Activin Signaling Pathway Research Articles

Molecular mechanisms of enhancing porcine granulosa cell proliferation and function by treatment in vitro with anti-inhibin alpha subunit antibody.

BackgroundThis study was conducted to clarify the effect of the inhibiting action of inhibin on porcine granulosa cell proliferation and function, and to investigate the underlying intracellular regulatory molecular mechanisms.MethodsPorcine granulosa cells were cultured in vitro, and were treated with an anti-inhibin alpha subunit antibody, with or without co-treatment of follicle-stimulating hormone (FSH) in the culture medium.ResultsTreatment with anti-inhibin alpha subunit antibody led to a significant increase in estradiol (E2) secretion and cell proliferation. Anti-inhibin alpha subunit antibody worked synergistically with FSH at low concentrations (25 microg/mL) to stimulate E2 secretion, but attenuated FSH action at high concentrations (50 microg/mL). Immunoneutralization of inhibin bioactivity increased FOXL2, Smad3, and PKA phosphorylation, and mRNA expression of the transcription factors CEBP and c-FOS. The expression of genes encoding gonadotropin receptors, FSHR and LHR, and of those involved in steroidogenesis, as well as IGFs and IGFBPs, the cell cycle progression factors cyclinD1 and cyclinD2, and the anti-apoptosis and anti-atresia factors Bcl2, TIMP, and ADAMTS were upregulated following anti-inhibin alpha-subunit treatment. Treatment with anti-inhibin alpha subunit down regulated expression of the pro-apoptotic gene encoding caspase3. Although expression of the pro-angiogenesis genes FN1, FGF2, and VEGF was upregulated, expression of the angiogenesis-inhibiting factor THBS1 was downregulated following anti-inhibin alpha subunit treatment.ConclusionsThese results suggest that immunoneutralization of inhibin bioactivity, through augmentation of the activin and gonadotropin receptor signaling pathways and regulation of gene expression, permits the development of healthy and viable granulosa cells. These molecular mechanisms help to explain the enhanced ovarian follicular development observed following inhibin immunization in animal models.

Efficient retina formation requires suppression of both Activin and BMP signaling pathways in pluripotent cells.

Retina formation requires the correct spatiotemporal patterning of key regulatory factors. While it is known that repression of several signaling pathways lead to specification of retinal fates, addition of only Noggin, a known BMP antagonist, can convert pluripotent Xenopus laevis animal cap cells to functional retinal cells. The aim of this study is to determine the intracellular molecular events that occur during this conversion. Surprisingly, blocking BMP signaling alone failed to mimic Noggin treatment. Overexpressing Noggin in pluripotent cells resulted in a concentration-dependent suppression of both Smad1 and Smad2 phosphorylation, which act downstream of BMP and Activin signaling, respectively. This caused a decrease in downstream targets: endothelial marker, xk81, and mesodermal marker, xbra. We treated pluripotent cells with dominant-negative receptors or the chemical inhibitors, dorsomorphin and SB431542, which each target either the BMP or Activin signaling pathway. We determined the effect of these treatments on retina formation using the Animal Cap Transplant (ACT) assay; in which treated pluripotent cells were transplanted into the eye field of host embryos. We found that inhibition of Activin signaling, in the presence of BMP signaling inhibition, promotes efficient retinal specification in Xenopus tissue, mimicking the affect of adding Noggin alone. In whole embryos, we found that the eye field marker, rax, expanded when adding both dominant-negative Smad1 and Smad2, as did treating the cells with both dorsomorphin and SB431542. Future studies could translate these findings to a mammalian culture assay, in order to more efficiently produce retinal cells in culture.

The differential anti-tumour effects of zoledronic acid in breast cancer - evidence for a role of the activin signaling pathway.

BackgroundNeo-adjuvant breast cancer clinical trials of zoledronic acid (ZOL) have shown that patients with oestrogen negative (ER-ve) tumours have improved disease outcomes. We investigated the molecular mechanism behind this differential anti-tumour effect according to ER status, hypothesising it may in part be mediated via the activin signaling pathway.MethodsThe effects of activin A, its inhibitor follistatin and zoledronic acid on proliferation of breast cancer cells was evaluated using either an MTS proliferation assay or trypan blue. Secretion of activin A and follistatin in conditioned medium (CM) from MDA-MB-231, MDA-MB-436, MCF7 and T47D cell lines were measured using specific ELISAs. The effects of ZOL on phosphorylation domains of Smad2 (pSmad2c + pSmad2L) were evaluated using immunofluorescence. Changes seen in vitro were confirmed in a ZOL treated subcutaneous ER-ve MDA-MB-436 xenograft model.ResultsActivin A inhibits proliferation of both ER-ve and oestrogen positive (ER + ve) breast cancer cells, an effect impaired by follistatin. ZOL significantly inhibits proliferation and the secretion of follistatin from ER-ve cells only, which increases the biological activity of the canonical activin A pathway by significantly increasing intracellular pSmad2c and decreasing nuclear accumulation of pSmad2L. In vivo, ZOL significantly decreases follistatin and pSmad2L expression in ER-ve subcutaneous xenografts compared to saline treated control animals.ConclusionsThis is the first report showing a differential effect of ZOL, according to ER status, on the activin pathway and its inhibitors in vitro and in vivo. These data suggest a potential molecular mechanism contributing to the differential anti-tumour effects reported from clinical trials and requires further evaluation in clinical samples.

Derivation of Mesenchymal Stromal Cells from Canine Induced Pluripotent Stem Cells by Inhibition of the TGFβ/Activin Signaling Pathway

In this study we have generated canine mesenchymal stromal cells (MSCs), also known as mesenchymal stem cells, from canine induced pluripotent stem cells (ciPSCs) by small-molecule inhibition of the transforming growth factor beta (TGFβ)/activin signaling pathway. These ciPSC-derived MSCs (ciPSC-MSCs) express the MSC markers CD73, CD90, CD105, STRO1, cPDGFRβ and cKDR, in addition to the pluripotency factors OCT4, NANOG and REX1. ciPSC-MSCs lack immunostaining for H3K27me3, suggesting that they possess two active X chromosomes. ciPSC-MSCs are highly proliferative and undergo robust differentiation along the osteo-, chondro- and adipogenic pathways, but do not form teratoma-like tissues in vitro. Of further significance for the translational potential of ciPSC-MSCs, we show that these cells can be encapsulated and maintained within injectable hydrogel matrices that, when functionalized with bound pentosan polysulfate, dramatically enhance chondrogenesis and inhibit osteogenesis. The ability to efficiently derive large numbers of highly proliferative canine MSCs from ciPSCs that can be incorporated into injectable, functionalized hydrogels that enhance their differentiation along a desired lineage constitutes an important milestone towards developing an effective MSC-based therapy for osteoarthritis in dogs, but equally provides a model system for assessing the efficacy and safety of analogous approaches for treating human degenerative joint diseases.

Conversion of human pancreatic acinar cells toward a ductal-mesenchymal phenotype and the role of transforming growth factor β and activin signaling.

Epithelial-mesenchymal transition may interfere with the differentiation of cultured pancreatic acinar cells toward endocrine cells. Therefore, it will be important to investigate into detail the reprogramming of human pancreatic acinar cells toward a mesenchymal phenotype: the association with acinoductal transdifferentiation, the influence of cell adhesion, and the regulation behind this process. Human exocrine cells, isolated from donor pancreata, were cultured in suspension or as monolayers. Non-genetic lineage tracing, using labeled ulex europaeus agglutinin 1 lectin, was performed, and the role of the transforming growth factor (TGF-β) superfamily was investigated. After 7 days in monolayer culture, the human acinar cells coexpressed the mesenchymal marker vimentin and the ductal marker Sox9. However, when the human exocrine cells were cultured in suspension, epithelial-mesenchymal transition was not observed. The spontaneous transition of the human acinar cells toward a ductal and mesenchymal phenotype was decreased by inhibition of the TGF-β and activin signaling pathways. The human acinar cells spontaneously undergo TGF-β- regulated reprogramming in the monolayer culture. These observations are helpful to develop culture methods for the in vitro reprogramming of pancreatic exocrine to endocrine cells. They are also of potential interest for studies on exocrine acinar cells in the development of pancreatic cancer.

PTP1B Is an Effector of Activin Signaling and Regulates Neural Specification of Embryonic Stem Cells

During embryogenesis, the Activin/Nodal pathway promotes the mesendodermal lineage and inhibits neural fate. The molecular mechanisms underlying this role of the Activin/Nodal pathway are not clear. In this study, we report a role for protein tyrosine phosphatase 1B (PTP1B) in Activin-mediated early fate decisions during ESC differentiation and show that PTP1B acts as an effector of the Activin pathway to specify mesendodermal or neural fate. We found that the Activin/ALK4 pathway directly recruits PTP1B and stimulates its release from the endoplasmic reticulum through ALK4-mediated cleavage. Subsequently, PTP1B suppresses p-ERK1/2 signaling to inhibit neural specification and promote mesendodermal commitment. These findings suggest that a noncanonical Activin signaling pathway functions in lineage specification of mouse and human embryonic stem cells.

The therapeutic potential of blocking the activin signalling pathway

Members of the transforming growth factor β (TGF-β) family regulate fundamental physiological process, such as cell growth, differentiation and apoptosis. As a result, defects in this pathway have been linked to uncontrolled proliferation and cancer progression. Here we explore the signal transduction mechanism of TGF-β focusing on therapeutic intervention in human diseases. Like TGF-β, another member of the TGF-β superfamily, activin has been proven to play an important role in maintenance of tissue homeostasis and dysregulation leads to disease. Several studies showed elevated levels of activin are responsible for the development of gonadal tumours and a cachexia-like weight loss syndrome. Discussing the recent advances in approaches developed to antagonise the activin pathway and the encouraging results obtained in animal models, this review presents a therapeutic rationale for targeting the activin pathway in conditions such as cachexia, neuromuscular and/or musculoskeletal disorders.

Characterization of TGFβ signaling during tail regeneration in the leopard Gecko (Eublepharis macularius)

The transforming growth factor beta (TGFβ)/activin signaling pathway has a number of documented roles during wound healing and is increasingly appreciated as an essential component of multi-tissue regeneration that occurs in amphibians and fish. Among amniotes (reptiles and mammals), less is known due in part to the lack of an appropriate model organism capable of multi-tissue regeneration. The leopard gecko Eublepharis macularius is able to spontaneously, and repeatedly, regenerate its tail following tail loss. We examined the expression and localization of several key components of the TGFβ/activin signaling pathway during tail regeneration of the leopard gecko. We observed a marked increase in phosphorylated Smad2 expression within the regenerate blastema indicating active TGFβ/activin signaling. Interestingly, during early regeneration, TGFβ1 expression is limited whereas activin-βA is strongly upregulated. We also observe the expression of EMT transcription factors Snail1 and Snail2 in the blastema. Combined, these observations provide strong support for the importance of different TGFβ ligands during multi-tissue regeneration and the potential role of TGFβ/activin-induced EMT programs during this process.

Research Resource: Small RNA-seq of Human Granulosa Cells Reveals miRNAs in FSHR and Aromatase Genes

The granulosa cells in the mammalian ovarian follicle respond to gonadotropin signaling and are involved in the processes of folliculogenesis and oocyte maturation. Studies on gene expression and regulation in human granulosa cells are of interest due to their potential for estimating the oocyte viability and in vitro fertilization success. However, the posttranscriptional gene expression studies on micro-RNA (miRNA) level in the human ovary have been scarce. The current study determined the miRNA profile by deep sequencing of the 2 intrafollicular somatic cell types: mural and cumulus granulosa cells (MGCs and CGCs, respectively) isolated from women undergoing controlled ovarian stimulation and in vitro fertilization. Altogether, 936 annotated and 9 novel miRNAs were identified. Ninety of the annotated miRNAs were differentially expressed between MGCs and CGCs. Bioinformatic prediction revealed that TGFβ, ErbB signaling, and heparan sulfate biosynthesis were targeted by miRNAs in both granulosa cell populations, whereas extracellular matrix remodeling, Wnt, and neurotrophin signaling pathways were enriched among miRNA targets in MGCs. Two of the nine novel miRNAs found were of intronic origin: one from the aromatase and the other from the FSH receptor gene. The latter miRNA was predicted to target the activin signaling pathway. In addition to revealing the genome-wide miRNA signature in human granulosa cells, our results suggest that posttranscriptional regulation of gene expression by miRNAs could play an important role in the modification of gonadotropin signaling. miRNA expression studies could therefore lead to new prognostic markers in assisted reproductive technologies.

Abstract 2735: Inactivation of activin signaling pathway accelerates the development of pancreatic intraductal papillary mucinous neoplasms in vivo.

Abstract The association of TGF-β pathway with human tumorigenesis has been rigorously demonstrated. However, little is known about activin signaling, part of the TGF-β family, in pancreatic tumorigenesis. We have previously reported sporadic mutations of the Activin receptor type 1B (ACVR1B) in human pancreatic ductal adenocarcinoma (PDAC). To investigate the significance of ACVR1B in pancreatic tumorigenesis, Acvr1bflox/flox; Pdx1-Cre mice were generated and examined. Chronic pancreatitis-like histological changes such as inflammatory cell infiltration, acinar to ductal metaplasia, and fibrosis were observed in Acvr1b mutant alone mice older than 8 months of age. In combination with mutant KrasG12D in the pancreas, Acvr1b deletion accelerated the development of pancreatic intraductal papillary mucious neoplasms (IPMN), but not the pancreatic intraepithelial neoplasias (PanINs). The IPMN progressed to invasive and metastatic cancer was observed in this model. The expressions of Cox-2 and phospho-PDK1 are dramatically increased in preneoplastic and neoplastic lesions, but activated Notch4 expression is exclusively observed in the IPMN lesions by immunohistochemistry, suggesting that Notch4 signaling may play a role in the formation or maintenance of the mucin-rich ductal phenotype in this model. Interestingly, the progression of IPMN to invasive cancer appears to require additional p16 loss in this double mutant mouse model. In human IPMN, loss of p16 expression is associated with increasing grade. Our data provide the first evidence that Acvr1b acts as a tumor-suppressor in vivo and that activin signaling plays a predominant role in the development of pancreatic IPMNs. Significance: PDAC can arise from PanINs or IPMN. It has been reported previously that disruption of TGF-β signaling promotes the progression of PanIN to PDAC in the TGFβRII knockout background. In contrast, here we present that activin signaling deficiency favors the development of IPMNs and enhances their progression to PDAC. This phenotype overlaps with those observed in the Smad4 knockout background, suggesting that the involvement of activin signaling in the IPMN to PDAC sequence is Smad4-dependent. Together these data demonstrate that TGF-β superfamily ligands play critical roles in regulating the IPMN-derived versus PanIN-derived carcinogenesis. Citation Format: Wanglong Qiu, Sophia Tang, Sohyae Lee, Andrew T. Turk, Anthony Sireci, Anne Qiu, Ralph H. Hruban, Helen E. Remotti, Gloria H. Su. Inactivation of activin signaling pathway accelerates the development of pancreatic intraductal papillary mucinous neoplasms in vivo. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2735. doi:10.1158/1538-7445.AM2013-2735

TGF-β Superfamily Gene Expression and Induction of the Runx1 Transcription Factor in Adult Neurogenic Regions after Brain Injury

Traumatic brain injury (TBI) increases neurogenesis in the forebrain subventricular zone (SVZ) and the hippocampal dentate gyrus (DG). Transforming growth factor-β (TGF-β) superfamily cytokines are important regulators of adult neurogenesis, but their involvement in the regulation of this process after brain injury is unclear. We subjected adult mice to controlled cortical impact (CCI) injury, and isolated RNA from the SVZ and DG at different post-injury time points. qPCR array analysis showed that cortical injury caused significant alterations in the mRNA expression of components and targets of the TGF-β, BMP, and activin signaling pathways in the SVZ and DG after injury, suggesting that these pathways could regulate post-injury neurogenesis. In both neurogenic regions, the injury also induced expression of Runt-related transcription factor-1 (Runx1), which can interact with intracellular TGF-β Smad signaling pathways. CCI injury strongly induced Runx1 expression in activated and proliferating microglial cells throughout the neurogenic regions. Runx1 protein was also expressed in a subset of Nestin- and GFAP-expressing putative neural stem or progenitor cells in the DG and SVZ after injury. In the DG only, these Runx1+ progenitors proliferated. Our data suggest potential roles for Runx1 in the processes of microglial cell activation and proliferation and in neural stem cell proliferation after TBI.

MicroRNA-181a Suppresses Mouse Granulosa Cell Proliferation by Targeting Activin Receptor IIA

Activin, a member of the transforming growth factor-β superfamily, promotes the growth of preantral follicles and the proliferation of granulosa cells. However, little is known about the role of microRNAs in activin-mediated granulosa cell proliferation. Here, we reported a dose- and time-dependent suppression of microRNA-181a (miR-181a) expression by activin A in mouse granulosa cells (mGC). Overexpression of miR-181a in mGC suppressed activin receptor IIA (acvr2a) expression by binding to its 3′-untranslated region (3′-UTR), resulting in down-regulation of cyclin D2 and proliferating cell nuclear antigen expression, leading to inhibition of the cellular proliferation, while overexpression of acvr2a attenuated the suppressive effect of miR-181a on mGC proliferation. Consistent with the inhibition of acvr2a expression, miR-181a prevented the phosphorylation of the activin intracellular signal transducer, mothers against decapentaplegic homolog 2 (Smad2), leading to the inactivation of activin signaling pathway. Interestingly, we found that miR-181a expression decreased in ovaries of mice at age of 8, 12, and 21 days, as compared with that in ovaries of 3-day old mice, and its level was reduced in preantral and antral follicles of mice compared with that in primary ones. Moreover, the level of miR-181a in the blood of patients with premature ovarian failure was significantly increased compared with that in normal females. This study identifies an interplay between miR-181a and acvr2a, and reveals an important role of miR-181a in regulating granulosa cell proliferation and ovarian follicle development.

Convergent animal and human evidence suggests the activin/inhibin pathway to be involved in antidepressant response

Despite the overt need for improved treatment modalities in depression, efforts to develop conceptually novel antidepressants have been relatively unsuccessful so far. Here we present a translational approach combining results from hypothesis-free animal experiments with data from a genetic association study in depression. Comparing genes regulated by chronic paroxetine treatment in the mouse hippocampus with genes showing nominally significant association with antidepressant treatment response in two pharmacogenetic studies, the activin pathway was the only one to show this dual pattern of association and therefore selected as a candidate. We examined the regulation of activin A and activin receptor type IA mRNA following antidepressant treatment. We investigated the effects of stereotaxic infusion of activin into the hippocampus and the amygdala in a behavioural model of depression. To analyse whether variants in genes in the activin signalling pathway predict antidepressant treatment response, we performed a human genetic association study. Significant changes in the expression of genes in the activin signalling pathway were observed following 1 and 4 weeks of treatment. Injection of activin A into the hippocampus exerts acute antidepressant-like effects. Polymorphisms in the betaglycan gene, a co-receptor mediating functional antagonism of activin signalling, significantly predict treatment outcome in our system-wide pharmacogenetics study in depression. We provide convergent evidence from mouse and human data that genes in the activin signalling pathway are promising novel candidates involved in the neurobiogical mechanisms underlying antidepressant mechanisms of action. Further, our data suggest this pathway to be a target for more rapid-acting antidepressants in the future.

Efficient derivation of bovine embryonic stem cells needs more than active core pluripotency factors

Pluripotency can be captured in vitro, providing that the culture environment meets the requirements that avoid differentiation while stimulating self-renewal. From studies in the mouse embryo, two kinds of pluripotent stem cells have been obtained from the early and late epiblast, embryonic stem cells (ESCs) and epiblast stem cells (EpiSCs), representing the naive and primed states, respectively. All attempts to derive convincing ESCs in ungulates have been unsuccessful, although all attempts were based on the assumption that the conditions used to derive mouse ESCs or human ESC could be applied in other species. Pluripotent cells derived in primates, rabbit, and pig strongly indicate that the state of pluripotency of these cells is, in fact, closer to EpiSCs than to ESCs, and thus depend on fibroblast growth factor (FGF) and Activin signaling pathways. Based on this observation, we have tried to derive EpiSC from the epiblast of bovine elongated embryos as well as ESCs from Day-8 blastocysts. We here show that the core transcription factors Oct4/Sox2/Nanog can be used as markers of pluripotency in the bovine since their expression was restricted to the developing epiblast after Day 8, and disappeared following differentiation of both the ESC-like and EpiSC-like cultures. Although FGF and Activin pathways are indeed present and active in the bovine, it is not sufficient/enough to maintain a long-term pluripotency ex vivo, as was reported for mouse and pig EpiSCs.

Growth Differentiation Factor 9 (GDF9) Suppresses Follistatin and Follistatin-Like 3 Production in Human Granulosa-Lutein Cells

BackgroundWe have demonstrated that growth differentiation factor 9 (GDF9) enhances activin A-induced inhibin β B-subunit mRNA levels in human granulosa-lutein (hGL) cells by regulating receptors and key intracellular components of the activin signaling pathway. However, we could not exclude its effects on follistatin (FST) and follistatin-like 3 (FSTL3), well recognized extracellular inhibitors of activin A.MethodologyhGL cells from women undergoing in vitro fertilization (IVF) treatment were cultured with and without siRNA transfection of FST, FSTL3 or GDF9 and then treated with GDF9, activin A, FST, FSTL3 or combinations. FST, FSTL3 and inhibin β B-subunit mRNA, and FST, FSTL3 and inhibin B protein levels were assessed with real-time RT-PCR and ELISA, respectively. Data were log transformed before ANOVA followed by Tukey's test.Principal FindingsGDF9 suppressed basal FST and FSTL3 mRNA and protein levels in a time- and dose-dependent manner and inhibited activin A-induced FST and FSTL3 mRNA and protein expression, effects attenuated by BMPR2 extracellular domain (BMPR2 ECD), a GDF9 antagonist. After GDF9 siRNA transfection, basal and activin A-induced FST and FSTL3 mRNA and protein levels increased, but changes were reversed by adding GDF9. Reduced endogenous FST or FSTL3 expression with corresponding siRNA transfection augmented activin A-induced inhibin β B-subunit mRNA levels as well as inhibin B levels (P values all <0.05). Furthermore, the enhancing effects of GDF9 in activin A-induced inhibin β B-subunit mRNA and inhibin B production were attenuated by adding FST.ConclusionGDF9 decreases basal and activin A-induced FST and FSTL3 expression, and this explains, in part, its enhancing effects on activin A-induced inhibin β B-subunit mRNA expression and inhibin B production in hGL cells.

Inhibition of FGF signaling converts dorsal mesoderm to ventral mesoderm in early Xenopus embryos

In early vertebrate development, mesoderm induction is a crucial event regulated by several factors including the activin, BMP and FGF signaling pathways. While the requirement of FGF in Nodal/activin-induced mesoderm formation has been reported, the fate of the tissue modulated by these signals is not fully understood. Here, we examined the fate of tissues when exogenous activin was added and FGF signaling was inhibited in animal cap explants of Xenopus embryos. Activin-induced dorsal mesoderm was converted to ventral mesoderm by inhibition of FGF signaling. We also found that inhibiting FGF signaling in the dorsal marginal zone, in vegetal-animal cap conjugates or in the presence of the activin signaling component Smad2, converted dorsal mesoderm to ventral mesoderm. The expression and promoter activities of a BMP responsive molecule, PV.1 and a Spemann organizer, noggin, were investigated while FGF signaling was inhibited. PV.1 expression increased, while noggin decreased. In addition, inhibiting BMP-4 signaling abolished ventral mesoderm formation induced by exogenous activin and FGF inhibition. Taken together, these results suggest that the formation of dorso-ventral mesoderm in early Xenopus embryos is regulated by a combination of FGF, activin and BMP signaling.

Alantolactone Inhibits Cell Proliferation by Interrupting the Interaction between Cripto-1 and Activin Receptor Type II A in Activin Signaling Pathway

It has been suggested that deregulation of activin signaling contributes to tumor formation. Activin signaling is blocked in cancer cells due to the complex formed by Cripto-1, activin, and activin receptor type II (ActRII). In this study, the authors used a mammalian two-hybrid system to construct a drug screening model to obtain a small molecular inhibitor capable of interrupting the interaction between Cripto-1 and ActRII. They screened 300 natural components and identified alantolactone. Data suggested that alantolactone induced activin/SMAD3 signaling in human colon adenocarcinoma HCT-8 cells. The authors also found that alantolactone exhibited antiproliferative function specific to tumor cells, with almost no toxicity to normal cells at a concentration of 5 µg/mL. Furthermore, they proved that the antiproliferative function of alantolactone was activin/SMAD3 dependent. These results suggest that alantolactone performs its antitumor effect by interrupting the interaction between Cripto-1 and the activin receptor type IIA in the activin signaling pathway. Moreover, screening for inhibitors of Cripto-1/ActRII is a potentially beneficial approach to aid in discovering novel cancer treatment.

High-Efficiency Induction of Neural Conversion in Human ESCs and Human Induced Pluripotent Stem Cells with a Single Chemical Inhibitor of Transforming Growth Factor Beta Superfamily Receptors

Chemical compounds have emerged as powerful tools for modulating ESC functions and deriving induced pluripotent stem cells (iPSCs), but documentation of compound-induced efficient directed differentiation in human ESCs (hESCs) and human iPSC (hiPSCs) is limited. By screening a collection of chemical compounds, we identified compound C (also denoted as dorsomorphin), a protein kinase inhibitor, as a potent regulator of hESC and hiPSC fate decisions. Compound C suppresses mesoderm, endoderm, and trophoectoderm differentiation and induces rapid and high-efficiency neural conversion in both hESCs and hiPSCs, 88.7% and 70.4%, respectively. Interestingly, compound C is ineffective in inducing neural conversion in mouse ESCs (mESCs). Large-scale kinase assay revealed that compound C targets at least seven transforming growth factor beta (TGF-β) superfamily receptors, including both type I and type II receptors, and thereby blocks both the Activin and bone morphogenesis protein (BMP) signaling pathways in hESCs. Dual inhibition of Activin and BMP signaling accounts for the effects of compound C on hESC differentiation and neural conversion. We also identified muscle segment homeobox gene 2 (MSX2) as a downstream target gene of compound C and a key signaling intermediate of the BMP pathway in hESCs. Our findings provide a single-step cost-effective method for efficient derivation of neural progenitor cells in adherent culture from human pluripotent stem cells. Therefore, it will be uniquely suitable for the production of neural progenitor cells in large scale and should facilitate the use of stem cells in drug screening and regenerative medicine and study of early human neural development.

Activin A and bone metastasis

Activin A, is a multifunctional cytokine of the transforming growth factor-beta superfamily of growth factors. This molecule has been shown to be implicated in the regulation of a broad range of important biological functions including bone remodelling. Therefore, a deregulation in the activin signalling pathway may result in disturbances of normal bone metabolism and, eventually, in the onset of severe pathological conditions associated with an altered bone resorption. These observations support the concept that Act A might also be implicated in the pathogenesis of bone metastasis. This review provides insight into the most recent advances in understanding the role of this growth factor in the pathogenesis of bone metastasis, and discusses the implications related to the biomedical applications of these findings.

Effects of Endogenous Growth Differentiation Factor 9 on Activin A-Induced Inhibin B Production in Human Granulosa-Lutein Cells

ABSTRACT Background We recently reported on the effects of exogenous growth differentiation factor 9 (GDF9) in enhancing activin A-induced inhibin βB-subunit mRNA and inhibin B levels in human granulosa-lutein (hGL) cells by modulating key components of the activin signaling pathway. We undertook the following study to characterize the role of endogenous GDF9 in this regard. Methods We compared inhibin subunit (α, βA, and βB) mRNA and inhibin B levels and activation of activin receptors (ACVRs) and Smad signaling pathway in hGL cells obtained from women undergoing in vitro fertilization and cultured with and without activin A treatment after GDF9-targeting small interfering RNA transfection. GDF9, inhibin subunits, ACVR2B/1B and Smad2/3/4/7 mRNA and/or protein levels, Smad phosphorylation, and inhibin B were assessed with RT-PCR, immunoblotting, and ELISA. Data were analyzed by ANOVA followed by Tukey’s test. Results GDF9 was detected as mRNA and protein in hGL cells and protein in follicular fluid from all 11 patients tested. Reduced endogenous GDF9 expression after targeting small interfering RNA transfection was associated with decreased ACVR2B/1B and Smad2/3/4 but increased inhibitory Smad7 mRNA and protein levels and, consequently, reduced activin A-induced βB-subunit mRNA and inhibin B levels. Conclusions We report here for the first time autocrine roles for endogenous GDF9 in hGL cells in enhancing activin A-induced βB-subunit mRNA and inhibin B levels via key components of the activin signaling pathway. However, the relative contributions of GDF9 in granulosa cells vs. oocyte as autocrine/paracrine regulators of βB-subunit and inhibin B production in normal and abnormal human ovarian functions remain to be determined.