Effects Of Activin Research Articles

Activin A regulates microRNAs and gene expression in LNCaP cells.

Prostate cancer (PCa) is an increasing health issue worldwide. For patients with advanced castration-resistant PCa (CRPC) treatment options are limited and overall survival is relatively short. Paired with this, non-invasive diagnostic options are yet to be established. Activins are members of the TGF-β superfamily and have been linked to prostate physiology. For instance, activin A is an inhibitor of growth in the prostate. A novel class of non-coding RNA, microRNAs (miRNAs) have been intrinsically linked to a range of cellular processes and carcinogenesis. No studies have investigated the impact of activin A on miRNA expression in PCa cell lines. Hence, the objective of this study was to determine the effect of activin A on miRNA expression and downstream target genes in PCa. Activin-sensitive (LNCaP) and insensitive (PC3) prostate cells were treated with 50 ng/ml of activin A for 72 hr. To examine miRNA expression following treatment, SYBR RT-qPCR miRNA arrays were used in conjunction with TaqMan RT-qPCR. MiRPath-TarBase analysis was conducted using the miRNAs that were significantly altered following activin A treatment of LNCaP cells to highlight enriched target genes within biological pathways. Highlighted target genes were assessed using pathway-focused TGF-β and cell cycle SYBR RT-qPCR arrays. Activin A treatment altered nine miRNAs in LNCaP cells: miR-222-3p, miR-15b-5p, miR-93-5p, miR-18a-5p, and let-7i-5p were significantly decreased, while miR-30a/30d-5p, let-7c, and miR-196b-5p were significantly increased versus media control. In PC3 cells five miRNAs were altered: miR-130a-3p, miR-7-5p, and miR-140-3p were significantly decreased while miR-191-5p and miR-26a-5p were significantly increased versus media control. MiRPath-TarBase analysis highlighted that the miRNAs significantly altered in LNCaP cells targeted genes contained in activin A-related KEGG pathways. Furthermore, when LNCaP cells were treated with activin A the expression of the targeted genes was the inverse of the expression of activin A-mediated miRNAs. This study demonstrated the ability of activin A to modulate miRNA expression in PCa cell lines and suggests a correlative relationship between miRNA expression and downstream target genes in LNCaP cells. This study provides impetus for further studies into activin A and miRNAs in PCa. Prostate 76:951-963, 2016. © 2016 Wiley Periodicals, Inc.

The effect of FSH and activin A on Akt and MAPK1/3 phosphorylation in cultured bovine ovarian cortical strips

BackgroundrhFSH and rhActA have been used in mammalian ovarian follicle culture systems for activation of follicular growth in vitro and suggested to be responsible for primordial follicle survival through MAPK and Akt pathways. The aim of our study was to determine the effects of rhFSH and rhActA on Akt, pAkt, MAPK1/3 and pMAPK1/3 protein levels in bovine ovarian cortical strips cultured in vitro.MethodsOvarian cortical strips from heifers were cultured in the presence of rhFSH (50 ng/mL), rhActA (100 ng/mL) or combination of these factors for 6 days. The strips were embedded in paraffin for histological observations and homogenized for western blot to determine Akt, pAkt, MAPK1/3 and pMAPK1/3 protein levels after the culture. Determination of primordial, primary and secondary follicle proportions at the end of culture as well as comparison of healthy follicle for each developmental stage after the culture was performed to quantify follicle survival and activation.ResultspAkt protein levels were significantly lower in rhFSH + rhActA group among the other groups, whereas pMAPK1/3 levels were not significantly changed. Follicular activation and survival was measured to be significantly lower in rhFSH + rhActA group. Percentage of healthy primordial follicles was higher in control group whereas healthy secondary follicle proportion was higher in both rhActA and rhFSH groups. rhActA alone had a better impact on follicular activation, since the percentage of the secondary follicles was significantly higher than other treatment groups.ConclusionsThe use of rhActA and rhFSH alone or in the combined form results in differential levels of Akt and MAPK proteins. Both rhActA and rhFSH alone has a remarkable contribution in survival and activation of the follicles in accordance with higher levels of these proteins. Thus, the manipulation of Akt and MAPK pathways with appropriate activators might contribute to proper activation and development of ovarian follicles in vitro.

Circulating Activin A is predictive of survival of cancer patients

Introduction and aim We demonstrated that human cancer cachexia is associated with increased circulating concentrations of Activin A. Given the cachectic and anorectic effect of Activin A demonstrated in animal models, our observation suggests that Activin A might play a role in the development of human cancer cachexia. Indeed, circulating Activin A was correlated positively with weight loss and negatively with skeletal muscle density, two well-established prognosis factors in cancer patients. Our goal was to investigate the value of circulating Activin A as a marker of survival in cancer patients. Material and methods Patients with colorectal or lung cancer were recruited at the time of diagnosis or at relapse and had clinical, nutritional (SNAQ score) and functional (ECOG, QLQC30) assessment. Body composition and skeletal muscle density were measured by CT-scan and plasma concentrations of Activin A were determined. Overall survival was estimated during 12 months (–1/ + 2 months) after inclusion. Results Among 152 patients included in the study, survival data was available for 149 patients. Patients with high levels of Activin A (> 665 pg/mL) had lower overall survival (68%) than those with levels in the normal range (84%; P Conclusion In cancer patients, a high circulating concentration of Activin A was associated with a shorter overall survival. Significant weight loss, low muscularity and low skeletal muscle density, were also associated with a poor prognosis.

Uric acid: a modulator of prostate cells and activin sensitivity.

Elevated serum uric acid (SUA) or urate is associated with inflammation and gout. Recent evidence has linked urate to cancers, but little is known about urate effects in prostate cancer. Activins are inflammatory cytokines and negative growth regulators in the prostate. A hallmark of prostate cancer progression is activin insensitivity; however, mechanisms underlying this are unclear. We propose that elevated SUA is associated with prostate cancer counteracting the growth inhibitory effects of activins. The expression of activins A and B, urate transporter GLUT9 and tissue urate levels were examined in human prostate disease. Intracellular and secreted urate and GLUT9 expression were assessed in human prostate cancer cell lines. Furthermore, the effects of urate and probenecid, a known urate transport inhibitor, were determined in combination with activin A. Activin A expression was increased in low-grade prostate cancer, whereas activin B expression was reduced in high-grade prostate cancer. Intracellular urate levels decreased in all prostate pathologies, while GLUT9 expression decreased in benign prostatic hyperplasia, prostatitis and high-grade prostate cancer. Activin responsive LNCaP cells had higher intracellular and lower secreted urate levels than activin-insensitive PC3 cells. GLUT9 expression in prostate cancer cells was progressively lower than in prostate epithelial cells. Elevated extracellular urate was growth promoting in vitro, which was abolished by the gout medication probenecid, and it antagonized the growth inhibitory effects of activins. This study shows for the first time that a change in plasma or intracellular urate levels, possibly involving GLUT9 and a urate efflux transporter, has an impact on prostate cancer cell growth, and that lowering SUA levels in prostate cancer is likely to be therapeutically beneficial.

Activin A Stimulates Aromatase via the ALK4-Smad Pathway in Endometriosis.

Endometriosis is an estrogen-dependent disease. We previously found that the expression of Activin A was upregulated in the peritoneal fluid of patients with endometriosis. The results of the present study indicated that Activin A induced estradiol secretion and P450arom expression in endometrial stromal cells (ESCs) derived from endometriosis patients. The mechanism of estrogenic synthesis was regulated by the Activin-Smad pathway in endometrial lesions. The data showed that the effect of Activin A on ESCs was partially abrogated by pretreatment with an inhibitor of ALK4 (the type I receptor, ActRIB) and Smad4-siRNA. Cumulatively, these data suggest that Activin A promotes the secretion of estradiol from ESCs by increasing the expression of P450arom via the ALK4-Smad pathway. These findings indicate the ALK4-Smad pathway may promote ectopic lesion survival and development.

A Simplified Method for Three-Dimensional (3-D) Ovarian Tissue Culture Yielding Oocytes Competent to Produce Full-Term Offspring in Mice.

In vitro growth of follicles is a promising technology to generate large quantities of competent oocytes from immature follicles and could expand the potential of assisted reproductive technologies (ART). Isolated follicle culture is currently the primary method used to develop and mature follicles in vitro. However, this procedure typically requires complicated, time-consuming procedures, as well as destruction of the normal ovarian microenvironment. Here we describe a simplified 3-D ovarian culture system that can be used to mature multilayered secondary follicles into antral follicles, generating developmentally competent oocytes in vitro. Ovaries recovered from mice at 14 days of age were cut into 8 pieces and placed onto a thick Matrigel drop (3-D culture) for 10 days of culture. As a control, ovarian pieces were cultured on a membrane filter without any Matrigel drop (Membrane culture). We also evaluated the effect of activin A treatment on follicle growth within the ovarian pieces with or without Matrigel support. Thus we tested four different culture conditions: C (Membrane/activin-), A (Membrane/activin+), M (Matrigel/activin-), and M+A (Matrigel/activin+). We found that the cultured follicles and oocytes steadily increased in size regardless of the culture condition used. However, antral cavity formation occurred only in the follicles grown in the 3-D culture system (M, M+A). Following ovarian tissue culture, full-grown GV oocytes were isolated from the larger follicles to evaluate their developmental competence by subjecting them to in vitro maturation (IVM) and in vitro fertilization (IVF). Maturation and fertilization rates were higher using oocytes grown in 3-D culture (M, M+A) than with those grown in membrane culture (C, A). In particular, activin A treatment further improved 3-D culture (M+A) success. Following IVF, two-cell embryos were transferred to recipients to generate full-term offspring. In summary, this simple and easy 3-D ovarian culture system using a Matrigel drop and activin A supplementation (M+A) provides optimal and convenient conditions to support growth of developmentally competent oocytes in vitro.

Oncogenic activin C interacts with decorin in colorectal cancer in vivo and in vitro

Activin C is a member of the transforming growth factor-β (TGF-β) superfamily with various biological activities. Decorin is a member of the small leucine-rich proteoglycan family, which can bind to TGF-β and modulate TGF-β-mediated signaling. In the decorin-deficient mouse model, we found that the expression of activin C was remarkably increased in the intestine of Dcn-/- mice compared to the expression of activin C in the intestine of Dcn+/+ mice. Addition of activin C protein to colorectal cancer cells or over-expression of activin C in these cells stimulated cell growth, migration, and invasion in vitro. Enhanced AP-1 expression in colorectal cancer cells was found to be associated with the oncoprotein-like effects of activin C through the JNK/AP-1 pathway, and not the Smad signaling pathway. However, these effects were abolished when decorin expression was restored by transfecting the cells with a decorin-expressing plasmid or by reducing the expression of activin C via interfering RNA. Further analysis demonstrated that decorin could directly bind to and accelerate the degradation of activin C. In conclusion, our data provided the first evidence demonstrating the oncogenic role of activin C in intestinal tumorigenesis of decorin-deficient mice and colorectal cancer cells. © 2015 Wiley Periodicals, Inc.

Apoptosis modulation by activin A and follistatin in human endometrial stromal cells

Activin A is a growth factor that stimulates decidualization and is abundantly expressed in endometrial proliferative disorders. Nevertheless, whether it directly affects endometrial cell survival is still unknown. This study investigated the effects of activin A on total death and apoptosis rates and on tumor necrosis factor (TNF) release by human endometrial stromal cells (HESC). We performed a controlled prospective in vitro study using primary HESC cultures obtained from healthy reproductive age women (n = 11). Cells were treated with medium alone (control) or activin A (25 ng/mL) or activin A (25 ng/mL) and its antagonist follistatin (250 ng/mL). Apoptosis and total cell death were measured by flow cytometry, while TNF concentrations in culture media were quantified by ELISA. Activin A decreased the percentage of apoptotic/dead cells from 31% to 22% (p < 0.05, paired t-test) and reduced TNF levels in culture medium by 14%, but there was no linear correlation between TNF release and apoptotic rates. Both effects of activin A were reversed by follistatin. These findings indicate that activin A promotes HESC survival, possibly by a TNF-independent pathway. This mechanism may be critical to the actions of activin A upon stromal cell growth and differentiation in physiology and disease.

Expression and role of the TGF-β family in glial cells infected with Borna disease virus

A previous study revealed that the expression of the Borna disease virus (BDV)-encoding phosphoprotein in glial cells was sufficient to induce neurobehavioral abnormalities resembling Borna disease. To evaluate the involvement of the TGF-β family in BDV-induced changes in cell responses by C6 glial cells, we examined the expression levels of the TGF-β family and effects of inhibiting the TGF-β family pathway in BDV-infected C6 (C6BV) cells. The expression of activin βA and BMP7 was markedly increased in BDV-infected cells. Expression of Smad7, a TGF-β family-inducible gene, was increased by BDV infection, and the expression was decreased by treatment with A-83-01 or LDN-193189, inhibitors of the TGF-β/activin or BMP pathway, respectively. These results suggest autocrine effects of activin A and BMP7 in C6BV cells. IGFBP-3 expression was also induced by BDV infection; it was below the detection limit in C6 cells. The expression level of IGFBP-3 was decreased by LDN-193189 in C6BV cells, suggesting that endogenous BMP activity is responsible for IGFBP-3 gene induction. Our results reveal the regulatory expression of genes related to the TGF-β family, and the role of the enhanced BMP pathway in modulating cell responses in BDV-infected glial cells.

Effects of normal and high circulating concentrations of activin A on vascular endothelial cell functions and vasoactive factor production.

Activin A, a TGFβ family member, circulates in the maternal blood at increasing concentrations throughout gestation during a healthy pregnancy. The circulating concentration of activin A is further increased in pre-eclampsia (PE), a hypertensive disorder of pregnancy that is marked by systemic maternal vascular endothelial cell dysfunction. The effect of increasing activin A concentrations on the maternal vascular endothelium is unknown. The study aim was to investigate the effect of physiological and pathological activin A concentrations observed in normotensive and PE pregnancies respectively, on vascular endothelial cell function. Immunostaining demonstrated the presence of the activin A receptor, ACVR2A, in SGHEC-7 cells used to model the vascular endothelium. SGHEC-7 cells were treated with activin A concentrations representative of concentrations throughout gestation in normotensive (0-10ng/ml) and PE (50ng/ml) pregnancies. xCELLigence functional assays revealed that normotensive activin A concentrations increased SGHEC-7 proliferation and migration, which was inhibited by PE concentrations. Additionally, fluorescence based assays showed that PE concentrations increased endothelial permeability. None of the tested activin A concentrations affected cell apoptosis. PE concentrations also resulted in an imbalance of the vasoactive factors eNOS, PTGIS and EDN1, as determined by quantitative real-time polymerase chain reaction and enzyme-linked immunosorbent assays. Compared with normotensive activin A concentrations, the higher PE activin A concentrations resulted in abnormal endothelial functions, which may contribute to the systemic maternal vascular endothelial cell dysfunction observed in the disorder.

The immunoregulatory and fibrotic roles of activin A in allergic asthma.

Activin A, a member of the TGF-β superfamily of cytokines, was originally identified as an inducer of follicle stimulating hormone release, but has since been ascribed roles in normal physiological processes, as an immunoregulatory cytokine and as a driver of fibrosis. In the last 10–15 years, it has also become abundantly clear that activin A plays an important role in the regulation of asthmatic inflammation and airway remodelling. This review provides a brief introduction to the activin A/TGF-β superfamily, focussing on the regulation of receptors and signalling pathways. We examine the contradictory evidence for generalized pro- vs. anti-inflammatory effects of activin A in inflammation, before appraising its role in asthmatic inflammation and airway remodelling specifically by evaluating data from both murine models and clinical studies. We identify key issues to be addressed, paving the way for safe exploitation of modulation of activin A function for treatment of allergic asthma and other inflammatory lung diseases.

Activin A Increases Human Trophoblast Invasion by Inducing SNAIL-Mediated MMP2 Up-Regulation Through ALK4

Activin A increases matrix metalloproteinase (MMP) 2 expression and cell invasion in human trophoblasts, but whether the expression of MMP2 is essential for the proinvasive effect of activin A has yet to be determined. Moreover, the identity of the activin receptor-like kinase (ALK; TGF-β type I receptors) and downstream transcription factors (eg, SNAIL and SLUG) mediating the effects of activin on MMP2 expression and trophoblast cell invasion remains unknown. To elucidate the role of MMP2 in activin A-induced human trophoblast cell invasion as well as the involvement of ALK4 and SNAIL. HTR8/SVneo immortalized human extravillous cytotrophoblast (EVT) cells and primary cultures of human first-trimester EVT cells were used as study models. Small interfering RNA (siRNA)-mediated knockdown approaches were used to investigate the molecular determinants of activin A-mediated functions. Levels of mRNA and protein were examined by reverse transcription-quantitative real-time PCR and Western blot, respectively. Cell invasiveness was measured by Matrigel-coated transwell assays. Treatment of HTR8/SVneo cells with activin A increased the production of SNAIL, SLUG, and MMP2 without altering that of MMP9, TIMP1, TIMP2, TWIST, RUNX2, ZEB1, or ZEB2. Similarly, activin A up-regulated the mRNA and protein levels of SNAIL and MMP2 in primary EVT cells. Knockdown of SNAIL attenuated activin A-induced MMP2 up-regulation in HTR8/SVneo and primary EVT cells. In HTR8/SVneo cells, activin A-induced production of SNAIL and MMP2 was abolished by pretreatment with the TGF-β type I receptor (ALK4/5/7) inhibitor SB431542 or siRNA targeting ALK4, SMAD2/3, or common SMAD4. Likewise, knockdown of ALK4 or SMAD4 abolished the stimulatory effects of activin A on SNAIL and MMP2 expression in primary EVT cells. Importantly, activin A-induced HTR8/SVneo and primary EVT cell invasion were attenuated by siRNA-mediated depletion of ALK4 or MMP2. Activin A induces human trophoblast cell invasion by inducing SNAIL-mediated MMP2 expression through ALK4 in a SMAD2/3-SMAD4-dependent manner.

Vascular Endothelial Growth Factor-A (VEGF-A) Mediates Activin A-Induced Human Trophoblast Endothelial-Like Tube Formation.

Remodeling of maternal spiral arteries during pregnancy requires a subpopulation of extravillous cytotrophoblasts (EVTs) to differentiate into endovascular EVTs. Activin A, which is abundantly expressed at the maternal-fetal interface, has been shown to promote trophoblast invasion, but its role in endovascular differentiation remains unknown. Vascular endothelial growth factor-A (VEGF-A) is well recognized as a key regulator in trophoblast endovascular differentiation. Whether and how activin A might regulate VEGF-A production in human trophoblasts and its relationship to endovascular differentiation have yet to be determined. In the present study, we found that activin A increased VEGF-A production in primary and immortalized (HTR8/SVneo) human EVT cells. In addition, activin A enhanced HTR8/SVneo endothelial-like tube formation, and these effects were attenuated by pretreatment with small interfering RNA targeting VEGF-A or the VEGF receptor 1/2 inhibitor SU4312. Pretreatment with the activin/TGF-β type 1 receptor (ALK4/5/7) inhibitor SB431542 abolished the stimulatory effects of activin A on phosphorylated mothers against decapentaplegic (SMAD)-2/3 phosphorylation, VEGF-A production, and endothelial-like tube formation. Moreover, small interfering RNA-mediated down-regulation of SMAD2, SMAD3, or common SMAD4 abolished the effects of activin A on VEGF-A production and endothelial-like tube formation. In conclusion, activin A may promote human trophoblast cell endothelial-like tube formation by up-regulating VEGF-A production in an SMAD2/3-SMAD4-dependent manner. These findings provide insight into the cellular and molecular events regulated by activin A during human implantation.

Activin suppresses LPS-induced Toll-like receptor, cytokine and inducible nitric oxide synthase expression in normal human melanocytes by inhibiting NF-κB and MAPK pathway activation.

Activins are dimeric growth and differentiation factors that belong to the transforming growth factor(TGF)-β superfamily of structurally related signaling proteins. In the present study, we examined the mechanisms through which activin regulates the lipopolysaccharide(LPS)-induced transcription of Toll-like receptors(TLRs), cytokines and inducible nitric oxide synthase(iNOS) in human melanocytes, as well as the involvement of nuclear factor(NF)-κB and mitogen-activated protein kinase(MAPK) signaling. Cell proliferation was analyzed by cell viability assay, mRNA expression was detected by RT-qPCR, and protein expression was measured by western blot analysis. LPS increased the mRNA expression of TLRs(TLR1-10) and cytokines [interleukin(IL)-1β, IL-6, IL-8 and TNF-α], as well as the mRNA and protein expression of iNOS. Activin decreased the LPS-induced TLR and cytokine mRNA expression, as well as the LPS-induced iNOS mRNA and protein expression. In addition, activin suppressed NF-κBp65 activation and blocked inhibitor of NF-κB(IκBα) degradation in LPS-stimulated melanocytes, and reduced LPS-induced p38MAPK and MEK/ERK activation. On the whole, our results demonstrated that activin inhibited TLR and cytokine expression in LPS-activated normal human melanocytes and suppressed LPS-induced iNOS gene expression. Moreover, the anti-inflammatory effects of activin were shown to be mediated through the suppression of NF-κB and MAPK signaling, resulting in reduced TLR and iNOS expression, and in the inhibition of inflammatory cytokine expression.

Abstract 174: Identification of activin A mediated microRNAs in a human prostate cancer cell line (LNCaP)

Abstract Prostate cancer (PCa) is an increasing worldwide health issue. Broadly, PCa is composed of two main forms, latent organ confined and aggressive metastatic. While therapeutic options for the latent form are available and effective, therapeutics for metastatic PCa are limited to androgen deprivation therapy (ADT), whose efficacy is short-lived. In the early stages, PCa cells regress upon androgen withdrawal, however, after a short period of time the cells acquire the ability to thrive in an environment of low androgens, known as androgen independence. This progression is one of the well recognised hallmarks of cancer, which also includes the acquisition of insensitivity to negative growth signals. A crucial negative growth regulator in the prostate is activin A, a member of the TGF-β superfamily. Activin A in low-grade PCa can inhibit cancer progression via promoting apoptosis and decreasing cell proliferation. However, in aggressive PCa the cells acquire insensitivity to the growth inhibitory effects of activin A, a situation resembling androgen independence. Hence, activin A insensitivity is an acquired capability in PCa, of which the underlying mechanisms are currently not well understood. Non-coding RNA, MicroRNAs (miRNA), play a crucial role in cellular processes via negatively regulating genes at the level of translation. No studies have investigated the effect of activin A on miRNA expression in PCa, creating a significant void in PCa biology. Therefore, the aim of this study was to investigate the effect of activin A on miRNA expression in LNCaP cells which are highly sensitive to activin A. Pathway-focused RT-qPCR arrays were utilized to assess miRNA expression following activin A treatment. Activin A significantly altered 9 miRNA; miR-222/15b/93/18a/let-7i were decreased while miR-30a/30d/let-7c/196b were increased compared to media control. To investigate the effect of the significantly altered miRNA on biological signaling pathways, miRPath pathway enrichment was conducted. These 9 miRNA were found to be targeting elements of pathways, including, PI3K-Akt, MAPK, cell cycle, PCa, and TGF-β. Western blots were conducted for proteins of the cell cycle pathway. Ki67 and PCNA have been shown to be dysregulated in PCa progression and are validated targets of miR-196b/let-7c and miR-30a/30d respectively. Protein levels of Ki67 and PCNA were significantly decreased upon activin A treatment, which correlated with increased levels of miR-196b/let-7c and miR-30a/30d. In conclusion, this study has identified for the first time activin A-mediated miRNAs in LNCaP cells. Like other TGF-β superfamily members where modulation of signaling has demonstrated therapeutic potential, alteration of activin A mediated miRNAs could provide a novel therapeutic for advanced prostate cancer. Citation Format: Edward Ottley, Elspeth Gold. Identification of activin A mediated microRNAs in a human prostate cancer cell line (LNCaP). [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 174. doi:10.1158/1538-7445.AM2015-174

Abstract 1438: Opposing influence of activin and TGFβ on PI3K and MEK/ERK signaling in colon cancer

Abstract Introduction: Colorectal cancer (CRC) is the second deadliest cancer in the US. The overall number of new cases and deaths has decreased, most likely due to increased screening. However, once CRC has metastasized, survival rate drops dramatically. In early stage CRC, the Transforming Growth Factor (TGF) β super family is growth suppressive; in advanced disease, high TGFβ serum levels are associated with poor prognosis. Activin and TGFβ regulate cell growth, apoptosis, and cell migration involving a poorly understood switch from growth suppressive to proinvasive actions. Understanding the switch to metastatic behavior and developing therapeutic strategies and biomarkers to target this process are clinical challenges. We recently observed opposing effects of TGFβ and activin on a downstream target, the cell cycle inhibitor p21. TGFβ induces SMAD4-dependent upregulation of p21, while activin downregulation of p21 is SMAD4-independent. Here, we dissect the unique contributions of mitogenic pathways to a TGFβ or activin-induced metastatic phenotype in colon cancer. Methods: Colon cancer cell lines (+/- SMAD4) were assessed for ligand-induced PI3K and MEK/ERK pathway activation. Protein/phospho-isoform expression and association were determined following knockdown and pharmacologic inhibition of pathway members. TGFβ and activin influence on cell migration via mitogenic pathways was assessed through use of small molecule inhibitors of signaling members. Epithelial mesenchymal transition (EMT) was assessed by altered expression of epithelial and mesenchymal markers. Pathway activation was compared in intestinal tumors from AOM/DSS treated ACVR2 knockout and ACVR2 wild type mice. Mitogenic signaling/growth factor status and p21 localization were correlated in primary colon cancers. Results: Activin, but not TGFβ, led to PI3K activation via interaction of ACVR1 and p85 independent of SMAD4, resulting in p21 downregulation. In contrast, TGFβ increased significantly p21 via MEK/ERK/SMAD4 synergy through a SMAD4-dependent mechanism. While activin induced EMT via PI3K, TGFβ induced EMT via MEK/ERK activation. In vivo, loss of ACVR2 resulted in loss of pAkt, consistent with activin-dependent PI3Ksignaling. In primary colon cancers, loss of nuclear p21 correlated with upstream activation of activin/PI3K while nuclear p21 was associated with TGFβ/MEK/ERK pathway activation. Conclusions: Activin and TGFβ diverge in their pro-migratory, SMAD4-independent signaling in colon cancer and utilize distinct mitogenic signaling to affect p21 localization. p21 localization in colon cancer may warrant further validation as a therapeutic biomarker for targeting TGFβ family receptors. Citation Format: Jessica I. Bauer, Naomi Akagi, Ozkan Ozden, Daniel R. Principe, Timothy Carroll, Seung Hyun Baik, Martina E. Spehlmann, Lars Eckmann, Paul J. Grippo, Barbara Jung. Opposing influence of activin and TGFβ on PI3K and MEK/ERK signaling in colon cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1438. doi:10.1158/1538-7445.AM2015-1438

Kdm6b and Pmepa1 as Targets of Bioelectrically and Behaviorally Induced Activin A Signaling.

The transforming growth factor-β (TGF-β) family member activin A exerts multiple neurotrophic and protective effects in the brain. Activin also modulates cognitive functions and affective behavior and is a presumed target of antidepressant therapy. Despite its important role in the injured and intact brain, the mechanisms underlying activin effects in the CNS are still largely unknown. Our goal was to identify the first target genes of activin signaling in the hippocampus in vivo. Electroconvulsive seizures, a rodent model of electroconvulsive therapy in humans, were applied to C57BL/6J mice to elicit a strong increase in activin A signaling. Chromatin immunoprecipitation experiments with hippocampal lysates subsequently revealed that binding of SMAD2/3, the intracellular effectors of activin signaling, was significantly enriched at the Pmepa1 gene, which encodes a negative feedback regulator of TGF-β signaling in cancer cells, and at the Kdm6b gene, which encodes an epigenetic regulator promoting transcriptional plasticity. Underlining the significance of these findings, activin treatment also induced PMEPA1 and KDM6B expression in human forebrain neurons generated from embryonic stem cells suggesting interspecies conservation of activin effects in mammalian neurons. Importantly, physiological stimuli such as provided by environmental enrichment proved already sufficient to engender a rapid and significant induction of activin signaling concomitant with an upregulation of Pmepa1 and Kdm6b expression. Taken together, our study identified the first target genes of activin signaling in the brain. With the induction of Kdm6b expression, activin is likely to gain impact on a presumed epigenetic regulator of activity-dependent neuronal plasticity.

Efficient endodermal induction of human adipose stem cells using various concentrations of Activin A for hepatic differentiation

Activin A, which is a signaling molecule similar to Nodal, rapidly promotes endoderm induction of both embryonic stem (ES) cells and MSCs. Protocols for hepatic induction exhibit differences in efficiency and reproducibility depending on the specific methods or sources of MSCs. We characterized the effects of Activin A concentration on induction efficiency during hepatic differentiation of MSCs. Human MSCs (hMSCs) were differentiated into a hepatic lineage via a three-step protocol. Cells were first cultured in fetal bovine serum-free MSCs conditioned medium supplemented with Activin A (20, 50, or 100 ng/mL) for 3 days followed by treatment with additional agents. RT-PCR analysis, immunocytochemistry assays, periodic acid and Schiff's solution staining, and ELISAs were performed to confirm hepatic induction of hMSCs. Expression of genes related to the primitive foregut endoderm was observed in cells treated with higher concentration of Activin A. Gene expression related to functional primitive hepatocytes was observed in the early phases of hepatic differentiation. During the early period of the differentiation protocol, greater albumin secretion was observed when cells were treated with higher concentrations of Activin A.Conclusion: Thus, Activin A concentration affects the rate of endoderm induction of hMSCs, and at higher concentrations in vitro.

Activin A inhibits BMP-signaling by binding ACVR2A and ACVR2B.

BackgroundActivins are members of the TGF-β family of ligands that have multiple biological functions in embryonic stem cells as well as in differentiated tissue. Serum levels of activin A were found to be elevated in pathological conditions such as cachexia, osteoporosis and cancer. Signaling by activin A through canonical ALK4-ACVR2 receptor complexes activates the transcription factors SMAD2 and SMAD3. Activin A has a strong affinity to type 2 receptors, a feature that they share with some of the bone morphogenetic proteins (BMPs). Activin A is also elevated in myeloma patients with advanced disease and is involved in myeloma bone disease.ResultsIn this study we investigated effects of activin A binding to receptors that are shared with BMPs using myeloma cell lines with well-characterized BMP-receptor expression and responses. Activin A antagonized BMP-6 and BMP-9, but not BMP-2 and BMP-4. Activin A was able to counteract BMPs that signal through the type 2 receptors ACVR2A and ACVR2B in combination with ALK2, but not BMPs that signal through BMPR2 in combination with ALK3 and ALK6.ConclusionsWe propose that one important way that activin A regulates cell behavior is by antagonizing BMP-ACVR2A/ACVR2B/ALK2 signaling.Electronic supplementary materialThe online version of this article (doi:10.1186/s12964-015-0104-z) contains supplementary material, which is available to authorized users.

Murine Inhibin α-Subunit Haploinsufficiency Causes Transient Abnormalities in Prepubertal Testis Development Followed by Adult Testicular Decline.

Activin production and signaling must be strictly regulated for normal testis development and function. Inhibins are potent activin inhibitors; mice lacking the inhibin-α gene (Inha-/- mice) cannot make inhibin and consequently have highly elevated activin and FSH serum concentrations and excessive activin signaling, resulting in somatic gonadal tumors and infertility. Dose-dependent effects of activin in testicular biology have been widely reported; hence, we hypothesized that male mice lacking one copy of the Inha gene would produce less inhibin and have an abnormal reproductive phenotype. To test this, we compared hormone concentrations, testis development, and sperm production in Inha+/+ and Inha+/- mice. Serum and testicular inhibin-α concentrations in adult Inha+/- mice were approximately 33% lower than wild type, whereas activin A, activin B, FSH, LH, and T were normal. Sixteen-day-old Inha+/- mice had a mixed phenotype, with tubules containing extensive germ cell depletion juxtaposed to tubules with advanced Sertoli and germ cell development. This abnormal phenotype resolved by day 28. By 8 weeks, Inha+/- testes were 11% larger than wild type and supported 44% greater daily sperm production. By 26 weeks of age, Inha+/- testes had distinct abnormalities. Although still fertile, Inha+/- mice had a 27% reduction in spermatogenic efficiency, a greater proportion of S-phase Sertoli cells and lower Leydig cell CYP11A1 expression. This study is the first to identify an intratesticular role for inhibin/inhibin-α subunit, demonstrating that a threshold level of this protein is required for normal testis development and to sustain adult somatic testicular cell function.