Bone morphogenetic protein signaling by hemojuvelin regulates hepcidin expression.

Hepcidin is a key regulator of systemic iron homeostasis. Hepcidin deficiency induces iron overload, whereas hepcidin excess induces anemia. Mutations in the gene encoding hemojuvelin (HFE2, also known as HJV) cause severe iron overload and correlate with low hepcidin levels, suggesting that hemojuvelin positively regulates hepcidin expression. Hemojuvelin is a member of the repulsive guidance molecule (RGM) family, which also includes the bone morphogenetic protein (BMP) coreceptors RGMA and DRAGON (RGMB). Here, we report that hemojuvelin is a BMP coreceptor and that hemojuvelin mutants associated with hemochromatosis have impaired BMP signaling ability. Furthermore, BMP upregulates hepatocyte hepcidin expression, a process enhanced by hemojuvelin and blunted in Hfe2-/- hepatocytes. Our data suggest a mechanism by which HFE2 mutations cause hemochromatosis: hemojuvelin dysfunction decreases BMP signaling, thereby lowering hepcidin expression.

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.

Dragon enhances BMP signaling and increases transepithelial resistance in kidney epithelial cells.

The neuronal adhesion protein Dragon acts as a bone morphogenetic protein (BMP) coreceptor that enhances BMP signaling. Given the importance of BMP signaling in nephrogenesis and its putative role in the response to injury in the adult kidney, we studied the localization and function of Dragon in the kidney. We observed that Dragon localized predominantly to the apical surfaces of tubular epithelial cells in the thick ascending limbs, distal convoluted tubules, and collecting ducts of mice. Dragon expression was weak in the proximal tubules and glomeruli. In mouse inner medullary collecting duct (mIMCD3) cells, Dragon generated BMP signals in a ligand-dependent manner, and BMP4 is the predominant endogenous ligand for the Dragon coreceptor. In mIMCD3 cells, BMP4 normally signaled through BMPRII, but Dragon enhanced its signaling through the BMP type II receptor ActRIIA. Dragon and BMP4 increased transepithelial resistance (TER) through the Smad1/5/8 pathway. In epithelial cells isolated from the proximal tubule and intercalated cells of collecting ducts, we observed coexpression of ActRIIA, Dragon, and BMP4 but not BMPRII. Taken together, these results suggest that Dragon may enhance BMP signaling in renal tubular epithelial cells and maintain normal renal physiology.

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.

Differential antagonism of activin, myostatin and growth and differentiation factor 11 by wild-type and mutant follistatin.

Follistatin binds and neutralizes members of the TGFbeta superfamily including activin, myostatin, and growth and differentiation factor 11 (GDF11). Crystal structure analysis of the follistatin-activin complex revealed extensive contacts between follistatin domain (FSD)-2 and activin that was critical for the high-affinity interaction. However, it remained unknown whether follistatin residues involved with myostatin and GDF11 binding were distinct from those involved with activin binding. If so, this would allow development of myostatin antagonists that would not inhibit activin actions, a desirable feature for development of myostatin antagonists for treatment of muscle-wasting disorders. We tested this hypothesis with our panel of point and domain swapping follistatin mutants using competitive binding analyses and in vitro bioassays. Our results demonstrate that activin binding and neutralization are mediated primarily by FSD2, whereas myostatin binding is more dependent on FSD1, such that deletion of FSD2 or adding an extra FSD1 in place of FSD2 creates myostatin antagonists with vastly reduced activin antagonism. However, these mutants also bind GDF11, indicating that further analysis is required for creation of myostatin antagonists that will not affect GDF11 activity that could potentially elicit GDF11-induced side effects 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.

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.

Heparin and activin-binding determinants in follistatin and FSTL3.

Local regulation of pituitary FSH secretion and many other cellular processes by follistatin (FS) can be ascribed to its potent ability to bind and bioneutralize activin, in conjunction with binding to cell surface heparan-sulfate proteoglycans through a basic heparin-binding sequence (HBS; residues 75-86) in the first of the three FS domains. The FS homolog, FSTL3, also binds activin, but lacks any HBS and cannot associate with cell surfaces. We have used mutational analyses to define the determinants for heparin binding and activin interaction in FS and to determine the effects of conferring heparin binding to FSTL3. Mutants expressed from 283F cells were tested for cell surface and heparin affinity binding, for competitive activin binding and for bioactivity by suppression of pituitary cell FSH secretion. Replacement of the HBS or the full-length FS-domain 1 abolished cell surface binding but enhanced activin binding 4- to 8-fold. Surface binding was partially reduced after mutation of either lysine pair 75/76 or 81/82 and eliminated after mutation of both pairs. The 75/76 mutation reduced activin binding and, therefore, pituitary cell bioactivity by 5-fold. However, insertion of the HBS into FSTL3 did not restore heparin binding or pituitary-cell bioactivity. These results show that 1) the residues within the HBS are necessary but not sufficient for heparin binding, and 2) the HBS also harbors determinants for activin binding. Introduction of the full domain from FS conferred heparin binding to FSTL3, but activin binding was abolished. This implies an evolutionary safeguard against surface binding by FSTL3, supporting other evidence for physiological differences between FS and FSTL3.

Follicular arrest in polycystic ovary syndrome is associated with deficient inhibin A and B biosynthesis.

CONTEXT: Previous studies suggest that inhibin subunit expression is decreased in granulosa cells of women with polycystic ovary syndrome (PCOS). OBJECTIVE: The objective of this study was to test the hypothesis that inhibin A and inhibin B protein concentrations are also decreased in PCOS follicles. DESIGN: The design was a parallel study. SETTING: The study was performed at an in vitro fertilization suite. PARTICIPANTS: We studied women with regular cycles (n = 36) and women with PCOS (n = 8). INTERVENTIONS: Follicular fluid was aspirated from the follicles of women with PCOS (n = 14 follicles) and from women with regular cycles at various times during the follicular phase (n = 50 follicles). MAIN OUTCOME MEASURE: Inhibin A and B concentrations from PCOS follicles were compared with those in size-matched follicles, dominant follicles (> or = 10 mm), and subordinate follicles from regularly cycling women. RESULTS: Inhibin A (220 +/- 38 vs. 400 +/- 72 IU/ml; P < 0.05) and inhibin B (75.4 +/- 10.4 vs. 139 +/- 26 ng/ml; P < 0.05) concentrations were lower in the follicular fluid of PCOS follicles compared with those of size-matched follicles from regularly cycling women. Inhibin A was also lower in the follicular fluid of PCOS compared with subordinate follicles from normal women (577 +/- 166 IU/ml; P < 0.05). Inhibin A concentrations increased with increasing follicle size, resulting in significantly higher follicular fluid concentrations in dominant follicles from normal women compared with PCOS follicles (2298 +/- 228 IU/ml; P < 0.05). CONCLUSIONS: These data demonstrate that inhibin A and inhibin B concentrations are significantly reduced in the follicular fluid of women with PCOS compared with those in the follicular fluid of size-matched follicles from normal women, consistent with the decreased inhibin subunit mRNA expression in previous studies. These findings point to the potential importance of inhibins in normal follicle development and suggest that inhibin deficiency may play a role in the follicle arrest associated with PCOS.

The role of follistatin domains in follistatin biological action.

Follistatin (FS) is an important regulator of pituitary FSH secretion through its potent ability to bind and bioneutralize activin. It also represents a prototype for binding proteins that control bioavailability of other TGFbeta-related growth factors such as the bone morphogenetic proteins. The 288-residue FS molecule has a distinctive structure comprised principally of three 10-cysteine FS domains. These are preceded by an N-terminal segment shown by us previously to contain hydrophobic residues essential for activin binding. To establish the contribution of the FS domains themselves to FS's bioactivity, we prepared mutants with deleted or exchanged domains and intradomain point mutations. Mutants were expressed from mammalian (Chinese hamster ovary) cells and evaluated for activin binding and for biological activity in assays measuring differing aspects of FS bioactivity: activin-mediated transcriptional activity and suppression of FSH secretion in primary pituitary cell cultures. The N-terminal domain (residues 1-63) alone could not bind activin or suppress activin-mediated transcription, either alone or combined in solution with the FS domain region (residues 64-288). Deletion of FS domains 1 or 2 abolished activin binding and biological activity in both assays, whereas deletion of domain 3 was tolerated. Bioactivity was also reduced or eliminated after exchange of domains (FS 2/1/3 and FS 3/1/2) or doubling of domain 1 (FS 1/1/3) or domain 2 (FS 2/2/3). Several hydrophobic residues clustered within the C-terminal region of FS domains 1 and 2 are highly conserved among all FS domains. Mutation of any of these to Asp or Ala either reduced or eliminated FS bioactivity and disrupted distant epitopes for heparin binding (FS domain 1) or antibody recognition (FS domain 2), suggesting their role in maintaining the conformational integrity of the domain and possibly the FS molecule as a whole. These results are consistent with the importance of domain conformation as well as the overall order of the domains in FS function. A continuous sequence comprising the N-terminal domain and followed by FS domains 1 and 2 fulfills the minimum structural requirement for activin binding and FS bioactivity.

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.

Follistatin-related protein and follistatin differentially neutralize endogenous vs. exogenous activin.

Follistatin-related protein (FSRP) is a new addition to the expanding follistatin (FS)-related gene family whose members contain at least one conserved 10-cysteine follistatin domain. In contrast to other members of this family, FSRP and follistatin also share a common exon/intron domain structure, substantial primary sequence homology, and an ability to irreversibly bind activin. In this study, we further explored the hypothesis that FSRP is a functional as well as structural homologue of FS. N-terminal sequencing of recombinant FSRP revealed that signal peptide cleavage occurs within exon 1, a significant structural difference from FS, in which cleavage occurs at the exon/intron boundary. Solid-phase radioligand competition assays revealed both FS and FSRP to preferentially bind activin with the next closest TGF-beta superfamily member, bone-morphogenic protein-7, being at least 500-fold less potent. Consistent with their similar activin-binding affinities, FSRP and FS both prevented exogenous (endocrine or paracrine) activin from accessing its receptor and inducing gene transcription in bioassays. However, FS was at least 100-fold more potent than FSRP in inhibiting gene transcription and FSH release mediated by endogenously produced (autocrine) activin-A or activin-B in multiple cell systems. Finally, FSRP lacks the heparin-binding sequence found in FS, and we found that it was also unable to bind cell surface heparin sulfated proteoglycans. These findings suggest that structural differences between FSRP and FS may underlie their different neutralizating capabilities with respect to exogenous vs. endogenous activin. Taken together with our previous studies showing that activin binding is essential for FS's biological activity, the differential activities of FSRP and FS further indicate that activin binding is necessary but not sufficient to account for all of FS's actions.

Differential distribution of follistatin isoforms: application of a new FS315-specific immunoassay.

Follistatin (FST) is a monomeric activin-binding and neutralization protein that has at least three isoforms in human tissues and fluids. The full-length FS315 protein has an acidic 26-residue C-terminal tail that is not present in the shortest form, FS288, due to alternative splicing. An intermediate form, FS303, was identified in follicular fluid that is presumably derived by proteolytic processing of this tail domain. Interestingly, the biochemistry of each of these three isoforms is distinct, including their ability to bind to cell surface proteoglycans, an activity that ranks in the order FS288 > FS303 > FS315. This would suggest that the soluble, circulating FST isoform is likely to be FS315, a hypothesis supported by previous determinations that the serum and follicular fluid forms of FST are biochemically distinct. To test this hypothesis, we developed an immunoassay that is specific for full-length FS315. This assay was validated for use with human serum and follicular fluid samples and then used to examine FST in these fluid compartments. Our results indicate that FS315 is indeed the major circulating FST isoform but is undetectable in follicular fluid samples aspirated from normal women or women with polycystic ovary syndrome. These observations confirm the compartmentalization of FST isoforms according to their biochemical properties and biological actions so that the most soluble form is found in the circulation, whereas the forms that bind to cell surface proteoglycans are found in tissue compartments such as the ovarian follicle. They also confirm that the source of FST in human serum is not the ovarian follicle.

Regulation of endometrial adenocarcinoma cell proliferation by Activin-A and its modulation by 17beta-estradiol.

A role for activins in regulating cellular transformation is suggested by the alpha-inhibin knockout mouse in which development of gonadal tumors is associated with elevated activin levels. It was the purpose of the current study to determine whether activin had similar actions on endometrial cell lines, specifically on a well differentiated estrogen-responsive endometrial adenocarcinoma cell line (ISH) and estrogen-unresponsive cells (HEC-50) obtained from a poorly differentiated endometrial adenocarcinoma. Activin was secreted by both adenocarcinoma cell lines. Using reverse transcription-PCR, messenger RNA type I and type II activin receptor subtypes were detected in both cell lines: expression of IB and IIB was approximately three- to fourfold greater in ISH cells than in HEC-50 cells, while activin receptor IA and IIA messenger RNA levels were approximately equal in both cell lines. Activin treatment (30-300 ng/ml) caused a dose- and time-dependent inhibition of ISH cells proliferation and resulted in a significant decrease in Bcl-2 protein and mRNA levels. No difference was observed in Bax expression. There was no significant effect of activin when the cultures of ISH cells were exposed to 17beta-estradiol. In contrast, activin showed a weak, but significant, mitogenic effect on HEC-50 cells without modifications in Bax and Bcl-2 mRNA and protein levels. The results demonstrate that activin is a regulator of endometrial cancer cell growth. 17beta-Estradiol may promote resistance of estrogen-responsive endometrial cancer cells to the growth-retarding effects of activin and one of the mechanisms might be a down-regulation of the activin receptors.

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.

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.

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.

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.

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.