010 review and adds to the knowledge base with new findings and information. The most substantial contributions in the past 10 years have been in the areas of human data, the investigation of TGFß family members other than activin [e.g., bone morphogenetic proteins (BMPs), growth and differentiation factor 11 (GDF11), nodal], and the expansion of ß-cell number through various mechanisms including transdifferentiation, which was previously believed to not be possible.

Betaglycan (TGFBR3) Functions as an Inhibin A, but Not Inhibin B, Coreceptor in Pituitary Gonadotrope Cells in Mice.

Inhibins are gonadal hormones that act on pituitary gonadotrope cells to suppress FSH synthesis and secretion. Inhibin A and B are heterodimers of the inhibin ⍺-subunit disulfide-linked to one of two inhibin ß-subunits. Homodimers or heterodimers of the inhibin ß-subunits form the activins, which stimulate FSH production. Activins signal through complexes of type I and II receptor serine/threonine kinases to increase transcription of the FSHß subunit gene. According to in vitro observations, inhibins impair FSH synthesis by competitively binding to activin type II receptors, particularly in the presence of the TGFß type III receptor (TGFBR3, or betaglycan). The role of TGFBR3 in inhibin action in vivo has not been determined. Here, we ablated Tgfbr3 specifically in murine gonadotropes. Conditional knockout females were supra-fertile, exhibiting enhanced folliculogenesis, numbers of ovulated eggs per cycle, and litter sizes relative to control mice. Despite these phenotypes, FSH levels appeared to be unaltered in knockout mice, and the mechanisms underlying their enhanced fertility remain unexplained. Inhibin B is the predominant form of the hormone in males and in females during most stages of the estrous cycle. Remarkably, inhibin A, but not inhibin B, suppression of FSH synthesis was impaired in cultured pituitaries of knockout mice, which may explain the absence of discernible changes in FSH levels in vivo. Collectively, these data challenge current dogma by demonstrating that TGFBR3 (betaglycan) functions as an inhibin A, but not an inhibin B, coreceptor in gonadotrope cells in vivo. Mechanisms of inhibin B action merit further investigation.

Repulsive Guidance Molecule b (RGMb) Is Dispensable for Normal Gonadal Function in Mice.

Bone morphogenetic protein (BMP) signaling plays an important role in spermatogenesis and follicle development. Our previous studies have shown that repulsive guidance molecule b (RGMb, also known as Dragon) is a coreceptor that enhances BMP2 and BMP4 signaling in several cell types and that RGMb is expressed in spermatocytes and spermatids in the testis and in oocytes of the secondary follicles in the ovary. Here, we demonstrated that specific deletion of Rgmb in germ cells in the testis and ovary did not alter Smad1/5/8 phosphorylation, gonadal structures, and fertility. In addition, ovaries from postnatal global Rgmb knockout mice showed similar structures to the wild-type ovaries. Our results suggest that RGMb is not essential for normal gonadal function.

Activin B Induces Noncanonical SMAD1/5/8 Signaling via BMP Type I Receptors in Hepatocytes: Evidence for a Role in Hepcidin Induction by Inflammation in Male Mice.

Induction of the iron regulatory hormone hepcidin contributes to the anemia of inflammation. Bone morphogenetic protein 6 (BMP6) signaling is a central regulator of hepcidin expression in the liver. Recently, the TGF-ß/BMP superfamily member activin B was implicated in hepcidin induction by inflammation via noncanonical SMAD1/5/8 signaling, but its mechanism of action and functional significance in vivo remain uncertain. Here, we show that low concentrations of activin B, but not activin A, stimulate prolonged SMAD1/5/8 signaling and hepcidin expression in liver cells to a similar degree as canonical SMAD2/3 signaling, and with similar or modestly reduced potency compared with BMP6. Activin B stimulates hepcidin via classical activin type II receptors ACVR2A and ACVR2B, noncanonical BMP type I receptors activin receptor-like kinase 2 and activin receptor-like kinase 3, and SMAD5. The coreceptor hemojuvelin binds to activin B and facilitates activin B-SMAD1/5/8 signaling. Activin B-SMAD1/5/8 signaling has some selectivity for hepatocyte-derived cells and is not enabled by hemojuvelin in other cell types. Liver activin B mRNA expression is up-regulated in multiple mouse models of inflammation associated with increased hepcidin and hypoferremia, including lipopolysaccharide, turpentine, and heat-killed Brucella abortus models. Finally, the activin inhibitor follistatin-315 blunts hepcidin induction by lipopolysaccharide or B. abortus in mice. Our data elucidate a novel mechanism for noncanonical SMAD activation and support a likely functional role for activin B in hepcidin stimulation during inflammation in vivo.

Activin Enhances α- to ß-Cell Transdifferentiation as a Source For ß-Cells In Male FSTL3 Knockout Mice.

Diabetes results from inadequate ß-cell number and/or function to control serum glucose concentrations so that replacement of lost ß-cells could become a viable therapy for diabetes. In addition to embryonic stem cell sources for new ß-cells, evidence for transdifferentiation/reprogramming of non-ß-cells to functional ß-cells is accumulating. In addition, de-differentiation of ß-cells observed in diabetes and their subsequent conversion to α-cells raises the possibility that adult islet cell fate is malleable and controlled by local hormonal and/or environmental cues. We previously demonstrated that inactivation of the activin antagonist, follistatin-like 3 (FSTL3) resulted in ß-cell expansion and improved glucose homeostasis in the absence of ß-cell proliferation. We recently reported that activin directly suppressed expression of critical α-cell genes while increasing expression of ß-cell genes, supporting the hypothesis that activin is one of the local hormones controlling islet cell fate and that increased activin signaling accelerates α- to ß-cell transdifferentiation. We tested this hypothesis using Gluc-Cre/yellow fluorescent protein (YFP) α-cell lineage tracing technology combined with FSTL3 knockout (KO) mice to label α-cells with YFP. Flow cytometry was used to quantify unlabeled and labeled α- and ß-cells. We found that Ins+/YFP+ cells were significantly increased in FSTL3 KO mice compared with wild type littermates. Labeled Ins+/YFP+ cells increased significantly with age in FSTL3 KO mice but not wild type littermates. Sorting results were substantiated by counting fluorescently labeled cells in pancreatic sections. Activin treatment of isolated islets significantly increased the number of YFP+/Ins+ cells. These results suggest that α- to ß-cell transdifferentiation is influenced by activin signaling and may contribute substantially to ß-cell mass.

Activins A and B Regulate Fate-Determining Gene Expression in Islet Cell Lines and Islet Cells From Male Mice.

TGFß superfamily ligands, receptors, and second messengers, including activins A and B, have been identified in pancreatic islets and proposed to have important roles regulating development, proliferation, and function. We previously demonstrated that Fstl3 (an antagonist of activin activity) null mice have larger islets with ß-cell hyperplasia and improved glucose tolerance and insulin sensitivity in the absence of altered ß-cell proliferation. This suggested the hypothesis that increased activin signaling influences ß-cell expansion by destabilizing the α-cell phenotype and promoting transdifferentiation to ß-cells. We tested the first part of this hypothesis by treating α- and ß-cell lines and sorted mouse islet cells with activin and related ligands. Treatment of the αTC1-6 α cell line with activins A or B suppressed critical α-cell gene expression, including Arx, glucagon, and MafB while also enhancing ß-cell gene expression. In INS-1E ß-cells, activin A treatment induced a significant increase in Pax4 (a fate determining ß-cell gene) and insulin expression. In sorted primary islet cells, α-cell gene expression was again suppressed by activin treatment in α-cells, whereas Pax4 was enhanced in ß-cells. Activin treatment in both cell lines and primary cells resulted in phosphorylated mothers against decapentaplegic-2 phosphorylation. Finally, treatment of αTC1-6 cells with activins A or B significantly inhibited proliferation. These results support the hypothesis that activin signaling destabilized the α-cell phenotype while promoting a ß-cell fate. Moreover, these results support a model in which the ß-cell expansion observed in Fstl3 null mice may be due, at least in part, to enhanced α- to ß-cell transdifferentiation.

Expression of repulsive guidance molecule b (RGMb) in the uterus and ovary during the estrous cycle in rats.

Repulsive guidance molecule b (RGMb; a.k.a. Dragon), initially identified in the embryonic dorsal root ganglion, is the first member of the RGM family shown to enhance bone morphogenetic protein (BMP) signaling by acting as a BMP co-receptor. BMP signaling has been demonstrated to play an important role in the reproductive organs. Our previous study found that RGMb was expressed in the reproductive axis, but whether RGMb expression in reproductive organs changes across the estrous cycle remains unknown. Here, we show in the rat that RGMb mRNA expression in the uterus was significantly higher during metesterus and diestrus than during proestrus and estrus. Western blotting indicated that RGMb protein was significantly lower during estrus compared with the other three stages. Immunohistochemistry revealed that RGMb protein was mainly localized to the uterine luminal and glandular epithelial cells of the endometrium. RGMb mRNA and protein in the ovary remained unchanged during the estrous cycle. RGMb protein was expressed in the oocytes of all follicles. Weak staining for RGMb protein was also found in corpora lutea. RGMb was not detected in granulosa cells and stromal cells. Taken together, RGMb expression in the uterus and ovary across the estrus cycle demonstrate that RGMb may be involved in the regulation of uterine function, follicular development as well as luteal activity.

Effects of activin A on survival, function and gene expression of pancreatic islets from non-diabetic and diabetic human donors.

Emerging evidence suggests that activin with its associated receptors, second messengers, and antagonists would be excellent targets for therapeutic drug development in the treatment of diabetes. We undertook the current study to investigate the ability to extrapolate findings from rodent studies to human islets in which data thus far has been scarce. We tested the hypothesis that human islets synthesize activin and that activin participates in the regulation of islet ß-cells. Human islets from 33 separate isolations were categorized based on functional status, culture status and diabetic status. Statistical comparisons were made by ANOVA with Tukey post-hoc adjustment for multiple comparisons. Experiments investigating activin utilized qPCR, FACS cell sorting, immunofluorescent antibody staining, functionality assays, viability assays and protein secretion assays. We have defined the transcript expression patterns of activin and the TGFß superfamily in human islets. We found INHBA (the gene encoding activin A) to be the most highly expressed of the superfamily in normal, cultured islets. We elucidated a link between the islet microenvironment and activin A. We found differential ligand expression based on diabetic, culture and functional status. Further, this is also the first report that links direct effects of activin A with the ability to restore glucose-stimulated insulin secretion in human islets from type 2 diabetic donors thereby establishing the relevance of targeting activin for therapeutic drug development.

Increased activin bioavailability enhances hepatic insulin sensitivity while inducing hepatic steatosis in male mice.

The development of insulin resistance is tightly linked to fatty liver disease and is considered a major health concern worldwide, although their mechanistic relationship remains controversial. Activin has emerging roles in nutrient homeostasis, but its metabolic effects on hepatocytes remain unknown. In this study, we investigated the effects of increased endogenous activin bioactivity on hepatic nutrient homeostasis by creating mice with inactivating mutations that deplete the circulating activin antagonists follistatin-like-3 (FSTL3) or the follistatin 315 isoform (FST315; FST288-only mice). We investigated liver histology and lipid content, hepatic insulin sensitivity, and metabolic gene expression including the HepG2 cell and primary hepatocyte response to activin treatment. Both FSTL3-knockout and FST288-only mice had extensive hepatic steatosis and elevated hepatic triglyceride content. Unexpectedly, insulin signaling, as assessed by phospho-Akt (a.k.a. protein kinase B), was enhanced in both mouse models. Pretreatment of HepG2 cells with activin A increased their response to subsequent insulin challenge. Gene expression analysis suggests that increased lipid uptake, enhanced de novo lipid synthesis, decreased lipolysis, and/or enhanced glucose uptake contribute to increased hepatic triglyceride content in these models. However, activin treatment recapitulated only some of these gene changes, suggesting that increased activin bioactivity may be only partially responsible for this phenotype. Nevertheless, our results indicate that activin enhances hepatocyte insulin response, which ultimately leads to hepatic steatosis despite the increased insulin sensitivity. Thus, regulation of activin bioactivity is critical for maintaining normal liver lipid homeostasis and response to insulin, whereas activin agonists may be useful for increasing liver insulin sensitivity.

Follistatin-like 3 (FSTL3) mediated silencing of transforming growth factor ß (TGFß) signaling is essential for testicular aging and regulating testis size.

Follistatin-like 3 (FSTL3) is a glycoprotein that binds and inhibits the action of TGFß ligands such as activin. The roles played by FSTL3 and activin signaling in organ development and homeostasis are not fully understood. The authors show mice deficient in FSTL3 develop markedly enlarged testes that are also delayed in their age-related regression. These FSTL3 knockout mice exhibit increased Sertoli cell numbers, allowing for increased spermatogenesis but otherwise showing normal testicular function. The data show that FSTL3 deletion leads to increased AKT signaling and SIRT1 expression in the testis. This demonstrates a cross-talk between TGFß ligand and AKT signaling and leads to a potential mechanism for increased cellular survival and antiaging. The findings identify crucial roles for FSTL3 in limiting testis organ size and promoting age-related testicular regression.

Activin B regulates islet composition and islet mass but not whole body glucose homeostasis or insulin sensitivity.

Based on the phenotype of the activin-like kinase-7 (ALK7)-null mouse, activins A and B have been proposed to play distinct roles in regulating pancreatic islet function and glucose homeostasis, with activin A acting to enhance islet function and insulin release while activin B antagonizes these actions. We therefore hypothesized that islets from activin B-null (BBKO) mice would have enhanced glucose-stimulated insulin secretion. In addition, we hypothesized that this enhanced islet function would translate into increased whole body glucose tolerance. We tested these hypotheses by analyzing glucose homeostasis, insulin secretion, and islet function in BBKO mice. No differences were observed in fasting glucose or insulin levels, glucose tolerance, or insulin sensitivity compared with weight-matched young or older males. Similarly, there were no significant differences in insulin secretion comparing islets from WT or BBKO males at either age. However, BBKO islets were more sensitive to activin A, myostatin (MSTN), and follistatin (FST) treatments, so that activin A and FST inhibited and MSTN enhanced glucose stimulated insulin secretion. While mean islet area and the distribution of islet areas were not different between the genotypes, islet mass, islet number, and the proportion of α-cells/islet were significantly reduced in BBKO islets. These results indicate that activin B does not antagonize activin A to influence whole body glucose homeostasis or ß-cell function but does influence islet mass and proportion of α-cells/islet. Therefore, loss of activin B signaling alone does not account for the ALK7-null phenotype, but activin B may have important roles in modulating islet mass, islet number, and the cellular composition of islets.

Differential synthesis and action of TGFß superfamily ligands in mouse and rat islets.

Members of the TGFß superfamily, including activins and TGFß, modulate glucose-stimulated insulin secretion (GSIS) in vitro using rat islets while genetic manipulations that reduce TGFß superfamily signaling in vivo in mice produced hypoplastic islets and/or hyperglycemia. Moreover, deletion of Fstl3, an antagonist of activin and myostatin, resulted in enlarged islets and ß-cell hyperplasia. These studies suggest that endogenous TGFß superfamily ligands regulate ß-cell generation and/or function. To test this hypothesis, we examined endogenous TGFß ligand synthesis and action in isolated rat and mouse islets. We found that activin A, TGFß1, and myostatin treatment enhanced rat islet GSIS but none of the ligands tested enhanced GSIS in mouse islets. However, follistatin inhibited GSIS, consistent with a role for endogenous TGFß superfamily ligands in regulating insulin secretion. Endogenous expression of TGFß superfamily members was different in rat and mouse islets with myostatin being highly expressed in mouse islets and not detectable in rats. These results indicate that TGFß superfamily members directly regulate islet function in a species-specific manner while the ligands produced by islets differ between mice and rats. The lack of in vitro actions of ligands on mouse islets may be mechanical or result from species-specific actions of these ligands.

Follistatin and follistatin like-3 differentially regulate adiposity and glucose homeostasis.

Transforming growth factor-ß superfamily ligands, including activin and myostatin, modulate body composition, islet function, and glucose homeostasis. Their bioactivity is controlled by the antagonists follistatin (FST) and FST like-3 (FSTL3). The hypothesis tested was that FST and FSTL3 have distinct roles in regulating body composition, glucose homeostasis, and islet function through regulation of activin and myostatin bioactivity. Three genetic mutant mouse lines were created. FSTL3 knockout (FSTL3 KO), a mouse line producing only the FST288 isoform (FST288-only) and a double mutant (2xM) in which the lines were crossed. FST288-only males were lighter that wild-type (WT) littermates while FSTL3 KO and 2xM males had reduced perigonadal fat pad weights. However, only 2xM mice had increased whole body fat mass and decreased lean mass by quantitative nuclear magnetic resonance (qNMR). Fasting glucose levels in FSTL3 WT and KO mice were lower than FST mice in younger animals but were higher in older mice. Serum insulin and pancreatic insulin content in 2xM mice was significantly elevated over other genotypes. Nevertheless, 2xM mice were relatively insulin resistant and glucose intolerant compared to FST288-only and WT mice. Fractional islet area and proportion of ß-cells/islet were increased in FSTL3 KO and 2xM, but not FST288-only mice. Despite their larger size, islets from FSTL3 KO and 2xM mice were not functionally enhanced compared to WT mice. These results demonstrate that body composition and glucose homeostasis are differentially regulated by FST and FSTL3 and that their combined loss is associated with increased fat mass and insulin resistance despite elevated insulin production.

The role of activin in mammary gland development and oncogenesis.

TGFß contributes to mammary gland development and has paradoxical roles in breast cancer because it has both tumor suppressor and tumor promoter activity. Another member of the TGFß superfamily, activin, also has roles in the developing mammary gland, but these functions, and the role of activin in breast cancer, are not well characterized. TGFß and activin share the same intracellular signaling pathways, but divergence in their signaling pathways are suggested. The purpose of this review is to compare the spatial and temporal expression of TGFß and activin during mammary gland development, with consideration given to their functions during each developmental period. We also review the contributions of TGFß and activin to breast cancer resistance and susceptibility. Finally, we consider the systemic contributions of activin in regulating obesity and diabetes; and the impact this regulation has on breast cancer. Elevated levels of activin in serum during pregnancy and its influence on pregnancy associated breast cancer are also considered. We conclude that evidence demonstrates that activin has tumor suppressing potential, without definitive indication of tumor promoting activity in the mammary gland, making it a good target for development of therapeutics.

Follistatin regulates germ cell nest breakdown and primordial follicle formation.

Follistatin (FST) is an antagonist of activin and related TGFß superfamily members that has important reproductive actions as well as critical regulatory functions in other tissues and systems. FST is produced as three protein isoforms that differ in their biochemical properties and in their localization within the body. We created FST288-only mice that only express the short FST288 isoform and previously reported that females are subfertile, but have an excess of primordial follicles on postnatal day (PND) 8.5 that undergo accelerated demise in adults. We have now examined germ cell nest breakdown and primordial follicle formation in the critical PND 0.5-8.5 period to test the hypothesis that the excess primordial follicles derive from increased proliferation and decreased apoptosis during germ cell nest breakdown. Using double immunofluorescence microscopy we found that there is virtually no germ cell proliferation after birth in wild-type or FST288-only females. However, the entire process of germ cell nest breakdown was extended in time (through at least PND 8.5) and apoptosis was significantly reduced in FST288-only females. In addition, FST288-only females are born with more germ cells within the nests. Thus, the excess primordial follicles in FST288-only mice derive from a greater number of germ cells at birth as well as a reduced rate of apoptosis during nest breakdown. These results also demonstrate that FST is critical for normal regulation of germ cell nest breakdown and that loss of the FST303 and/or FST315 isoforms leads to excess primordial follicles with accelerated demise, resulting in premature cessation of ovarian function.

Sarcopenia associated with portosystemic shunting is reversed by follistatin.

BACKGROUND & AIMS: The distinct role of portosystemic shunting (PSS) in the pathogenesis of sarcopenia (skeletal muscle loss) that occurs commonly in cirrhosis is unclear. We have previously shown increased expression of myostatin (inhibitor of skeletal muscle mass) in the portacaval anastamosis (PCA) rat model of sarcopenia of PSS. The present study was performed to examine the mechanisms of sarcopenia following PCA. METHODS: In PCA and sham operated pair fed control rats, the phenylalanine flooding dose method was used to quantify the fractional and absolute protein synthesis rates in the skeletal muscle over time and in response to follistatin, a myostatin antagonist. The expression of myostatin and markers of satellite cell (myocyte precursors) proliferation and differentiation were quantified by real-time PCR and Western blot analyses. RESULTS: The absolute synthesis rate (ASR) was lower at 2, 4, and 6 weeks (p<0.05) and the fractional synthesis rate (FSR) of skeletal muscle protein was significantly lower (p<0.05) at week 2 in the PCA rats compared to control rats. Expression of myostatin was elevated while markers of satellite cell proliferation and differentiation were lower at 4 and 6 weeks after PCA. Follistatin increased skeletal muscle mass, muscle FSR and ASR, decreased expression of myostatin protein, and increased expression of markers of satellite cell function. CONCLUSIONS: Sarcopenia associated with PSS is caused by impaired protein synthesis and reduced satellite cell function due to increased myostatin expression. Confirming these alterations in human patients with cirrhosis will provide novel therapeutic targets for sarcopenia of liver disease.

Emerging roles for the TGFbeta family in pancreatic beta-cell homeostasis.

Loss of functional beta-cells is the primary cause of type 2 diabetes, so that there is an acute need to understand how beta-cell number and function are regulated in the adult under normal physiological conditions. Recent studies suggest that members of the transforming growth factor (TGF)-beta family regulate beta-cell function and glucose homeostasis. These factors are also likely to influence beta-cell proliferation and/or the incorporation of new beta-cells from progenitors in adults. Soluble TGFbeta antagonists also appear to have important roles in maintaining homeostasis, and the coordinated activity of TGFbeta family members is likely to regulate the differentiation and function of adult beta-cells, raising the possibility of developing new diabetes therapies based on TGFbeta agonists or antagonists.