Endogenous Activin Research Articles

Zygotic activin a is dispensable for the mouse preimplantation embryo development and for the derivation and pluripotency of embryonic stem cells†.

In this work, we aimed to determine the role of activin A during crucial events of mouse embryogenesis and distinguish the function of the protein of zygotic origin and the one secreted by the maternal reproductive tract. To this end, we recorded the progression of development and phenotype of Inhba knockout embryos and compared them with the heterozygotes and wild-type embryos using time-lapse imaging and detection of lineage-specific markers. We revealed that the zygotic activin A deficiency does not impair the course and rate of development of embryos to the blastocyst stage. Inhba knockout embryos form functional epiblast, as evidenced by their ability to give rise to embryonic stem cells. Our study is the first to show that derivation, maintenance in culture, and pluripotency of embryo-derived embryonic stem cells are exogenous and endogenous activin A-independent. However, the implantation competence of activin A-deficient embryos may be compromised as indicated in the outgrowth assay.

Gonadotropin elevation is ootoxic to ovulatory oocytes and inhibits oocyte maturation, and activin decoy receptor ActRIIB:Fc therapeutically restores maturation

BackgroundElevated FSH often occurs in women of advanced maternal age (AMA, age ≥ 35) and in infertility patients undergoing controlled ovarian stimulation (COS). There is controversy on whether high endogenous FSH contributes to infertility and whether high exogenous FSH adversely impacts patient pregnancy rates.MethodsThe senescence-accelerated mouse-prone-8 (SAMP8) model of female reproductive aging was employed to assess the separate impacts of age and high FSH activity on the percentages (%) of viable and mature ovulated oocytes recovered after gonadotropin treatment. Young and midlife mice were treated with the FSH analog equine chorionic gonadotropin (eCG) to model both endogenous FSH elevation and exogenous FSH elevation. Previously we showed the activin inhibitor ActRIIB:Fc increases oocyte quality by preventing chromosome and spindle misalignments. Therefore, ActRIIB:Fc treatment was performed in an effort to increase % oocyte viability and % oocyte maturation.ResultsThe high FSH activity of eCG is ootoxic to ovulatory oocytes, with greater decreases in % viable oocytes in midlife than young mice. High FSH activity of eCG potently inhibits oocyte maturation, decreasing the % of mature oocytes to similar degrees in young and midlife mice. ActRIIB:Fc treatment does not prevent eCG ootoxicity, but it restores most oocyte maturation impeded by eCG.ConclusionsFSH ootoxicity to ovulatory oocytes and FSH maturation inhibition pose a paradox given the well-known pro-growth and pro-maturation activities of FSH in the earlier stages of oocyte growth. We propose the FOOT Hypothesis (“FSH OoToxicity Hypothesis), that FSH ootoxicity to ovulatory oocytes comprises a new driver of infertility and low pregnancy success rates in DOR women attempting spontaneous pregnancy and in COS/IUI patients, especially AMA women. We speculate that endogenous FSH elevation also contributes to reduced fecundity in these DOR and COS/IUI patients.Restoration of oocyte maturation by ActRIB:Fc suggests that activin suppresses oocyte maturation in vivo. This contrasts with prior studies showing activin A promotes oocyte maturation in vitro. Improved oocyte maturation with agents that decrease endogenous activin activity with high specificity may have therapeutic benefit for COS/IVF patients, COS/IUI patients, and DOR patients attempting spontaneous pregnancies.

Activin A, a Novel Chemokine, Induces Mouse NK Cell Migration via AKT and Calcium Signaling.

Natural killer (NK) cells can migrate quickly to the tumor site to exert cytotoxic effects on tumors, and some chemokines, including CXCL8, CXCL10 or and CXCL12, can regulate the migration of NK cells. Activin A, a member of the transforming growth factor β (TGF-β) superfamily, is highly expressed in tumor tissues and involved in tumor development and immune cell activation. In this study, we focus on the effects of activin A on NK cell migration. In vitro, activin A induced NK cell migration and invasion, promoted cell polarization and inhibited cell adhesion. Moreover, activin A increased Ca2+, p-SMAD3 and p-AKT levels in NK cells. An AKT inhibitor and Ca2+ chelator partially blocked activin A-induced NK cell migration. In vivo, exogenous activin A increased tumor-infiltrating NK cells in NS-1 cell solid tumors and inhibited tumor growth, and blocking endogenous activin A with anti-activin A antibody reduced tumor-infiltrating NK cells in 4T-1 cell solid tumors. These results suggest that activin A induces NK cell migration through AKT signaling and calcium signaling and may enhance the antitumor effect of NK cells by increasing tumor-infiltrating NK cells.

Increase in activin A may counteract decline in synaptic plasticity with age.

Activin A, a member of the transforming growth factor β (TGF-β) family, is widely recognized for its neurotrophic and neuroprotective function in the developing and injured brain, respectively. Moreover, in the healthy adult brain, activin A has been shown to tune signal processing at excitatory synapses in a fashion that improves cognitive performance. Because its level in human cerebrospinal fluid rises with age, we wondered whether activin A has a role in mitigating the gradual cognitive decline that healthy individuals experience in late-life. To interrogate the role of activin A in synaptic plasticity in the aging brain, we used an established transgenic mouse line, in which expression of a dominant-negative mutant of activin receptor IB (dnActRIB) serves to disrupt activin receptor signaling in a forebrain-specific fashion. In brain slices of young adult dnActRIB mice (2-4 months old), the NMDA receptor-dependent and -independent forms of long-term potentiation (LTP) at the Schaffer collateral-CA1 pyramidal cell synapse of the hippocampus were equally impaired relative to the extent of LTP measured in the wild-type preparation. Unexpectedly, the difference between the genotypes disappeared when the two forms of LTP were re-examined in slices from middle-aged mice (13-16 months old). Since the level of activin A and endogenous ActRIB both displayed a significant elevation in middle-aged hippocampus, we reasoned that with such a rise, the dominant-negative effect of the mutant receptors could be overcome. Substantiating this idea, we found that administration of recombinant activin A was indeed capable of restoring full-blown LTP in slices from young dnActRIB mice. Our data suggest that, beginning in the middle-aged brain, endogenous activin receptor signaling appears to become strengthened in an attempt to stave off cognitive decline. If further corroborated, this concept would also hold promise for new therapeutic venues to preserve cognitive functions in the aged brain.

Palovarotene Action Against Heterotopic Ossification Includes a Reduction of Local Participating Activin A-Expressing Cell Populations.

Heterotopic ossification (HO) consists of extraskeletal bone formation. One form of HO is acquired and instigated by traumas or surgery, and another form is genetic and characterizes fibrodysplasia ossificans progressiva (FOP). Recently, we and others showed that activin A promotes both acquired and genetic HO, and in previous studies we found that the retinoid agonist palovarotene inhibits both HO forms in mice. Here, we asked whether palovarotene's action against HO may include an interference with endogenous activin A expression and/or function. Using a standard mouse model of acquired HO, we found that activin A and its encoding RNA (Inhba) were prominent in chondrogenic cells within developing HO masses in untreated mice. Single-cell RNAseq (scRNAseq) assays verified that Inhba expression characterized chondroprogenitors and chondrocytes in untreated HO, in addition to its expected expression in inflammatory cells and macrophages. Palovarotene administration (4 mg/kg/d/gavage) caused a sharp inhibition of both HO and amounts of activin A and Inhba transcripts. Bioinformatic analyses of scRNAseq data sets indicated that the drug had reduced interactions and cross-talk among local cell populations. To determine if palovarotene inhibited Inhba expression directly, we assayed primary chondrocyte cultures. Drug treatment inhibited their cartilaginous phenotype but not Inhba expression. Our data reveal that palovarotene markedly reduces the number of local Inhba-expressing HO-forming cell populations. The data broaden the spectrum of HO culprits against which palovarotene acts, accounting for its therapeutic effectiveness. © 2023 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Dorsal-Ventral Gradient of Activin Regulates Strength of GABAergic Inhibition along Longitudinal Axis of Mouse Hippocampus in an Activity-Dependent Fashion.

The functional and neurophysiological distinction between the dorsal and ventral hippocampus affects also GABAergic inhibition. In line with this notion, ventral CA1 pyramidal cells displayed a more dynamic and effective response to inhibitory input compared to their dorsal counterparts. We posit that this difference is effected by the dorsal-ventral gradient of activin A, a member of the transforming growth factor-β family, which is increasingly recognized for its modulatory role in brain regions involved in cognitive functions and affective behavior. Lending credence to this hypothesis, we found that in slices from transgenic mice expressing a dominant-negative mutant of activin receptor IB (dnActRIB), inhibitory transmission was enhanced only in CA1 neurons of the dorsal hippocampus, where the basal activin A level is much higher than in the ventral hippocampus. We next asked how a rise in endogenous activin A would affect GABAergic inhibition along the longitudinal axis of the hippocampus. We performed ex vivo recordings in wild-type and dnActRIB mice after overnight exposure to an enriched environment (EE), which engenders a robust increase in activin A levels in both dorsal and ventral hippocampi. Compared to control mice from standard cages, the behaviorally induced surge in activin A produced a decline in ventral inhibition, an effect that was absent in slices from dnActRIB mice. Underscoring the essential role of activin in the EE-associated modulation of ventral inhibition, this effect was mimicked by acute application of recombinant activin A in control slices. In summary, both genetic and behavioral manipulations of activin receptor signaling affected the dorsal-ventral difference in synaptic inhibition, suggesting that activin A regulates the strength of GABAergic inhibition in a region-specific fashion.

#5591 CIRCULATING ACTIVIN A REFLECTS THE SEVERITY OF RENAL FIBROSIS IN BIOPSY-PROVEN KIDNEY DISEASES—THE TAIPEI RENAL BIOPSY COHORT STUDY

Abstract Background and Aims Despite considerable evidence indicating circulating activin A as a novel renal biomarker, its performance for predicting the severity of renal fibrosis and major adverse renal events (MARE) has not yet been extensively studied. Method We sought to assess the relationship between plasma activin A, renal fibrosis severity, and incidence of MARE in 339 patients undergoing clinically indicated native renal biopsies. To determine the primary source of circulating activin A, RNA-sequencing and histological analyses were performed on kidney biopsy specimens from patients with chronic kidney disease (CKD). We also conducted in vitro experiments to investigate whether inhibiting endogenous activin A could attenuate TGF-β-mediated activation of cultured fibroblasts. Results The median baseline eGFR and proteinuria were 36 mL/min/1.73 m2 and 2.9 mg/mg creatinine, respectively. After multivariable adjustment, elevated plasma activin A was associated with the extent of renal fibrosis. Histological analysis showed increased activin A expression in kidney tissues from patients with CKD, mainly in interstitial myofibroblasts. RNA-sequencing of tubulointerstitial tissue from human biopsy samples also revealed a direct correlation between tissue activin A mRNA expression and plasma activin A levels. During a median follow-up of 22 months, 113 participants suffered MARE. Cox proportional hazards analysis revealed that plasma activin A was associated with higher risk of renal events; however, the association became insignificant after correcting for confounders. Results from in vitro studies demonstrated that knocking down activin A expression could prevent TGF-β-induced activation of NRK49F fibroblasts. Conclusion The findings of this study support activin A as a potential diagnostic and therapeutic target in fibrotic kidney disease.

Correlation analysis of NT-proBNP (N-terminal probrain natriuretic peptide), 25-Hydroxyvitamin D, HMGB1(High-mobility group box 1), ACTA (endogenous activin A), blood glucose level, and electrolyte level with developmental quotient scores in neonates with hypoxic-ischemic encephalopathy

BackgroundTo investigate the correlation between N-terminal probrain natriuretic peptide (NT-proBNP), 25-hydroxyvitamin D (25-(OH) D), high-mobility group box 1(HMGB1), endogenous activin A (ACTA), blood glucose level, electrolyte levels and developmental quotient (DQ) scores of Hypoxic-ischemic encephalopathy (HIE).MethodsIn this retrospective study, a total of 90 neonates diagnosed with HIE who were admitted to our hospital from January 2018 to June 2021 were retrospectively enrolled, and 40 healthy full-term neonates born in our hospital during the same period were randomly selected. Neonates with HIE and healthy conditions were set as the study group and control group, respectively. Neonates with HIE are divided into three subgroups, mild, moderate, and severe, based on the severity of HIE. The Gesell Developmental Scale (GDS) was used to assess neural development of neonates at 9 to 12 months postnatal. Biomarkers of peripheral venous blood were measured and collected in all neonates, including NT-proBNP, (25-(OH) D), HMGB1, ACTA, electrolyte levels and blood glucose levels. General demographic information and Apgar score were compared between the two groups. The differences between the two groups of biomarkers were compared and the correlation between these biomarkers and DQ scores was evaluated.ResultsThere was no significant difference in gestational age, maternal age, gender, way of birth, birth weight, gestational age and whether the mother was a primipara between the two groups (P>0.05). The 10 min Apgar score of the study group (5.87±0.36) was lower than that of the control group (9.37±0.32) with significant difference (P<0.05). The levels of NT-proBNP, HMGB1, and ACTA in the study group were higher than that in the control group (243.87±21.29 pmol/L vs. 116.98±22.19 pmol/L; 8.92±1.87 μg/L vs. 3.28±1.08 μg/L; 23.78±0.89 ng/ml vs. 2.98±0.38 ng/ml), while the levels of 25-(OH) D and electrolyte levels were lower than that in the control group (24.28±1.87 vs. 31.29±1.93; K+: 4.49±0.23 mmol/L vs. 4.73±0.21 mmol/L; Na+: 118.76±13.02 mmol/L vs. 134.28±12.29 mmol/L; Ca2+: 1.77±0.23 mmol/L vs. 2.35±0.26 mmol/L; Mg2+: 0.61±0.17 mmol/L vs. 0.91±0.17 mmol/L), with statistically significant differences (P<0.001). The levels of NT-probNP, HMGB1, ACTA and the incidence of hypoglycemia were the highest in the severe group, which were significantly higher than those in the moderate group and mild group (P<0.05). The levels of NT-probNP, HMGB1, ACTA and the incidence of hypoglycemia were the lowest in the mild group. The 25-(OH) D level, the incidence of hyperglycemia and electrolyte levels were the lowest in the severe group, which were significantly lower than those in the moderate and mild groups (all P<0.05). Meanwhile, the 25-(OH) D level, the incidence of hyperglycemia and electrolyte levels in the moderate group were lower than those in the mild group, and the differences were statistically significant (all P<0.05).The incidence of hyperglycemia in severe group (16 cases) was the lowest, significantly lower than that in moderate group (17 cases) and mild group (22 cases), and the difference was statistically significant (all P<0.05).The DQ scores of HIE neonates were negatively correlated with NT-proBNP, HMGB1, and ACTA (r=-0.671, -0.421, -0.518, all P< 0.001). The DQ scores was positively correlated with levels of 25-(OH) D and blood glucose level (r =0.621, 0.802, all P< 0.001). The DQ scores was also positively correlated with levels of potassium, sodium, calcium and magnesium (0.367, 0.782, 0.218, 0.678, all P<0.001). ConclusionThe NT-proBNP, HMGB1, ACTA, 25-(OH) D, blood glucose levels and electrolyte levels are correlated with the severity of HIE, and developmental quotient scores in neonates with HIE. These biomarkers are suggestive for assessing the prognosis of neonate with HIE.

ActivinA promotes cell proliferation, invasion and migration and predicts poor prognosis in patients with colorectal cancer.

Activin A is a member of the transforming growth factor-β superfamily of cytokines and displays various pathophysiological activities, including regulation of muscle catabolism and atrophy. Activin A expression is upregulated in several human cancer types and in certain pathologies, its expression is associated with poor prognosis. In the present study, activin A expression was assessed in colorectal cancer (CRC) tissue specimens from 157 patients with primary CRC and the relationship between activin A levels and clinicopathological characteristics, including skeletal muscle mass, and prognosis, was determined. Furthermore, the effects of knockdown of endogenous or exposure to exogenous activin A on the malignant behavior of human CRC cell lines were investigated in vitro. The results indicated that activin A mRNA was significantly upregulated in CRC tumor tissues compared with normal intestinal epithelium. High activin A expression was significantly associated with shorter cancer-specific survival (P=0.047) and overall survival (P=0.014). According to a multivariate analysis, tumor activin A levels were an independent prognostic factor for overall survival (P=0.001). However, activin A mRNA levels were not associated with the skeletal muscle index. The in vitro experiments demonstrated that exposure to exogenous activin A increased the proliferation, invasion and migration of CRC cell lines, whereas knockdown of endogenous activin A had the opposite effects. In conclusion, activin A is an autocrine and paracrine regulator of CRC cell proliferation and high tumor expression of activin A is associated with poor prognosis in patients with CRC.

Activin A promotes the development of acquired heterotopic ossification and is an effective target for disease attenuation in mice.

Heterotopic ossification (HO) is a common, potentially debilitating pathology that is instigated by inflammation caused by tissue damage or other insults, which is followed by chondrogenesis, osteogenesis, and extraskeletal bone accumulation. Current remedies are not very effective and have side effects, including the risk of triggering additional HO. The TGF-β family member activin A is produced by activated macrophages and other inflammatory cells and stimulates the intracellular effectors SMAD2 and SMAD3 (SMAD2/3). Because HO starts with inflammation and because SMAD2/3 activation is chondrogenic, we tested whether activin A stimulated HO development. Using mouse models of acquired intramuscular and subdermal HO, we found that blockage of endogenous activin A by a systemically administered neutralizing antibody reduced HO development and bone accumulation. Single-cell RNA-seq analysis and developmental trajectories showed that the antibody treatment reduced the recruitment of Sox9+ skeletal progenitors, many of which also expressed the gene encoding activin A (Inhba), to HO sites. Gain-of-function assays showed that activin A enhanced the chondrogenic differentiation of progenitor cells through SMAD2/3 signaling, and inclusion of activin A in HO-inducing implants enhanced HO development in vivo. Together, our data reveal that activin A is a critical upstream signaling stimulator of acquired HO in mice and could represent an effective therapeutic target against forms of this pathology in patients.

Endothelial Cells Express Activin A which may serve as an antagonist to TGFβ binding to Activin‐Like Kinase 1 Receptor

Transforming growth factor (TGFβ) family and their receptors have been shown to be involved in angiogenesis as well as tumor cells. Members of the family that have been reported to act on endothelial cells are TGFβ, Bone Morphogenetic protein 9 and 10 (BMP‐9, BMP‐10), and Activin A. They all can signal through the type I receptor Activin‐Like Kinase 1 (ALK‐1). We have screened endothelial cells vs tumor cells for ALK‐1, Endoglin, and potential ligands BMP‐10 and Activin A to determine which ligands and receptors may be active in signaling in angiogenesis. We show that endothelial cells express ALK‐1 and endoglin as well as BMP‐10 and Activin A by quantitative RT‐PCR. Transfecting Human Umbilical Vein Endothelial Cells (HUVEC) compared to human 293 cells and Hela cells with an ALK‐1 plasmid resulted in increased expression of ALK‐1 in all cell lines. ALK‐1 overexpression did not affect Smad 1 or Smad 5 expression but increased BMP‐10 and Activin A expression in HUVEC. This data suggests these factors may function as endogenous ligands to control endothelial function and angiogenesis. TGFβ can only bind ALK‐1 on endothelial cells at 37°C but not 23 or 4° C. But our results indicate that endogenous Activin A is bound to ALK‐1 and can be displaced by added TGFβ. This supports the finding above that HUVEC express Activin A. Thus Activin A may function as an endogenous regulator of TGFβ function in endothelial cells.Support or Funding InformationResearch support was from an SFSC Innovation Grant (MP), Portz National Honors Fellowships (GW and NM), and funds from Saint Leo University (ID).

Induction of experimental autoimmune orchitis in mice: responses to elevated circulating levels of the activin-binding protein, follistatin

Experimental autoimmune orchitis (EAO) is a rodent model of chronic testicular inflammation that mimics the pathology observed in some types of human infertility. In a previous study, testicular expression of the inflammatory/immunoregulatory cytokine, activin A, was elevated in adult mice during the onset of EAO, indicating a potential role in the regulation of the disease. Consequently, we examined the development of EAO in mice with elevated levels of follistatin, an endogenous activin antagonist, as a potential therapeutic approach to testicular inflammation. Prior to EAO induction, mice received a single intramuscular injection of a non-replicative recombinant adeno-associated viral vector carrying a gene cassette of the circulating form of follistatin, FST315 (FST group). Serum follistatin levels were increased 5-fold in the FST group compared with the control empty vector (EV) group at 30 and 50 days of EAO, but intra-testicular levels of follistatin or activin A were not significantly altered. Induction of EAO was reduced, but not prevented, with mild-to-severe damage in 75% of the EV group and 40% of the FST group, at 50 days following immunisation with testicular homogenate. However, the EAO damage score (based on disruption of the blood-testis barrier, apoptosis, testicular damage and fibrosis) and extent of intratesticular inflammation (expression of inflammatory mediators) were directly proportional to the levels of activin A measured in the testis at 50 days. These data implicate activin A in the progression of EAO, thereby providing a potential therapeutic target; however, elevating circulating follistatin levels were not sufficient to prevent EAO development.

Activin/follistatin system in grass carp pituitary cells: - Regulation by local release of growth hormone and luteinizing hormone and its functional role in growth hormone synthesis and secretion.

Gonadotrophin regulation by activin/follistatin system is well-documented, but the corresponding effect on growth hormone (GH) has not been fully characterized and with little information available in lower vertebrates, especially in fish models. In grass carp, local interactions of GH and luteinizing hormone (LH) can induce GH release and gene expression at pituitary level via autocrine/paracrine mechanisms. To shed light on the role of activin/follistatin system in GH regulation by local actions of GH and LH, grass carp activin βA and βB were cloned, shown to be single-copy genes expressed in the pituitary, and confirmed to encode activin proteins capable of transactivating promoter with activin-responsive elements. In grass carp pituitary cells, activin A and B were effective in reducing GH secretion and GH cell content with concurrent drop in GH mRNA level whereas the opposite was true for follistatin, the activin-binding protein known to neutralize the effects of endogenous activin. Treatment with activin A and B not only could suppress basal but also inhibit GH mRNA expression induced by GH and human chorionic gonadotropin (hCG), a functional analogue of LH in fish model. Apparently, down-regulation of GH mRNA by activin was mediated by reducing GH transcript stability with concurrent inhibition on GH promoter activity via the SMAD pathway. In reciprocal experiments, GH treatment was found to up-regulate activin βA, activin βB and follistatin mRNA levels in carp pituitary cells but the opposite was noted by removing endogenous GH with GH antiserum. Interestingly, parallel treatment with hCG could also inhibit basal as well as GH-induced activin βA, activin βB and follistatin gene expression. These results, as a whole, indicate that the pituitary activin/follistatin system can serve as a regulatory target for local interactions of GH and LH and contribute to GH regulation by autocrine/paracrine mechanisms in the carp pituitary.

Abstract B20: Activin receptor-like kinase 1 complexes with endoglin in TGFß-mediated signaling in angiogenesis

Abstract Background: Activin Receptor-Like Kinase 1 (ALK1), a type I transforming growth factor (TGFß) family receptor, and endoglin, a type III receptor, are expressed in endothelial cells (EC) and involved in angiogenesis. Control of angiogenesis is critical in diseases such as cancer. Four ligands, TGFß, activin, bone morphogenetic protein 9 (BMP-9), and BMP-10, have been shown to bind ALK-1. Thus angiogenesis may be mediated by different combinations of ligands and receptors. Purpose: We investigated the role of TGFß and ALK-1 in tube formation. We also screened EC and also cancer cell lines for expression of various TGFß family receptors to begin to address which ligands and receptors are involved in angiogenesis and endothelial-tumor interactions,. Results: We also show that fibroblast growth factor 2 (FGF-2) + TGFß increase ALK-1 but not ALK 5 type I receptor expression in endothelial cells after tube formation. We show data that the type I receptor Activin-Like Kinase 1 and the co-receptor endoglin can signal in endothelial cells via the TGFß ligand. Antisense RNA that targets ALK 1 (and also ALK5) also disrupts tube formation. We further show that endogenous activin binds ALK 1 and can be displaced by exogenous TGFß. This suggests it may function to regulate control of ALK 1 activation. Further, ALK-1 and endoglin may also be involved in cell-cell interactions between endothelial and other cells. Thus we screened endothelial and cancer cells for TGFß family receptor expression by RT-PCR. Endothelial cells show similar expression of ALK-1, ALK-5, and endoglin and lower expression of BMP receptor II and TGFßRII. Cancer cells show much lower expression of type II receptors. Citation Format: James Hawker, Patricia Varacallo, Iain Duffy. Activin receptor-like kinase 1 complexes with endoglin in TGFß-mediated signaling in angiogenesis. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Angiogenesis and Vascular Normalization: Bench to Bedside to Biomarkers; Mar 5-8, 2015; Orlando, FL. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl):Abstract nr B20.

Mutation switches ligand specificity

Genetic Disorder Fibrodysplasia ossificans progressiva (FOP) is a rare but deadly genetic condition that causes bone growth in place of soft tissues. The causal mutation in the bone morphogenetic protein (BMP) receptor ACVR1 is thought to boost receptor activity and trigger bone formation. Now, Harsell et al. suggest a different mechanism. They found that the mutated receptor (mACVR1) responds to activin. Normally, activin blocks BMP from binding to ACVR1. Adult mice expressing mACVR1 developed heterotopic ossification that required stimulation by endogenous activin. Small sponges soaked with activin ossified when implanted into mACVR1 mice, and animals treated with a monoclonal antibody to activin were protected. This unusual mechanism may explain why ossification in FOP patients is triggered by tissue trauma, which induces activin. Sci. Transl. Med. 7 , 303ra137 (2015).

Recombinant BMP4 and BMP7 increase activin A production by up-regulating inhibin βA subunit and furin expression in human granulosa-lutein cells.

Granulosa cell-derived activins play important roles in the regulation of ovarian functions. To date, there is limited information pertaining to the intracellular regulation, assembly, and secretion of endogenous activin A in human granulosa cells. The aim of this study was to examine the effects of BMP4 and BMP7 on furin expression and activin A production as well as the underlying mechanisms of action in human granulosa cells. An established immortalized human granulosa cell line (SVOG) and primary granulosa-lutein cells were used as study models. Expression of inhibin subunits and furin as well as activin A accumulation were examined after exposure to recombinant human BMP4 or BMP7. A BMP type I receptor inhibitor (dorsomorphin), a furin inhibitor (Decanoyl-Arg-Val-Lys-Arg-chloromethylketone), and small interfering RNAs targeting SMAD4 and furin were used to verify the specificity of the effects and investigate potential mechanisms. The study was conducted in an academic center. Specific mRNA and protein levels were examined using real time qPCR and Western blot. Activin A levels were measured using enzyme immunoassay. Treatment with bone morphogenetic protein (BMP) 4 and BMP7 significantly increased furin mRNA and protein, inhibin βA mRNA, and activin A accumulation. Pre-treatment with dorsomorphin or SMAD4 knockdown reversed the stimulatory effects of BMP4 and BMP7 on furin and inhibin βA expression. In addition, furin knockdown or pre-treatment with a furin inhibitor attenuated the BMP4- and BMP7-induced accumulation of activin A. Recombinant BMP4 and BMP7 increase the production of bioactive mature activin A by up-regulating both the production and proteolytic processing of inhibin βA subunit in human granulosa cells. The enhancement of inhibin βA subunit processing is attributable to a SMAD-dependent up-regulation of its proprotein convertase, furin. These findings provide a potential mechanism by which theca cells can regulate neighboring granulosa cells in the ovary.

Acute modulation of synaptic plasticity of pyramidal neurons by activin in adult hippocampus.

Activin A is known as a neuroprotective factor produced upon acute excitotoxic injury of the hippocampus (in pathological states). We attempt to reveal the role of activin as a neuromodulator in the adult male hippocampus under physiological conditions (in healthy states), which remains largely unknown. We showed endogenous/basal expression of activin in the hippocampal neurons. Localization of activin receptors in dendritic spines (=postsynapses) was demonstrated by immunoelectron microscopy. The incubation of hippocampal acute slices with activin A (10 ng/mL, 0.4 nM) for 2 h altered the density and morphology of spines in CA1 pyramidal neurons. The total spine density increased by 1.2-fold upon activin treatments. Activin selectively increased the density of large-head spines, without affecting middle-head and small-head spines. Blocking Erk/MAPK, PKA, or PKC prevented the activin-induced spinogenesis by reducing the density of large-head spines, independent of Smad-induced gene transcription which usually takes more than several hours. Incubation of acute slices with activin for 2 h induced the moderate early long-term potentiation (moderate LTP) upon weak theta burst stimuli. This moderate LTP induction was blocked by follistatin, MAPK inhibitor (PD98059) and inhibitor of NR2B subunit of NMDA receptors (Ro25-6981). It should be noted that the weak theta burst stimuli alone cannot induce moderate LTP. These results suggest that MAPK-induced phosphorylation of NMDA receptors (including NR2B) may play an important role for activin-induced moderate LTP. Taken together, the current results reveal interesting physiological roles of endogenous activin as a rapid synaptic modulator in the adult hippocampus.

The type I TGFß receptor ALK‐1 and it’s ligands promote angiogenesis (544.2)

Control of angiogenesis is critical in diseases such as cancer, heart disease, or wound repair. Growth factors such as transforming growth factor beta (TGFß) and fibroblast growth factor 2 (FGF‐2) contribute to angiogenesis. The TGFß family signaling receptor Activin‐Like Kinase 1 Receptor (ALK‐1) is expressed in coronary venular endothelial cells (CVEC) at near confluent cell densities on gelatin, but not collagen but it is induced by TGFß + FGF‐2 during tube formation in collagen. Other genes induced during tube formation were Smad 6 and Fibroblast Growth Factor Receptor 1 (FGFR1). By contrast, ALK‐5, a related TGFß type 1 receptor, and TGFßRII were expressed in these cells in both gelatin and collagen. ALK‐1 is also expressed in other EC types, smooth muscle cells, cardiac fibroblasts, embryonic fibroblasts (3T3) but not epithelial cells. Three ligands: TGFß, activin, and bone morphogenetic protein 9 (BMP‐9), have been shown to bind ALK‐1. We show that endogenous activin binds ALK 1 and can be displaced by exogenous TGFß. It may function to regulate control of ALK 1 activation. The response of endothelial cells to various ALK‐1 ligands may depend on the cell matrix of endothelial cells and which other co‐receptors (type II receptors and endoglin) are expressed. We are: 1) testing expression of various ALK‐1 ligands and co‐receptors in endothelial cells under various conditions; and 2) we are generating mutants of ALK‐1, ALK‐5, and endoglin to further test their roles in angiogenesis.Grant Funding Source: Research support was from AHA‐TX and Dept Veteran’s Affairs grants (JH)

Inhibition of Activin A Ameliorates Skeletal Muscle Injury and Rescues Contractile Properties by Inducing Efficient Remodeling in Female Mice

Activin A, a member of the transforming growth factor-β superfamily, provides pleiotropic regulation of fibrosis and inflammation. We aimed at determining whether selective inhibition of activin A would provide a regenerative benefit. The introduction of activin A into normal muscle increased the expression of inflammatory and muscle atrophy genes Tnf, Tnfrsf12a, Trim63, and Fbxo32 by 3.5-, 10-, 2-, and 4-fold, respectively. The data indicate a sensitive response of muscle to activin A. Two hours after cardiotoxin-induced muscle damage, local activin A protein expression increased by threefold to ninefold. Neutralization of activin A with a specific monoclonal antibody in this muscle injury model decreased the muscle protein levels of lymphotoxin α and Il17a by 32% and 42%, respectively. Muscle histopathological features showed that activin A antibody-treated mice displayed an increase in muscle degradation, with the concomitant 9.2-fold elevation in F4/80-positive cells 3 days after injury. At the same time, the number of Pax7/Myod1-positive cells also increased, indicative of potentiated muscle precursor activation. Ultimately, activin A inhibition resulted in rapid recovery of muscle contractile properties indicated by a restoration of maximum and specific force. In summary, selective inhibition of activin A with a monoclonal antibody in muscle injury leads to the early onset of tissue degradation and subsequent enhanced myogenesis, thereby accelerating muscle repair and functional recovery.

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.