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.

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.

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.

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.

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.

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.

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.

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.

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.

Biological activity of follistatin isoforms and follistatin-like-3 is dependent on differential cell surface binding and specificity for activin, myostatin, and bone morphogenetic proteins.

Follistatin (FST) and FST-like-3 (FSTL3) are activin-binding and neutralization proteins that also bind myostatin. Three FST isoforms have been described that differ in tissue distribution and cell-surface binding activity, suggesting that the FST isoforms and FSTL3 may have some nonoverlapping biological actions. We produced recombinant FST isoforms and FSTL3 and compared their biochemical and biological properties. Activin-binding affinities and kinetics were comparable between the isoforms and FSTL3, whereas cell-surface binding differed markedly (FST288 > FST303 > FST315 > FSTL3). Inhibition of endogenous activin bioactivity, whether the FST isoforms were administered endogenously or exogenously, correlated closely with surface binding activity, whereas neutralization of exogenous activin when FST and FSTL3 were also exogenous was consistent with their equivalent activin-binding affinities. This difference in activin inhibition was also evident in an in vitro bioassay because FST288 suppressed, whereas FST315 enhanced, activin-dependent TT cell proliferation. Moreover, when FSTL3, which does not associate with cell membranes, was expressed as a membrane-anchored protein, its endogenous activin inhibitory activity was dramatically increased. In competitive binding assays, myostatin was more potent than bone morphogenetic proteins (BMPs) 6 and 7, and BMPs 2 and 4 were inactive in binding to FST isoforms, whereas none of the BMPs tested competed with activin for binding to FSTL3. Neutralization of exogenous BMP or myostatin bioactivity correlated with the relative abilities of the isoforms to bind cell-surface proteoglycans. These results indicate that the differential biological actions among the FST isoforms and FSTL3 are primarily dependent on their relative cell-surface binding ability and ligand specificity.

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.

Differential biosynthesis and intracellular transport of follistatin isoforms and follistatin-like-3.

Follistatin (FST) and FST-like-3 (FSTL3) are structurally related proteins that bind and neutralize activin and closely related members of the TGFbeta superfamily. Three FST isoforms (FST288, FST303, and FST315) are produced from the Fst gene that are primarily secreted proteins. FSTL3 is secreted, but is also observed within the nucleus of most cells. We used pulse-chase (35)S labeling to examine the biosynthetic and intracellular transport patterns that lead to differential secretion and intracellular retention of these proteins. Among the FST isoforms, FST315 was secreted fastest and FST288 was secreted more slowly, with some remaining intracellular. In contrast, FSTL3 was secreted the slowest, with newly synthesized proteins being both secreted and trafficked to the nucleus. This nuclear FSTL3 was N-glycosylated, although not to the same degree as secreted FSTL3. Both FST and FSTL3 have two Mets in their signal sequence. Mutation of the first Met in FST288 eliminated protein translation, whereas FSTL3 could be translated from either Met. However, although FSTL3 translated from the second Met, which had no signal sequence, was confined to the nucleus, it was not glycosylated. Interestingly, this FSTL3 retained activin-antagonizing activity. Thus, although bioactive, nuclear FSTL3 can be translated from the second Met when the first Met is mutated, the glycosylated nuclear FSTL3 produced endogenously indicates that a different mechanism must be used under natural conditions that apparently includes N-glycosylation. Moreover, the differential biosynthetic and intracellular transport patterns for FST288 and FSTL3 suggest that these two activin-binding proteins may have distinct intracellular roles.

Localization and action of Dragon (repulsive guidance molecule b), a novel bone morphogenetic protein coreceptor, throughout the reproductive axis.

Bone morphogenetic proteins (BMPs) play important roles in reproduction including primordial germ cell formation, follicular development, spermatogenesis, and FSH secretion. Dragon, a recently identified glycosylphosphatidylinositol-anchored member of the repulsive guidance molecule family, is also a BMP coreceptor. In the present study, we determined the tissue and cellular localization of Dragon in reproductive organs using immunohistochemistry and in situ hybridization. Among reproductive organs, Dragon was expressed in testis, epididymis, ovary, uterus, and pituitary. In the testis of early postnatal mice, Dragon was found in gonocytes and spermatogonia, whereas in immature testes, Dragon was only weakly expressed in spermatogonia. Interestingly, pregnant mare serum gonadotropin treatment of immature mice robustly induced Dragon production in spermatocytes. In adult testis, Dragon was found in spermatocytes and round spermatids. In the ovary, Dragon was detected exclusively within oocytes and primarily those within secondary follicles. In the pituitary, Dragon-expressing cells overlapped FSH-expressing cells. Dragon was also expressed in a number of cell lines originating from reproductive tissues including Ishikawa, Hela, LbetaT2, MCF-7, and JEG3 cells. Immunocytochemistry and gradient sucrose ultracentrifugation studies showed Dragon was localized in lipid rafts within the plasma membrane. In reproductive cell lines, Dragon expression enhanced signaling of exogenous BMP2 or BMP4. The present studies demonstrate that Dragon expression is dynamically regulated throughout the reproductive tract and that Dragon protein modulates BMP signaling in cells from reproductive tissues. The overlap between Dragon expression and the functional BMP signaling system suggests that Dragon may play a role in mammalian reproduction.

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.

Overexpression of follistatin-like 3 in gonads causes defects in gonadal development and function in transgenic mice.

Activin has numerous biological activities including regulation of follicular development, spermatogenesis, and steroidogenesis within the gonads. Activities of activin are regulated by follistatin (FST), an activin binding protein, and perhaps follistatin-like 3 (FSTL3; also known as FLRG and FSRP). FSTL3 is a recently described member of the FST family having an overall structure and activity profile similar to that of FST, including binding and neutralization of activin. FSTL3 is most highly expressed in the placenta and testis, whereas FST is highest in the ovary and kidney, suggesting that FSTL3 has biological actions that do not entirely overlap those of FST. To investigate the role of local FSTL3 as a potential regulator of activin action in gonad development and function, we examined FSTL3 expression in the mouse testis. FSTL3 protein was localized to Leydig cells, spermatagonia, and mature spermatids in normal male mice. We then created transgenic mice using a human FSTL3 cDNA driven by the mouse alpha-inhibin promoter. Three of five transgenic founders were fertile and were bred to establish lines. In the highest expressing line 3, transgene expression was largely restricted to gonads, with pituitary, adrenal, brain, and uterine expression being substantially lower. Gonad weights, sperm counts, and fertility were significantly reduced in transgenic males, and reduced litter size was evident in line 3 females. Within the testis, highest transgene expression was observed in Sertoli cells, and although most tubules showed evidence of normal spermatogenic development, degenerating tubules devoid of germ cells and Leydig cell hyperplasia were also evident in every line 3 animal examined. Ovaries from line 3 females contained fewer antral follicles and more apparent follicular atresia. Although circulating human FSTL3 levels were undetectable, FSH and LH levels in adult transgenic mice were not significantly different from wild-type animals. However, testosterone levels were significantly increased at d 21 and significantly reduced at d 60 compared with wild-type males. These results suggest that FSTL3 is likely to be a local regulator of activin action in gonadal development and gametogenesis and, further, that activin appears to have important actions in gonadal development and function that are critical for normal reproduction.

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.