Activin Treatment Research Articles

Smad2 and Smad3 differentially modulate chordin transcription via direct binding on the distal elements in gastrula Xenopus embryos

Transforming growth factor (TGF)β/activin superfamily regulates diverse biological processes including germ layer specification and axis patterning in vertebrates. TGFβ/activin leads to phosphorylation of Smad2 and Smad3, followed by regulation of their target genes. Activin treatment also induces the essential organizer gene chordin (chrd). The involvement of Smad2/3 in chrd expression has been unclear as to whether Smad2/3 involvement is direct or indirect and whether any cis-acting response elements for Smad2/3 are present in the proximal or distal regions of its promoter. In the present study, we isolated the −2250 bps portion of the chrd promoter, showing that it contained Smad2/3 direct binding sites at its distal portion, separate from the proximal locations of other organizer genes, goosecoid and cerberus. The pattern of transcription activation for the promoter (−2250 bps) was indistinguishable from that of the endogenous chrd in gastrula Xenopus embryos. Reporter gene assays and site-directed mutagenesis analysis of the chrd promoter mapped two active activin/Smad response elements (ARE1 and ARE2) for Smad2 and Smad3. For a differential chrd induction, Smad2 acted on both ARE1 and ARE2, but Smad3 was only active for ARE2. Collectively, the results demonstrate that the distal region of chrd promoter contains the direct binding cis-acting elements for Smad2 and Smad3, which differentially modulate chrd transcription in gastrula Xenopus embryos.

Involvement of CHOP in activin A‑induced myeloma NS‑1 cell apoptosis.

Activin A, a multifunctional cytokine, is a member of transforming growth factor-β (TGF-β) superfamily. It is associated with a variety of pathophysiological processes, including inflammation, fibrosis, and tumorigenesis. Chronic or prolonged endoplasmic reticulum (ER) stress can lead to cells apoptosis. However, whether ER stress-related proteins, such as CHOP, GADD34 are involved in activin A-induced myeloma cell apoptosis remains unknown. In the present study, it was revealed that activin A inhibited the proliferation of myeloma cell line NS-1 cells and induced NS-1 cell apoptosis. Activin A upregulated the expression of CHOP, GADD34, caspase-3, and caspase-12. Moreover, both Smad3 and p-Smad3 levels were increased with treatment of activin A. Further studies revealed that the overexpression of activin signaling protein Smad3 in NS-1 cells increased the levels of CHOP, caspase-3, and p-Smad3. These data indicated that the CHOP protein of the ER stress pathway may be involved in activin A-induced NS-1 cell apoptosis, and also indicated the potential therapy of activin A-induced apoptosis via CHOP signaling for multiple myeloma.

H3K18ac Primes Mesendodermal Differentiation upon Nodal Signaling.

SummaryCellular responses to transforming growth factor β (TGF-β) depend on cell context. Here, we explored how TGF-β/nodal signaling crosstalks with the epigenome to promote mesendodermal differentiation. We find that expression of a group of mesendodermal genes depends on both TRIM33 and nodal signaling in embryoid bodies (EBs) but not in embryonic stem cells (ESCs). Only in EBs, TRIM33 binds these genes in the presence of expanded H3K18ac marks. Furthermore, the H3K18ac landscape at mesendodermal genes promotes TRIM33 recruitment. We reveal that HDAC1 binds to active gene promoters and interferes with TRIM33 recruitment to mesendodermal gene promoters. However, the TRIM33-interacting protein p300 deposits H3K18ac and further enhances TRIM33 recruitment. ATAC-seq data demonstrate that TRIM33 primes mesendodermal genes for activation by maintaining chromatin accessibility at their regulatory regions. Altogether, our study suggests that HDAC1 and p300 are key factors linking the epigenome through TRIM33 to the cell context-dependent nodal response during mesendodermal differentiation.

Abstract B73: Activin A and activin C have opposing effects on pathways involved in cancer progression in Ovcar3 cells

Abstract Activins are members of the transforming growth factor-β (TGFB) family that play important roles in the development, growth, and function of the ovary. These proteins consist of dimers and hetero-dimers of inhibin-βA, -βB, and -βC subunits that form activin A, B, C, AB, or AC. The few studies that have examined the effects of activin A on ovarian cancer cell growth have produced conflicting results, with some showing stimulation and others inhibition of growth in the same cell lines. Little has been published on the function of activin C. Mice lacking inhibin develop ovarian granulosa cell tumors that secrete increased amounts of activin A; they also develop cachexia. Overexpression of activin C in these mice inhibits the growth of these tumors and prevents cachexia development, possibly by inhibiting activin A signaling. We hypothesized that activin C would inhibit the effects of activin A on proliferation in epithelial ovarian cancer cell lines. The objectives of our study were a) to determine the effect of activin A, activin C, or activin A and C in combination on epithelial ovarian cancer cell proliferation and b) to identify the downstream pathways altered by activin treatment. Ovcar3 cells were treated with and without different concentrations of recombinant activin A, activin C, and activin A and C together for 96h and proliferation was measured using the CellTiter 96 MTS cell proliferation assay in replicate wells in 5 to 6 different cell passages. Following treatment for 18h, RNA was isolated from Ovcar3 cells from three separate passages and gene expression changes were measured with the NanoString PanCancer Pathway Code Set. Activin A at both 10ng/ml (P < 0.05) and 50ng/ml (P < 0.0001) inhibited proliferation in Ovcar3 cells, as did activin C at 100ng/ml (P < 0.01). The combination of activin A + C inhibited growth of the cells (P < 0.0001), but this was not significantly different to the effect of activin A alone. NanoString analysis revealed the number of genes that were significantly differentially expressed (Benjamini-Yekutieli p-value adjusted) with a log2 fold change greater than 0.5 or less than -0.5 was 34, 10, and 40 for activin A, activin C, and activin A + C treatment, respectively. NanoString directed global significance gene set analysis revealed activin A treatment upregulated the TGFB pathway along with gene pathways commonly misregulated in cancer, while the DNA repair and chromatin modulation pathways were downregulated by activin A. This suggests a potential pro-oncogenic role for activin A. In contrast, activin C downregulated the TGFB and Hedgehog pathways, upregulated chromatin modulation, and had a small positive effect on the DNA repair pathway. When Ovcar3 cells were treated with both activin A and C, pathway regulation was generally similar to that of activin A alone (with slightly higher scores), except for chromatin modulation, which was upregulated, as with activin C treatment. In conclusion, activin A and activin C have opposing effects on several pathways known to be important in the progression of cancer. We believe that activin C has potential as a therapeutic agent for the treatment of ovarian cancer, and further research to better understand the role of activin C in ovarian cancer progression is warranted. Citation Format: Karen L. Reader, David G. Mottershead, Helen D. Nicholson. Activin A and activin C have opposing effects on pathways involved in cancer progression in Ovcar3 cells. [abstract]. In: Proceedings of the AACR Conference: Addressing Critical Questions in Ovarian Cancer Research and Treatment; Oct 1-4, 2017; Pittsburgh, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(15_Suppl):Abstract nr B73.

FSTL3 Inhibition Restores Glucose Responsive Insulin Secretion in Nonfunctional Human Islets

Type 2 diabetes results from inadequate insulin production to control blood glucose. To address this need for replacing lost insulin-producing beta cells, most therapeutic development strategies are focused on enhancing functional beta cell number. Fairbanks Pharmaceuticals is pursuing therapies that enhance beta cell function and regeneration and is based on prior research in which inactivation of follistatin-like-3 (FSTL3) resulted in enlarged islets, increased beta cell number, and enhanced insulin secretion. At least some of these new beta cells arose from alpha to beta cell transdifferentiation in FSTL3 knockout mice. Fairbanks has produced a monoclonal antibody (FP-101) that can completely block FSTL3 from binding activin A and B, GDF11, and myostatin that could also disrupt pre-formed and otherwise irreversible complexes. FP-101 was also able to restore glucose-responsive insulin secretion in otherwise non-functional islets taken from HFD-fed mice. To determine if FSTL3 neutralization could be effective in humans, human islets from normal donors were first exposed to elevated glucose (25 nM) for 48-72 hours, a commonly utilized model for glucotoxicity. As expected, this caused loss of glucose response to subsequent GSIS. However, when treated for 24 hours with FP-101, GSIS was restored, indicating that neutralization of FSTL3 might be an effective strategy for restoring function to human non-functional islets. This observation agrees with previous research in which activin treatment directly restored GSIS in human islets from diabetic donors. Taken together with the potential for induction of alpha to beta cell transdifferentiation, these results suggest that neutralization of FSTL3 may have a dual benefit with acute restoration of GSIS in non-functional islets and chronic treatment leading to restoration of beta cell number through enhancement of alpha to beta cell transdifferentiation. In vivo studies will be initiated shortly. Disclosure A. Schneyer: Stock/Shareholder; Self; Fairbanks Pharmaceuticals, Inc.. Stock/Shareholder; Spouse/Partner; Fairbanks Pharmaceuticals, Inc.. Employee; Self; Fairbanks Pharmaceuticals, Inc.. M. Brown: None.

Chromatin status and transcription factor binding to gonadotropin promoters in gonadotrope cell lines

BackgroundProper expression of key reproductive hormones from gonadotrope cells of the pituitary is required for pubertal onset and reproduction. To further our understanding of the molecular events taking place during embryonic development, leading to expression of the glycoproteins luteinizing hormone (LH) and follicle-stimulating hormone (FSH), we characterized chromatin structure changes, imparted mainly by histone modifications, in model gonadotrope cell lines.MethodsWe evaluated chromatin status and gene expression profiles by chromatin immunoprecipitation assays, DNase sensitivity assay, and RNA sequencing in three developmentally staged gonadotrope cell lines, αT1–1 (progenitor, expressing Cga), αT3–1 (immature, expressing Cga and Gnrhr), and LβT2 (mature, expressing Cga, Gnrhr, Lhb, and Fshb), to assess changes in chromatin status and transcription factor access of gonadotrope-specific genes.ResultsWe found the common mRNA α-subunit of LH and FSH, called Cga, to have an open chromatin conformation in all three cell lines. In contrast, chromatin status of Gnrhr is open only in αT3–1 and LβT2 cells. Lhb begins to open in LβT2 cells and was further opened by activin treatment. Histone H3 modifications associated with active chromatin were high on Gnrhr in αT3–1 and LβT2, and Lhb in LβT2 cells, while H3 modifications associated with repressed chromatin were low on Gnrhr, Lhb, and Fshb in LβT2 cells. Finally, chromatin status correlates with the progressive access of LHX3 to Cga and Gnrhr, followed by PITX1 binding to the Lhb promoter.ConclusionOur data show the gonadotrope-specific genes Cga, Gnrhr, Lhb, and Fshb are not only controlled by developmental transcription factors, but also by epigenetic mechanisms that include the modulation of chromatin structure, and histone modifications.

NFkB is essential for activin-induced colorectal cancer migration via upregulation of PI3K-MDM2 pathway.

Colorectal cancer (CRC) remains a common and deadly cancer due to metastatic disease. Activin and TGFB (TGFβ) signaling are growth suppressive pathways that exert non-canonical pro-metastatic effects late in CRC carcinogenesis. We have recently shown that activin downregulates p21 via ubiquitination and degradation associated with enhanced cellular migration independent of SMADs. To investigate the mechanism of metastatic activin signaling, we examined activated NFkB signaling and activin ligand expression in CRC patient samples and found a strong correlation. We hypothesize that activation of the E3 ubiquitin ligase MDM2 by NFkB leads to p21 degradation in response to activin treatment. To dissect the link between activin and pro-carcinogenic NFkB signaling and downstream targets, we found that activin but not TGFB induced activation of NFkB leading to increased MDM2 ubiquitin ligase via PI3K. Further, overexpression of wild type p65 NFkB increased MDM2 expression while the NFkB inhibitors NEMO-binding domain (NBD) and Bay11-7082 blocked the activin-induced increase in MDM2. In conclusion, in colon cancer cell migration, activin utilizes NFkB to induce MDM2 activity leading to the degradation of p21 in a PI3K dependent mechanism. This provides new mechanistic knowledge linking activin and NFkB signaling in advanced colon cancer which is applicable to targeted therapeutic interventions.

Abstract A43: Activin receptor type IA in pancreatic cancer and its implications in tumor progression

Abstract Several studies have shown the importance of the Transforming Growth Factor Beta (TGFβ) pathway in pancreatic tumorigenesis and cancer progression, displaying a seemingly paradoxical role in tumor development. However, Activin, a TGFβ family member and its contributions to tumor development and progression are largely understudied in most cancers, particularly pancreatic cancer. The functions of Activin through its receptors are context and cell-type specific, as they exert different effects in various cancer types, including colon, breast and prostate. In normal pancreas, Activin A isoform levels in serum are low, while in pancreatic cancer Activin A levels positively correlate with shorter survival and metastasis. Activin, like TGFβ, signals through a complex containing Activin A type I and type II receptors. A type II homodimer comprised of Activin receptor type IIA (ACVRIIA) or IIB (ACVRIIB) binds ligand via an extracellular domain and recruits a homodimer of Activin receptor type IA (ACVRIA) or IB (ACVRIB), leading to formation of a tetrameric complex and phosphorylation of type I receptor by the type II serine/threonine kinase activity. ACVRIA and ACVRIB differ in amino acid sequence within the activation region, called the GS region, presumably giving receptor specificity and differences in regulation of canonical (SMAD-dependent) and non-canonical (SMAD-independent) downstream signaling. Human pancreatic ductal adenocarcinomas contain somatic mutations in the ACVR1B gene, and one recent study in vivo showed that pancreatic-specific loss of ACVR1B accelerates development of mutant KRAS-induced intraductal papillary mucinous neoplasms in mice. Our in silico data compiled from human pancreatic cancer patients demonstrates that there is a significant 50% loss of ACVR1B mRNA in cancer patients compared to normal tissue from the same cohort. Moreover, pancreatic cancer patients show a significant 80% upregulation of ACVR1A mRNA compared to normal. Yet, Activin effects through ACVRIA in pancreatic cancer have not yet been established. Our studies aim to characterize the effects of ACVR1A activation in pancreatic tumor development and progression. We hypothesize that the opposing effects of Activin signaling in pancreatic cancer may be due to ACVRIA activation and its subsequent pro-survival and pro-tumorigenic functions. Pancreatic cancer cell lines were analyzed for levels of ACVR1A expression, MIA PaCa-2 showed the highest level of expression followed by AsPC-1, with PANC-1 showing the lowest expression. MIA PaCa-2 and AsPC-1 cell lines showed increased migration in scratch assays compared to control following treatment with Activin A. We are currently establishing MIA PaCa-2 and AsPC-1 stable cell lines with a knockdown of ACVR1A to assess migratory and proliferative changes upon Activin treatment when ACVR1A is downregulated. Human and murine tissue immunostaining of ACVR1A showed expression of the receptor in the normal pancreas as well as tumor tissue samples. Human pancreas staining for ACVR1A was stronger in the carcinoma sections of tumor tissue samples compared to a lighter staining in normal tissue samples. Murine tissues showed staining in the neoplastic lesions of KC and EL-KRAS transgenic mice, while no strong staining was observed in the wild type mouse. We conclude that not only TGFβ exerts dual effects in regards to pro and anti-tumor promoting effects in the context of pancreatic cancer, but rather Activin signaling pathway is also important in pancreatic carcinogenesis and a novel target that can be useful for therapeutic interventions in pancreatic cancer. Determining Activin signaling through ACVR1A can be essential in deciphering the role of Activin in pancreatic tumor development and progression. Citation Format: Georgina E. Mancinelli, Jonas Staudacher, Barbara Jung, Paul J. Grippo.{Authors}. Activin receptor type IA in pancreatic cancer and its implications in tumor progression. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2016 May 12-15; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(24 Suppl):Abstract nr A43.

Activin pathway enhances colorectal cancer stem cell self-renew and tumor progression

Activin belongs to transforming growth factor (TGF)-β super family of growth and differentiation factors and activin pathway participated in broad range of cell process. Studies elaborated activin pathway maintain pluripotency in human stem cells and suggest that the function of activin/nodal signaling in self-renew would be conserved across embryonic and adult stem cells. In this study, we tried to determine the effect of activin signaling pathway in regulation of cancer stem cells as a potential target for cancer therapy in clinical trials. A population of colorectal cancer cells was selected by the treatment of activin A. This population of cell possessed stem cell character with sphere formation ability. We demonstrated activin pathway enhanced the colorectal cancer stem cells self-renew and contribute to colorectal cancer progression in vivo. Targeting activin pathway potentially provide effective strategy for colorectal cancer therapy.

Activin A secreted by human mesenchymal stem cells induces neuronal development and neurite outgrowth in an in vitro model of Alzheimer's disease: neurogenesis induced by MSCs via activin A.

Alzheimer's disease (AD) is characterized by progressive loss of memory in addition to cortical atrophy. Cortical atrophy in AD brains begins in the parietal and temporal lobes, which are near the subventricular zone (SVZ). The aim of this study was to activate the neurogenesis in the SVZ of AD brains by human mesenchymal stem cells (hMSCs). Neural stem cells (NSCs) were isolated from SVZ of 4-month-old 5XFAD mice. Co-culture of hMSCs with SVZ-derived NSCs from 5XFAD mice induced neuronal development and neurite outgrowth. To examine the inducing factor of neurogenesis, human cytokine array was performed with co-cultured media, and revealed elevated release of activin A from hMSCs. Also, we confirmed that the mRNA levels of activin A and activin receptor in the SVZ of 5XFAD mice were significantly lower than normal mice. Treatment of human recombinant activin A in SVZ-derived NSCs from 5XFAD mice induced neuronal development and neurite outgrowth. These data suggest that use of hMSCs and activin A to recover neurogenesis in future studies of cortical regeneration to treat AD.

Activin Enhances α- to β-Cell Transdifferentiation as a Source For β-Cells In Male FSTL3 Knockout Mice.

Diabetes results from inadequate β-cell number and/or function to control serum glucose concentrations so that replacement of lost β-cells could become a viable therapy for diabetes. In addition to embryonic stem cell sources for new β-cells, evidence for transdifferentiation/reprogramming of non-β-cells to functional β-cells is accumulating. In addition, de-differentiation of β-cells observed in diabetes and their subsequent conversion to α-cells raises the possibility that adult islet cell fate is malleable and controlled by local hormonal and/or environmental cues. We previously demonstrated that inactivation of the activin antagonist, follistatin-like 3 (FSTL3) resulted in β-cell expansion and improved glucose homeostasis in the absence of β-cell proliferation. We recently reported that activin directly suppressed expression of critical α-cell genes while increasing expression of β-cell genes, supporting the hypothesis that activin is one of the local hormones controlling islet cell fate and that increased activin signaling accelerates α- to β-cell transdifferentiation. We tested this hypothesis using Gluc-Cre/yellow fluorescent protein (YFP) α-cell lineage tracing technology combined with FSTL3 knockout (KO) mice to label α-cells with YFP. Flow cytometry was used to quantify unlabeled and labeled α- and β-cells. We found that Ins+/YFP+ cells were significantly increased in FSTL3 KO mice compared with wild type littermates. Labeled Ins+/YFP+ cells increased significantly with age in FSTL3 KO mice but not wild type littermates. Sorting results were substantiated by counting fluorescently labeled cells in pancreatic sections. Activin treatment of isolated islets significantly increased the number of YFP+/Ins+ cells. These results suggest that α- to β-cell transdifferentiation is influenced by activin signaling and may contribute substantially to β-cell mass.

Abstract 42: The NF-κB/MDM2 pathway is engaged in activin/ PI3 kinase induced degradation of p21 in colon cancer cells

Abstract Introduction: The signaling pathways for activin and TGFβ are frequently disrupted in colorectal cancers (CRC) and these mutations are associated with metastatic disease. Understanding the cellular consequences wrought by mutations to these signaling pathways may provide opportunities for the development of new therapeutic approaches. Our lab has previously shown opposing effects on a downstream target of TGFβ and activin, namely the cyclin-dependent kinase (CDK) inhibitor p21. TGFβ induces SMAD4-dependent upregulation of p21, while activin downregulates p21 in a SMAD4-independent mechanism via ubiquitination and degradation allowing for enhanced cell migration. However, the mode of regulation leading to p21 degradation is not completely known. We hypothesize that regulation of the E3 ubiquitin ligase MDM2 by NF-κB is involved in p21 degradation. Here we explore the role of MDM2 in the regulation of p21 in response to activin and TGFβ treatment in FET CRC cells. Methods: Colon cancer cell lines were assessed for ligand-induced PI3K/NF-κB/MDM2/p21 pathway engagement. FET cells were transfected with increasing concentrations of either wild type p65 or a dominant-negative mutant of p65 and treated with activin or TGFβ prior to immunoblot analysis for IκB-alpha, MDM2 and p21. DNA-binding activity of NF-κB was assessed by electrophoretic mobility shift assay (EMSA). The requirement for NF-κB activation was determined through use of the selective inhibitor peptide NBD (NEMO-binding domain), and PI3 kinase activity was confirmed by inhibition with LY294002. Results: Because activin induces ubiquitination of p21, we investigated the potential involvement of MDM2 and further the regulation of MDM2 by NF-κB. NF-κB has been reported to regulate MDM2 through direct binding to an NF-κB binding site in the promoter of MDM2. Activin induced activation of NF-κB as measured by increased DNA binding activity to an NF-κB consensus-binding site as well as by a reduction in IκB-alpha protein. Furthermore, we found this regulation to be dependent on activation of PI3 kinase. Overexpression of wild type p65 plasmid but not dominant negative p65 was found to increase MDM2 expression while the NF-κB specific inhibitor NBD blocked the activin-induced increase in MDM2. By contrast, TGFβ treatment did not contribute to an increase in MDM2 levels via NF-κB pathway. Conclusions: Here, we provide evidence to indicate that in colon cancer cells, activin may be working through NF-κB to promote MDM2 activity leading to the degradation of p21 in a PI3 kinase dependent mechanism. Future experiments will explore if pharmaceutical intervention in this pathway can inhibit the metastatic activity of activin as a therapeutic approach for advanced colon cancer. Citation Format: Arundhati Jana, Seung Hyua Baik, Timothy Carroll, Ozkan Ozden, Nancy L. Krett, Barbara Jung. The NF-κB/MDM2 pathway is engaged in activin/ PI3 kinase induced degradation of p21 in colon cancer cells. [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 42. doi:10.1158/1538-7445.AM2015-42

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.

Activins A and B Regulate Fate-Determining Gene Expression in Islet Cell Lines and Islet Cells From Male Mice.

TGFβ superfamily ligands, receptors, and second messengers, including activins A and B, have been identified in pancreatic islets and proposed to have important roles regulating development, proliferation, and function. We previously demonstrated that Fstl3 (an antagonist of activin activity) null mice have larger islets with β-cell hyperplasia and improved glucose tolerance and insulin sensitivity in the absence of altered β-cell proliferation. This suggested the hypothesis that increased activin signaling influences β-cell expansion by destabilizing the α-cell phenotype and promoting transdifferentiation to β-cells. We tested the first part of this hypothesis by treating α- and β-cell lines and sorted mouse islet cells with activin and related ligands. Treatment of the αTC1-6 α cell line with activins A or B suppressed critical α-cell gene expression, including Arx, glucagon, and MafB while also enhancing β-cell gene expression. In INS-1E β-cells, activin A treatment induced a significant increase in Pax4 (a fate determining β-cell gene) and insulin expression. In sorted primary islet cells, α-cell gene expression was again suppressed by activin treatment in α-cells, whereas Pax4 was enhanced in β-cells. Activin treatment in both cell lines and primary cells resulted in phosphorylated mothers against decapentaplegic-2 phosphorylation. Finally, treatment of αTC1-6 cells with activins A or B significantly inhibited proliferation. These results support the hypothesis that activin signaling destabilized the α-cell phenotype while promoting a β-cell fate. Moreover, these results support a model in which the β-cell expansion observed in Fstl3 null mice may be due, at least in part, to enhanced α- to β-cell transdifferentiation.

The role of Dickkopf-3 overexpression in esophageal adenocarcinoma

ObjectivesNinety percent of patients with esophageal adenocarcinoma ultimately die of their disease, highlighting the need for novel therapeutic targets. The goal of this study was to define the functional significance of overexpression of Dickkopf-3 (DKK3) in esophageal adenocarcinoma. MethodsDKK3 expression was analyzed by real-time polymerase chain reaction in 95 chemonaive and 21 chemoresistant esophageal adenocarcinomas. The esophageal adenocarcinoma cell line OE33 was stably transfected with DKK3 (OE33/DKK3) and evaluated using WST-1 (Roche, Basel, Switzerland), Matrigel (BD Biosciences, San Jose, Calif), endothelial tube formation, and chemosensitivity assays. Tumorigenesis was evaluated by injecting 1 × 106 OE33/DKK3 and vector cells in NOD/SCIDγ mice. ResultsDKK3 was overexpressed (>2-fold) in 75.8% (72/95) of esophageal adenocarcinomas. DKK3 protein was present at moderate to high levels in 46.8% (29/62) of esophageal adenocarcinomas on tissue microarray. Stable transfection of DKK3 significantly increased proliferation (P < .05) and Matrigel invasion (P < .001). Levels of SMAD4, a key mediator of the transforming growth factor-ß pathway, increased after activin treatment of OE33/DKK3, and siSMAD4 significantly decreased Matrigel invasion, suggesting that DKK3 acts through the transforming growth factor-β pathway. OE33/DKK3 cells increased endothelial tube formation and were significantly more resistant to 5-fluorouracil and cisplatin, and DKK3 expression was significantly higher in chemoresistant esophageal adenocarcinomas (P < .005). In NOD/SCIDγ mice, OE33/DKK3 cells resulted in tumors at all sites (8/8), whereas vector cells grew in only 1 of 8 sites. Nodal metastases were also significantly increased in patients with esophageal adenocarcinomas highly overexpressing DKK3, 28 of 32 (88%) versus 42 of 63 (68%) (P < .05). ConclusionsThese findings suggest that DKK3 may be important in mediating invasion in esophageal adenocarcinoma and could be a novel target in the treatment and prevention of metastatic disease.

Mutual effects of melatonin and activin on induction of aldosterone production by human adrenocortical cells

Melatonin has been reported to suppress adrenocorticotropin (ACTH) secretion in the anterior pituitary and cortisol production in the adrenal by different mechanisms. However, the effect of melatonin on aldosterone production has remained unknown. In this study, we investigated the role of melatonin in the regulation of aldosterone production using human adrenocortical H295R cells by focusing on the activin system expressed in the adrenal. Melatonin receptor MT1 mRNA and protein were expressed in H295R cells and the expression levels of MT1 were increased by activin treatment. Activin increased ACTH-induced, but not angiotensin II (Ang II)-induced, aldosterone production. Melatonin alone did not affect basal synthesis of either aldosterone or cortisol. However, melatonin effectively enhanced aldosterone production induced by co-treatment with ACTH and activin, although melatonin had no effect on aldosterone production induced by Ang II in combination with activin. These changes in steroidogenesis became apparent when the steroid production was evaluated by the ratio of aldosterone/cortisol. Melatonin also enhanced dibutyryl-AMP-induced aldosterone/cortisol levels in the presence of activin, suggesting a functional link to the cAMP-PKA pathway for induction of aldosterone production by melatonin and activin. In accordance with the data for steroids, ACTH-induced, but not Ang II-induced, cAMP synthesis was also amplified by co-treatment with melatonin and activin. Furthermore, the ratio of ACTH-induced mRNA level of CYP11B2 compared with that of CYP17 was amplified in the condition of treatment with both melatonin and activin. In addition, melatonin increased expression of the activin type-I receptor ALK-4 but suppressed expression of inhibitory Smads6/7, leading to the enhancement of Smad2 phosphorylation. Collectively, the results showed that melatonin facilitated aldosterone production induced by ACTH and activin via the cAMP-PKA pathway. The results also suggested that mutual enhancement of melatonin and activin receptor signaling is involved in the induction of aldosterone output by adrenocortical cells.

Abstract 3449: Overexpression of activin A and B as well as altered activation of canonical and non-canonical signaling pathways in malignant mesothelioma

Abstract Activin A and B, members of the TGF-β superfamily, are regulators of differentiation, inflammation and wound healing. Dimeric activins bind to transmembrane activin type I and type II receptors and induce signaling through activation of the canonical Smad2/3 pathway as well as through activation of MAPK pathways. Depending on the tissue type activins can promote or inhibit cellular growth and/or migration. Activin A has recently been reported to have growth promoting properties in malignant mesothelioma (MM). MM is a rare and aggressive tumor originating most commonly from pleural mesothelial cells. It is asbestos exposure related malignancy that takes decades to develop. MM tumors are highly invasive and extremely resistant to conventional cancer therapy. Novel diagnostic markers and drug targets are urgently needed. We have characterized the expression of activins, follistatins and activin receptors in mesothelioma tumors and cultured mesothelioma cells. In addition, we have identified signaling pathways induced in mesothelioma cells by activin treatment. Expression of activins and receptors in mesothelioma tumors were analyzed by immunohistochemistry. Activins, follistatins and receptors in primary mesothelioma cells and in mesothelioma cell lines were analyzed at the level of mRNA and protein expression. Activation of Smad2/3 and Smad1/5/8 signaling pathways were analyzed by luciferase reporter assays and Western blotting. Activation of MAPK/JNK and MAPK/ERK pathways were analyzed by Western blotting. Immortalized but nonmalignant Met5A cells served as a control cell line. Activin A and B were strongly expressed in mesothelioma tumor tissue. Cultured primary mesothelioma cells and mesothelioma cell lines also expressed higher levels of activin A and B compared to Met5A cells. All activin receptors (ALK3, ALK4, ALK7, ACVR2A, ACVR2B) were found expressed in cultured mesothelioma cells. ACVR2A and ALK7 were significantly overexpressed in all mesothelioma cells. Majority of the mesothelioma cell lines had attenuated Smad2/3 activation upon activin stimulation. In Met5A and H28 cells there was activation of Smad2/3 as well as MAPK/JNK pathways. On the contrary, activation of MAPK/ERK pathway was detected in cells displaying attenuated Smad2/3 activation.Our results show that activin A and B are overexpressed in MM. This together with upregulation of specific activin receptors points to a role for activins in MM progression. Furthermore, we observed that mesothelioma cells have altered responses to activin stimulation, namely attenuation of canonical Smad2/3 signaling and increase in MAPK/ERK signaling. This is likely to have an impact on mesothelioma cell behavior and tumor progression. Citation Format: Jenni A. Tamminen, Mikko Rönty, Eva Sutinen, Arja Pasternack, Olli Ritvos, Marjukka Myllärniemi, Katri Koli. Overexpression of activin A and B as well as altered activation of canonical and non-canonical signaling pathways in malignant mesothelioma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3449. doi:10.1158/1538-7445.AM2014-3449

Combined Treatment of Activin A and Heparin Binding-EGF (HB-EGF) Enhances In Vitro Production of Bovine Embryos

Combined Treatment of Activin A and Heparin Binding-EGF (HB-EGF) Enhances <i>In Vitro</i> Production of Bovine Embryos

Hanging drop cultures of human testis and testis cancer samples: a model used to investigate activin treatment effects in a preserved niche.

Background:Testicular germ cell tumours of young adults, seminoma or non-seminomas, are preceded by a pre-invasive precursor, carcinoma in situ (CIS), understood to arise through differentiation arrest of embryonic germ cells. Knowledge about the malignant transformation of germ cells is currently limited by the lack of experimental models. The aim of this study was to establish an experimental tissue culture model to maintain normal and malignant germ cells within their niche and allow investigation of treatment effects.Methods:Human testis and testis cancer specimens from orchidectomies were cultured in ‘hanging drops' and effects of activin A and follistatin treatment were investigated in seminoma cultures.Results:Testis fragments with normal spermatogenesis or CIS cells were cultured for 14 days with sustained proliferation of germ cells and CIS cells and without increased apoptosis. Seminoma cultures survived 7 days, with proliferating cells detectable during the first 5 days. Activin A treatment significantly reduced KIT transcript and protein levels in seminoma cultures, thereby demonstrating a specific treatment response.Conclusions:Hanging drop cultures of human testis and testis cancer samples can be employed to delineate mechanisms governing growth of normal, CIS and tumorigenic germ cells retained within their niche.

Activin-Directed Differentiation of Human Embryonic Stem Cells Differentially Modulates Alveolar Epithelial Wound Repair via Paracrine Mechanism

Differentiated embryonic stem cells (ESC) can ameliorate lung inflammation and fibrosis in animal lung injury models; therefore, ESC, or their products, could be candidates for regenerative therapy for incurable lung diseases, such as idiopathic pulmonary fibrosis (IPF). In this study, we have investigated the paracrine effect of differentiated and undifferentiated human ESC on alveolar epithelial cell (AEC) wound repair. hESC line, SHEF-2 cells were differentiated with Activin treatment for 22 days in an embryoid body (EB) suspension culture. Conditioned media (CM) which contain cell secretory factors were collected at different time points of differentiation. CM were then tested onin vitro wound repair model with human type II AEC line, A549 cells (AEC). Our study demonstrated that CM originated from undifferentiated hESC significantly inhibited AEC wound repair when compared to the control. Whereas, CM originated from Activin-directed hESC differentiated cell population demonstrated a differential reparative effect on AEC wound repair model. CM obtained from Day-11 of differentiation significantly enhanced AEC wound repair in comparison to CM collected from pre- and post-Day-11 of differentiation. Day-11 CM enhanced AEC wound repair through significant stimulation of cell migration and cell proliferation. RT-PCR and immunocytochemistry confirmed that Day-11 CM was originated form a mixed population of endodermal/mesodermal differentiated hESC. This report suggests a putative paracrine-mediated epithelial injury healing mechanism by hESC secreted products, which is valuable in the development of novel stem cell-based therapeutic strategies.