The most prevalent neurological disorders of myelin include perinatal brain injury leading to cerebral palsy in infants and multiple sclerosis in adults. Although these disorders have distinct etiologies, they share a common neuropathological feature of failed progenitor differentiation into myelin-producing oligodendrocytes and lack of myelin, for which there is an unmet clinical need. Here, we reveal that a molecular pathology common to both disorders is dysregulation of activin receptors and that activin receptor signaling is required for the majority of myelin generation in development and following injury. Using a constitutive conditional knockout of all activin receptor signaling in oligodendrocyte lineage cells, we discovered this signaling to be required for myelination via regulation of oligodendrocyte differentiation and myelin compaction. These processes were found to be dependent on the activin receptor subtype Acvr2a, which is expressed during oligodendrocyte differentiation and axonal ensheathment in development and following myelin injury. During efficient myelin regeneration, Acvr2a upregulation was seen to coincide with downregulation of Acvr2b, a receptor subtype with relatively higher ligand affinity; Acvr2b was shown to be dispensable for activin receptor-driven oligodendrocyte differentiation and its overexpression was sufficient to impair the abovementioned ligand-driven responses. In actively myelinating or remyelinating areas of human perinatal brain injury and multiple sclerosis tissue, respectively, oligodendrocyte lineage cells expressing Acvr2a outnumbered those expressing Acvr2b, whereas in non-repairing lesions Acvr2b+ cells were increased. Thus, we propose that following human white matter injury, this increase in Acvr2b expression would sequester ligand and consequently impair Acvr2a-driven oligodendrocyte differentiation and myelin formation. Our results demonstrate dysregulated activin receptor signaling in common myelin disorders and reveal Acvr2a as a novel therapeutic target for myelin generation following injury across the lifespan.

Activins transduce their signals by binding to activin type I receptors and activin type II receptors, both of which contain a serine/threonine kinase domain. In this study, we established stable transfectants expressing two types of activin receptors, ActRI and ActRIB, to clarify the role of these receptors in activin signalling for growth inhibition in HS-72 mouse B-cell hybridoma cells. Over-expression of ActRI suppressed activin A-induced cell-cycle arrest in the G1 phase caused by inhibition of retinoblastoma protein phosphorylation through induction of p21CIP1/WAF1, a cyclin-dependent kinase inhibitor, and subsequent apoptosis. In contrast, HS-72 clones that over-expressed ActRIB significantly facilitated activin A-induced apoptosis. These results indicate that ActRI and ActRIB are distinct from each other and that the ActRI/ActRIB expression ratio could regulate cell-cycle arrest in the G1 phase and subsequent apoptosis in HS-72 cells induced by activin A.

Activin A functions in BMP signaling in two ways: it either engages ACVR1B to activate Smad2/3 signaling or binds ACVR1 to form a non-signaling complex (NSC). Although the former property has been studied extensively, the roles of the NSC remain unexplored. The genetic disorder fibrodysplasia ossificans progressiva (FOP) provides a unique window into ACVR1/Activin A signaling because in that disease Activin can either signal through FOP-mutant ACVR1 or form NSCs with wild-type ACVR1. To explore the role of the NSC, we generated 'agonist-only' Activin A muteins that activate ACVR1B but cannot form the NSC with ACVR1. Using one of these muteins, we demonstrate that failure to form the NSC in FOP results in more severe disease pathology. These results provide the first evidence for a biological role for the NSC in vivo and pave the way for further exploration of the NSC's physiological role in corresponding knock-in mice.

Activins are selective regulators of FSH production by pituitary gonadotrope cells. In a gonadotrope-like cell line, LβT2, activins stimulate FSH via the activin type IIA receptor (ACVR2A) and/or bone morphogenetic protein type II receptor (BMPR2). Consistent with these observations, FSH is greatly reduced, though still present, in global Acvr2a knockout mice. In contrast, FSH production is unaltered in gonadotrope-specific Bmpr2 knockout mice. In light of these results, we questioned whether an additional type II receptor might mediate the actions of activins or related TGF-β ligands in gonadotropes. We focused on the activin type IIB receptor (ACVR2B), even though it does not mediate activin actions in LβT2 cells. Using a Cre-lox strategy, we ablated Acvr2a and/or Acvr2b in murine gonadotropes. The resulting conditional knockout (cKO) animals were compared with littermate controls. Acvr2a cKO (cKO-A) females were subfertile (~70% reduced litter size), cKO-A males were hypogonadal, and both sexes showed marked decreases in serum FSH levels compared with controls. Acvr2b cKO (cKO-B) females were subfertile (~20% reduced litter size), cKO-B males had a moderate decrease in testicular weight, but only males showed a significant decrease in serum FSH levels relative to controls. Simultaneous deletion of both Acvr2a and Acvr2b in gonadotropes led to profound hypogonadism and FSH deficiency in both sexes; females were acyclic and sterile. Collectively, these data demonstrate that ACVR2A and ACVR2B are the critical type II receptors through which activins or related TGF-β ligands induce FSH production in mice in vivo.

A new crystal structure of activin in complex with the extracellular domain of its type II receptor (ActRIIb-ECD) shows that the ligand exhibits an unexpected flexibility. The motion in the activin dimer disrupts its type I receptor interface, which may account for the disparity in its affinity for type I versus type II receptors. We have measured the affinities of activin and its antagonist inhibin for ActRIIb-ECD and found that the affinity of the 2-fold symmetric homodimer activin for ActRIIb-ECD depends on the availability of two spatially coupled ActRIIb-ECD molecules, whereas the affinity of the heterodimer inhibin does not. Our results indicate that activin's affinity for its two receptor types is greatly influenced by their membrane-restricted setting. We propose that activin affinity is modulated by the ligand flexibility and that cooperativity is achieved by binding to two ActRII chains that immobilize activin in a type I binding-competent orientation.

Long-lasting forms of synaptic plasticity and memory require de novo protein synthesis. Yet, how learning triggers this process to form memory is unclear. Translin/trax is a candidate to drive this learning-induced memory mechanism by suppressing microRNA-mediated translational silencing at activated synapses. We find that mice lacking translin/trax display defects in synaptic tagging, which requires protein synthesis at activated synapses, and long-term memory. Hippocampal samples harvested from these mice following learning show increases in several disease-related microRNAs targeting the activin A receptor type 1C (ACVR1C), a component of the transforming growth factor-β receptor superfamily. Furthermore, the absence of translin/trax abolishes synaptic upregulation of ACVR1C protein after learning. Finally, synaptic tagging and long-term memory deficits in mice lacking translin/trax are mimicked by ACVR1C inhibition. Thus, we define a new memory mechanism by which learning reverses microRNA-mediated silencing of the novel plasticity protein ACVR1C via translin/trax.

Activin-stimulated FSH synthesis and release by the pituitary gonadotrope is antagonized by gonadally derived inhibins. The two isoforms of inhibin, inhibin A and B, bind to the activin type II receptors, though at a lower affinity than the activins, but do not stimulate intracellular signaling. Theoretically, therefore, inhibins can prevent activin signaling through competitive binding if present at higher concentrations than the activins. In reality, the inhibins have been shown to antagonize activin signaling when the two ligand types are present at equimolar concentrations. These observations led to the hypothesis that inhibin binding proteins or co-receptors exist that either increase the affinity of the inhibins for the activin receptors or propagate inhibin-specific intracellular signals. Two candidate inhibin co-receptors, betaglycan and InhBP/p120, interact with activin receptors and augment inhibin antagonism of activin action. Here, we report the effect of betaglycan and InhBP/p120 on both inhibin A and inhibin B binding to the activin receptors ActRIIA and ActRIIB2. InhBP/p120 did not bind inhibin A or B when expressed alone or in combination with activin receptors, requiring a re-examination of the role of this protein in inhibin biology. Both inhibins bound the activin type II receptor, ActRIIB2. Inhibin B had a higher affinity for this receptor than inhibin A but an approximately 10-fold lower affinity than that of activin A. Inhibin A and B bound betaglycan with high affinity; however, only inhibin A binding to ActRIIB2 was significantly enhanced in the presence of betaglycan. Both inhibin isoforms showed slight but significant binding to ActRIIA, yet this binding was potentiated in the presence of betaglycan. Additionally, the complex formed between the inhibins, ActRIIA, and betaglycan was resistant to disruption by activin A, whereas activin A potently competed for inhibin binding to ActRIIB2 and betaglycan. Collectively, these data show that the inhibin isoforms have different affinities for the activin type II receptors but bind betaglycan with high affinity. A recently developed model of inhibin action proposes that inhibins form a high affinity, activin-resistant ternary complex with activin type II receptors and betaglycan, thereby providing a mechanism for inhibin antagonism of activin signaling. Importantly, the results presented here clearly show that this model does not apply equally to both forms of inhibin nor to the different activin type II receptor isoforms. Thus, it appears that the mechanisms of inhibin action may vary depending on the ligand and receptor types involved.

Activin and Bone Morphogenetic Protein (BMP) signaling plays crucial roles in vertebrate organ formation, including osteo- and angiogenesis, and tissue homeostasis, such as neuronal maintenance. Activin and BMP signaling needs to be precisely controlled by restricted expression of shared receptors, stoichiometric composition of receptor-complexes and presence of regulatory proteins. A R206H mutation in the human (hs) BMP type I receptor hsACVR1, on the other hand, leads to excessive phosphorylation of Sons of mothers against decapentaplegic (SMAD) 1/5/8. This in turn causes increased inflammation and heterotopic ossification in soft tissues of patients suffering from Fibrodysplasia Ossificans Progressiva (FOP). Several animal models have been established to understand the spontaneous and progressive nature of FOP, but often have inherent limitations. The Japanese medaka (Oryzias latipes, ola) has recently emerged as popular model for bone research. To assess whether medaka is suitable as a potential FOP animal model, we determined the expression of Activin receptor type I (ACVR1) orthologs olaAcvr1 and olaAcvr1l with that of Activin type II receptors olaAcvr2ab, olaAcvr2ba and olaAcvr2bb in embryonic and adult medaka tissues by in situ hybridization. Further, we showed that Activin A binding properties are conserved in olaAcvr2, as are the mechanistic features in the GS-Box of both olaAcvr1 and olaAcvr1l. This consequently leads to FOP-typical elevated SMAD signaling when the medaka type I receptors carry the R206H equivalent FOP mutation. Together, this study therefore provides experimental groundwork needed to establish a unique medaka model to investigate mechanisms underlying FOP.

The 30+ unique ligands of the TGFβ family signal by forming complexes using different combinations of type I and type II receptors. Therapeutically, the extracellular domain of a single receptor fused to an Fc molecule can effectively neutralize subsets of ligands. Increased ligand specificity can be accomplished by using the extracellular domains of both the type I and type II receptor to mimic the naturally occurring signaling complex. Here, we report the structure of one "type II-type I-Fc" fusion, ActRIIB-Alk4-Fc, in complex with two TGFβ family ligands, ActA, and GDF11, providing a snapshot of this therapeutic platform. The study reveals that extensive contacts are formed by both receptors, replicating the ternary signaling complex, despite the inherent low affinity of Alk4. Our study shows that low-affinity type I interactions support altered ligand specificity and can be visualized at the molecular level using this platform.

Activins belong to the transforming growth factor (TGF)-β family of multifunctional cytokines and signal via the activin receptors ALK4 or ALK7 to activate the SMAD2/3 pathway. In some cases, activins also signal via the bone morphogenetic protein (BMP) receptor ALK2, causing activation of the SMAD1/5/8 pathway. In this study, we aimed to dissect how activin A and activin B homodimers, and activin AB and AC heterodimers activate the two main SMAD branches. We compared the activin-induced signaling dynamics of ALK4/7-SMAD2/3 and ALK2-SMAD1/5 in a multiple myeloma cell line. Signaling via the ALK2-SMAD1/5 pathway exhibited greater differences between ligands than signaling via ALK4/ALK7-SMAD2/3. Interestingly, activin B and activin AB very potently activated SMAD1/5, resembling the activation commonly seen with BMPs. As SMAD1/5 was also activated by activins in other cell types, we propose that dual specificity is a general mechanism for activin ligands. In addition, we found that the antagonist follistatin inhibited signaling by all the tested activins, whereas the antagonist cerberus specifically inhibited activin B. Taken together, we propose that activins may be considered dual specificity TGF-β family members, critically affecting how activins may be considered and targeted clinically.

Activins and bone morphogenetic proteins (BMPs) play critical, sometimes opposing roles, in multiple physiological and pathological processes and diseases. They signal to distinct Smad branches; activins signal mainly to Smad2/3, while BMPs activate mainly Smad1/5/8. This gives rise to the possibility that competition between the different type I receptors through which activin and BMP signal for common type II receptors can provide a mechanism for fine-tuning the cellular response to activin/BMP stimuli. Among the transforming growth factor-β superfamily type II receptors, ACVR2A/B are highly promiscuous, due to their ability to interact with different type I receptors (e.g., ALK4 vs. ALK2/3/6) and with their respective ligands [activin A (ActA) vs. BMP9/2]. However, studies on complex formation between these full-length receptors situated at the plasma membrane, and especially on the potential competition between the different activin and BMP type I receptors for a common activin type II receptor, were lacking.

BMPR2 is a type II Transforming Growth Factor (TGF)-β family receptor that is fundamentally associated with pulmonary arterial hypertension (PAH) in humans. BMPR2 shares functional similarities with the type II activin receptors ACVR2A and ACVR2B, as it interacts with an overlapping group of TGF-β family growth factors (GFs). However, how BMPR2 recognizes GFs remains poorly understood. Here, we solved crystal structures of BMPR2 in complex with the GF activin B and of ACVR2A in complex with the related GF activin A. We show that both BMPR2 and ACVR2A bind GFs with nearly identical geometry using a conserved hydrophobic hot spot, while differences in contacting residues are predominantly found in loop areas. Upon further exploration of the GF-binding spectrum of the two receptors, we found that although many GFs bind both receptors, the high-affinity BMPR2 GFs comprise BMP15, BMP10, and Nodal, whereas those of ACVR2A are activin A, activin B, and GDF11. Lastly, we evaluated GF-binding domain BMPR2 variants found in human PAH patients. We demonstrate that mutations within the GF-binding interface resulted in loss of GF binding, while mutations in loop areas allowed BMPR2 to retain the ability to bind cognate GFs with high affinity. In conclusion, the in vitro activities of BMPR2 variants and the crystal structures reported here indicate biochemically relevant complexes that explain how some GF-binding domain variants can lead to PAH.

The developing testis provides an environment that nurtures germ cell development, ultimately ensuring spermatogenesis and fertility. Impacts on this environment are considered to underlie aberrant germ cell development and formation of germ cell tumour precursors. The signaling events involved in testis formation and male fetal germ cell development remain largely unknown. Analysis of knockout mice lacking single Tgfβ family members has indicated that Tgfβ's are not required for sex determination. However, due to functional redundancy, it is possible that additional functions for these ligands in gonad development remain to be discovered. Using FACS purified gonadal cells, in this study we show that the genes encoding Activin's, TGFβ's, Nodal and their respective receptors, are expressed in sex and cell type specific patterns suggesting particular roles in testis and germ cell development. Inhibition of signaling through the receptors ALK4, ALK5 and ALK7, and ALK5 alone, demonstrated that TGFβ signaling is required for testis cord formation during the critical testis-determining period. We also show that signaling through the Activin/NODAL receptors, ALK4 and ALK7 is required for promoting differentiation of male germ cells and their entry into mitotic arrest. Finally, our data demonstrate that Nodal is specifically expressed in male germ cells and expression of the key pluripotency gene, Nanog was significantly reduced when signaling through ALK4/5/7 was blocked. Our strategy of inhibiting multiple Activin/NODAL/TGFβ receptors reduces the functional redundancy between these signaling pathways, thereby revealing new and essential roles for TGFβ and Activin signaling during testis formation and male germ cell development.

Skeletal muscle mass is decreased under a wide range of pathologic conditions. In particular, chemotherapy is well known for inducing muscle loss and atrophy. Previous studies using tonsil-derived mesenchymal stem cells (T-MSCs) or a T-MSC-conditioned medium showed effective recovery of total body weight in the chemotherapy-preconditioned bone marrow transplantation mouse model. This study investigated whether extracellular vesicles of T-MSCs, such as exosomes, are a key player in the recovery of body weight and skeletal muscle mass in chemotherapy-treated mice. T-MSC exosomes transplantation significantly decreased loss of total body weight and muscle mass in the busulfan-cyclophosphamide conditioning regimen in BALB/c recipient mice containing elevated serum activin A. Additionally, T-MSC exosomes rescued impaired C2C12 cell differentiation in the presence of activin A in vitro. We found that T-MSC exosomes possess abundant miR-145-5p, which targets activin A receptors, ACVR2A, and ACVR1B. Indeed, T-MSC exosomes rescue muscle atrophy both in vivo and in vitro via miR-145-5p dependent manner. These results suggest that T-MSC exosomes have therapeutic potential to maintain or improve skeletal muscle mass in various activin A elevated pathologic conditions.

Mortality from prostate cancer (PCa) is due to the formation of metastatic disease. Understanding how that process is regulated is therefore critical. We previously demonstrated that endoglin, a type III transforming growth factor β (TGFβ) superfamily receptor, suppresses human PCa cell invasion and metastasis. Endoglin-mediated suppression of invasion was also shown by us to be dependent upon the type I TGFβ receptor, activin receptor-like kinase 2 (ALK2), and the downstream effector, Smad1. In this study we demonstrate for the first time that two type II TGFβ receptors are required for endoglin-mediated suppression of invasion: activin A receptor type IIA (ActRIIA) and bone morphogenetic protein receptor type II (BMPRII). Downstream signaling through these receptors is predominantly mediated by Smad1. ActRIIA stimulates Smad1 activation in a kinase-dependent manner, and this is required for suppression of invasion. In contrast BMPRII regulates Smad1 in a biphasic manner, promoting Smad1 signaling through its kinase domain but suppressing it through its cytoplasmic tail. BMPRII's Smad1-regulatory effects are dependent upon its expression level. Further, its ability to suppress invasion is independent of either kinase function or tail domain. We demonstrate that ActRIIA and BMPRII physically interact, and that each also interacts with endoglin. The current findings demonstrate that both BMPRII and ActRIIA are necessary for endoglin-mediated suppression of human PCa cell invasion, that they have differential effects on Smad1 signaling, that they make separate contributions to regulation of invasion, and that they functionally and physically interact.

Although equine gestation is unique from the standpoint of fetal gonadal enlargement and regression, the activator of this process is still unknown. The present study aimed to show a possible role of activin during equine gestation. In the first experiment, weekly plasma samples from six pregnant mares were used to measure activin A. In the second experiment, eight pregnant mares carrying female (gestational days 110, 140, 180, and 270) and male fetuses (gestational days 120, 180, 225, and 314) were used for immunohistochemistry of activin receptors (IA, IB, IIA, IIB), and their intracellular mediators (Smad2, Smad3, Smad4). Activin A levels in maternal circulation remained low until fourth weeks of gestation, thereafter, started to increase, and peaked first at 11 weeks of gestation. The second significant peak was observed on the day of parturition. Activin receptors type IA, IB, IIA, and IIB were immunostained in interstitial and germ cells of fetal ovaries and testes along with utero-placental tissues. Smad2, Smad3, and Smad4 were also immunolocalized in all these organs. These results demonstrated the activin-producing capacity of utero-placental tissues, and also evidenced the existence of activin receptors and functional signaling molecules in these organs. The first increment in circulating activin A in maternal circulation coinciding with the timing of initiation of fetal gonadal enlargement suggests that activin from the utero-placental tissues may have a stimulatory role in fetal gonad enlargement and utero-placental development in mares, whereas the second peak could be important to follicular development in the maternal ovary for foal heat.

Glucagon-like peptide 1 (GLP-1) receptor agonists reduce food intake, producing remarkable weight loss in overweight and obese individuals. While much of this weight loss is fat mass, there is also a loss of lean mass, similar to other approaches that induce calorie deficit. Targeting signaling pathways that regulate skeletal muscle hypertrophy is a promising avenue to preserve lean mass and modulate body composition. Myostatin and Activin A are TGFβ-like ligands that signal via the activin type II receptors (ActRII) to antagonize muscle growth. Pre-clinical and clinical studies demonstrate that ActRII blockade induces skeletal muscle hypertrophy and reduces fat mass. In this manuscript, we test the hypothesis that combined ActRII blockade and GLP-1 receptor agonism will preserve muscle mass, leading to improvements in skeletomuscular and metabolic function and enhanced fat loss.

The adipokine, leptin (LEP), is a hormonal gateway, signaling energy stores to appetite-regulatory neurons, permitting reproduction when stores are sufficient. Dual-labeling for LEP receptors (LEPRs) and gonadotropins or GH revealed a 2-fold increase in LEPR during proestrus, some of which was seen in LH gonadotropes. We therefore investigated LEPR functions in gonadotropes with Cre-LoxP technology, deleting the signaling domain of the LEPR (Lepr-exon 17) with Cre-recombinase driven by the rat LH-β promoter (Lhβ-cre). Selectivity of the deletion was validated by organ genotyping and lack of LEPR and responses to LEP by mutant gonadotropes. The mutation had no impact on growth, body weight, the timing of puberty, or pregnancy. Mutant females took 36% longer to produce their first litter and had 50% fewer pups/litter. When the broad impact of the loss of gonadotrope LEPR on all pituitary hormones was studied, mutant diestrous females had reduced serum levels of LH (40%), FSH (70%), and GH (54%) and mRNA levels of Fshβ (59%) and inhibin/activin β A and β B (25%). Mutant males had reduced serum levels of GH (74%), TSH (31%), and prolactin (69%) and mRNA levels of Gh (31%), Ghrhr (30%), Fshβ (22%), and glycoprotein α-subunit (Cga) (22%). Serum levels of LEP and ACTH and mRNA levels of Gnrhr were unchanged. However, binding to GnRH receptors was reduced in LEPR-null LH or FSH gonadotropes by 82% or 89%, respectively, in females (P < .0001) and 27% or 53%, respectively, in males (P < .03). This correlated with reductions in GnRH receptor protein immunolabeling, suggesting that LEP's actions may be posttranscriptional. Collectively, these studies highlight the importance of LEP to gonadotropes with GnRH-binding sites and activin as potential targets. LEP may modulate population growth, adjusting the number of offspring to the availability of food supplies.

Activin ligands are formed from two disulfide-linked inhibin β (Inhβ) subunit chains. They exist as homodimeric proteins, as in the case of activin A (ActA; InhβA/InhβA) or activin C (ActC; InhβC/InhβC), or as heterodimers, as with activin AC (ActAC; InhβA:InhβC). While the biological functions of ActA and activin B (ActB) have been well characterized, little is known about the biological functions of ActC or ActAC. One thought is that the InhβC chain functions to interfere with ActA production by forming less active ActAC heterodimers. Here, we assessed and characterized the signaling capacity of ligands containing the InhβC chain. ActC and ActAC activated SMAD2/3-dependent signaling via the type I receptor, activin receptor-like kinase 7 (ALK7). Relative to ActA and ActB, ActC exhibited lower affinity for the cognate activin type II receptors and was resistant to neutralization by the extracellular antagonist, follistatin. In mature murine adipocytes, which exhibit high ALK7 expression, ActC elicited a SMAD2/3 response similar to ActB, which can also signal via ALK7. Collectively, these results establish that ActC and ActAC are active ligands that exhibit a distinct signaling receptor and antagonist profile compared to other activins.

The health of neurons is critically dependent on the relative signaling intensities of survival-promoting synaptic and death-inducing extrasynaptic NMDA receptors. Here, we show that BDNF is a regulator of this balance and promotes neuroprotection by reducing toxic NMDA receptor signaling. BDNF acts by initiating synaptic NMDA-receptor/nuclear-calcium-driven adaptogenomics, leading to increased expression of inhibin β-A (inhba). Inhibin β-A (its homodimer is known as activin A) in turn reduces neurotoxic extrasynaptic NMDA-receptor-mediated calcium influx, thereby shielding neurons against mitochondrial dysfunction, a major cause of excitotoxicity. Thus, BDNF induces acquired neuroprotection by enhancing synaptic activity and lowering extrasynaptic NMDA receptor death signaling through a nuclear calcium-inhibin β-A pathway. This process, which confers protection against ischemic brain damage in a mouse stroke model, may be compromised in Huntington's disease, Alzheimer's disease, or aging-related neurodegenerative conditions that are associated with reduced BDNF levels and/or enhanced extrasynaptic NMDA receptor signaling.