Activin Receptor Type IA Research Articles

Gemigliptin Inhibits Interleukin-1β-Induced Endothelial-Mesenchymal Transition via Canonical-Bone Morphogenetic Protein Pathway.

BackgroundEndothelial-to-mesenchymal transition (EndMT) contributes to inflammatory conditions inducing conversion of endothelial cells (ECs) into activated fibroblasts, promoting fibrotic diseases. Pro-inflammatory cytokine is the most potent inducer of EndMT. We investigated inhibition of interleukin-1β (IL-1β)-induced EndMT by gemigliptin, a dipeptidyl peptidase-IV inhibitor.MethodsWe exposed human umbilical vein endothelial cells (HUVECs) to 10 ng/mL IL-1β/20 μM gemigliptin and analyzed the expression of endothelial, smooth muscle, mesenchymal, and osteoblastic markers, bone morphogenetic protein (BMP), Smad, and non-Smad signaling pathway proteins.ResultsMorphological changes showed gemigliptin blocked IL-1β-induced EndMT, upregulated EC markers, and downregulated smooth muscle and mesenchymal markers. IL-1β activation of HUVECs is initiated by the BMP/Smad and non-smad BMP signaling pathways. Gemigliptin inhibited IL-1β induction of BMP2 and 7, activin receptor type IA, BMP receptor type IA, and BMP receptor type II. Reversal of IL-1β-mediated inhibition of BMP-induced Smad1/5/8, Smad2, and Smad3 phosphorylation by gemigliptin suggests involvement of the Smad pathway in gemigliptin action. In the non-Smad BMP pathway, gemigliptin treatment significantly increased the deactivation of extracellular regulated protein kinase (ERK), p38, and JNK by IL-1β. Gemigliptin treatment suppressed BMP-2-induced expression of key osteoblastic markers including osterix, runt-related transcription factor 2, and hepcidin during IL-1β-induced EndMT.ConclusionWe demonstrated a novel protective mechanism of gemigliptin against fibrosis by suppressing IL-1β-induced EndMT.

Gemigliptin Inhibits IL-1ß–Induced Endothelial-Mesenchymal Transition via BMP2/Smad/MAPK/Runx2 Pathway

Objective: Endothelial-to-mesenchymal transition (EndMT) contributes to inflammatory conditions inducing conversion of endothelial cells (ECs) into activated fibroblasts, promoting fibrotic diseases. Pro-inflammatory cytokine interleukin (IL)-1ß is the most potent inducer of EndMT. We investigated inhibition of IL-1ß-induced EndMT by gemigliptin, a DPP-4 inhibitor. Methods: We exposed human umbilical vein ECs (HUVECs) to 10 ng/mL IL-1ß/20 uM gemigliptin and analyzed the expression of endothelial, smooth muscle, mesenchymal, and osteoblastic markers, bone morphogenetic protein (BMP), Smad, and non-Smad signaling pathway proteins. Results: Morphological changes showed gemigliptin blocked IL-1ß-induced EndMT, upregulated EC markers, and downregulated smooth muscle and mesenchymal markers. IL-1ß activation of HUVECs is initiated by the BMP/Smad and canonical BMP signaling pathways. Gemigliptin inhibited IL-1ß induction of BMP2 and 7, activin receptor type IA, BMP receptor type IA, and BMP receptor II. Reversal of IL-1ß-mediated inhibition of BMP-induced Smad 1/5/8, Smad2, and Smad3 phosphorylation by gemigliptin suggests involvement of the Smad pathway in gemigliptin action. In the canonical BMP pathway, gemigliptin treatment significantly increased the deactivation of ERK, p38, and JNK by IL-1ß. Gemigliptin treatment suppressed BMP-2-induced expression of key osteoblastic markers including osterix and runt-related transcription factor 2 during IL-1ß-induced EndMT. Conclusions: We demonstrated a novel protective mechanism of gemigliptin against fibrosis by suppressing IL-1ß-induced EndMT. Disclosure H. Kwon: None. O. Hong: None. Y. Kang: None. H. Kwon: None. S. Yoo: None.

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.

ACVR1-Fc suppresses BMP signaling and chondro-osseous differentiation in an in vitro model of Fibrodysplasia ossificans progressiva

Fibrodysplasia ossificans progressiva (FOP) is a rare and devastating genetic disease of heterotopic endochondral ossification (HEO), and currently no effective therapies are available for this disease. A recurrent causative heterozygous mutation (c.617 G>A; R206H) for FOP was identified in activin receptor type IA (ACVR1), a bone morphogenetic protein (BMP) type I receptor. This mutation aberrantly activates the BMP-Smad1/5/8 signaling pathway and leads to HEO in FOP patients. Here we report development of a soluble recombinant ACVR1-Fc fusion protein by combining the extracellular domain of human wild type ACVR1 and the Fc portion of human immunoglobulin gamma 1 (IgG1). The ACVR1-Fc fusion protein significantly down-regulated the dysregulated BMP signaling caused by the FOP ACVR1 mutation and effectively suppressed chondro-osseous differentiation in a previously described cellular FOP model, human umbilical vein endothelial cells (HUVECs) that were infected with adenovirus-ACVR1R206H (HUVECR206H). This ACVR1-Fc fusion protein holds great promise for prevention and treatment of HEO in FOP and related diseases.

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.

T.O.2 Allele-specific silencing against the ALK2 mutants, R206H and G356D, in fibrodysplasia ossificans progressiva (FOP)

Abstract Fibrodysplasia ossificans progressiva (FOP) is an autosomal dominant congenital disorder characterized by postnatal progressive heterotopic ossification in soft tissues, especially skeletal muscle. In FOP, acute heterotopic ossification is induced by muscle injury, such as accidental trauma or surgical operations. Currently, no definitive treatment exists for FOP. The activin receptor type IA/ activin-like kinase 2 ( ACVR1/ALK2 ) gene has been identified as the responsible gene for both familial and sporadic cases of FOP, and disease-associated ALK2 mutations have been found. ALK2 protein, which is one of the signaling receptors for bone morphogenetic proteins (BMPs) and which induce heterotopic bone formation in skeletal muscle in vivo and initiate the differentiation pathway through which myoblasts convert to osteoblastic cells in vitro . Chemical inhibitors such as dorsomorphin to the pathogenic ALK2 receptors are considered possible medical agents for FOP, but their adverse effects on normal ALK2 and other receptors cannot be excluded. Here we describe another treatment strategy for FOP using allele-specific RNA interference (ASP-RNAi), and show modified small interfering RNAs (siRNAs) conferring allele-specific silencing against disease-causing ALK2 mutants (R206H and G356D) found in FOP, without affecting normal ALK2 allele. Thus, the siRNAs presented here may become novel therapeutic agents for FOP, and their induced ASP-RNAi may pave the way for the achievement of radical treatment of FOP and/or for the relief of its severe symptoms. Takahashi M et al. PNAS 2010;107(50):21731–6; Takahashi M et al. Gene Therapy 2011, in press.

Genetic abnormalities in Fibrodysplasia Ossificans Progressiva

Fibrodysplasia ossificans progressiva (FOP), characterized by congenital malformation of bones, is an autosomal dominant disorder. This is a rare genetic disorder and its worldwide prevalence is approximately 1/2,000,000. There is no ethnic, racial, gender, or geographic predilection to FOP. It is regarded as one of the intractable disorders, which is not only an extremely disabling disease but also a condition of considerably shortened lifespan. Although the genetic defects of FOP are not completely known, several clinical and animal model studies have implicated that mutations in bone morphogenetic proteins, their receptors, and activin receptor type IA (ACVR1) genes are associated with FOP primarily. The noggin (NOG) gene has also been reported in some studies. In most of the cases of FOP, the mutation was found as 'de novo' however there is paternal age effect on mutations. Unfortunately, at present there is no efficient treatment for FOP. The recent discoveries of genetic basis of FOP provide a clue to the underlying pathophysiology and potential therapy. This review article focuses on the genetic mutations in FOP, their usage as diagnostic markers, and possible target specific drug development to treat FOP patients.

Disease-causing allele-specific silencing against the ALK2 mutants, R206H and G356D, in fibrodysplasia ossificans progressiva

Fibrodysplasia ossificans progressiva (FOP) is an autosomal dominant congenital disorder characterized by progressive heterotopic bone formation. Currently, no definitive treatment exists for FOP. The activin receptor type IA / activin-like kinase 2 (ACVR1/ALK2) gene has been identified as the responsible gene for FOP, and disease-associated ALK2 mutations have been found. Chemical inhibitors to the pathogenic ALK2 receptors are considered possible medical agents for FOP, but their adverse effects on normal ALK2 and other receptors cannot be excluded. Here we describe another treatment strategy for FOP using allele-specific RNA interference (ASP-RNAi), and show modified small interfering RNAs (siRNAs) conferring allele-specific silencing against disease-causing ALK2 mutants found in FOP, without affecting normal ALK2 allele. Thus, the siRNAs presented here may become novel therapeutic agents for FOP, and their induced ASP-RNAi may pave the way for the achievement of radical treatment of FOP and/or for the relief of its severe symptoms.

Aberrantly increased mRNA expression of betaglycan, an inhibin co‐receptor in the ovarian tissues in women with polycystic ovary syndrome

To compare the gene expressional levels of receptors for activins and inhibins in ovarian tissues between women with polycystic ovary syndrome (PCOS) and normal controls, and to analyze their biologically relevant associations with serum hormone levels. Total RNA of ovarian tissues from PCOS (n = 14) and normal controls (n = 21) were isolated during the follicular phase of the menstrual cycle. Real-time PCRs were performed to examine the relative mRNA expression levels of the activin receptors, including activin receptor type IA (ActRIA), type IB (ActRIB), type IIA (ActRIIA), and type IIB (ActRIIB), and the inhibin receptors, betaglycan and an inhibin binding protein (InhBP/p120). At the same time, the serum levels of estradiol (E(2)), testosterone (T), follicle-stimulating hormone (FSH), luteinizing hormone (LH), inhibin B and activin A were measured. The PCOS patients showed endocrine characteristics with higher concentrations of LH, T and inhibin B, and a lower concentration of activin A. Real-time PCR demonstrated that the relative expression level of betaglycan against that of GAPDH was increased 1.5-fold in the ovarian tissues during the follicular phase of PCOS patients when compared with normal controls, while the activin receptors ActRIA, ActRIB, ActRIIA, ActRIIB, and the inhibin co-receptor InhBP/p120 were unchanged. Moreover, the betaglycan mRNA expression showed biologically relevant associations with serum FSH, LH, E(2) and inhibin B levels in both the PCOS and normal controls. This is the first report to demonstrate the aberrantly increased expression of betaglycan mRNA in PCOS ovaries. The mechanism by which betaglycan contributes to the pathologic process of PCOS remains to be clarified.

The ACVR1 617G>A mutation is also recurrent in three Japanese patients with fibrodysplasia ossificans progressiva

Fibrodysplasia ossificans progressiva (FOP) is a rare autosomal dominant disorder of skeletal malformations and presents progressive extra-skeletal ossification. The 617G>A (R206H) mutation in the activin receptor type IA (ACVR1) gene has been identified in all examined individuals with FOP of various ethnic groups, including Caucasian and Chinese descents. Here, we examined three Japanese patients with FOP for ACVR1 mutations. We identified the 617G>A mutation in all three patients. Our results suggest that the mutation in the ACVR1 gene is common and recurrent in the global population.

The autocrine effect of activin A on human ovarian clear cell adenocarcinoma cells

The functions of activin, a member of TGF-beta superfamily, in ovarian clear cell adenocarcinoma remain unsolved, although we recently found that inhibin betaA-subunit, activin A, activin receptor type IA, type IB, type IIA, type IIB, Smad2, Smad3 and Smad4 were localized in tumor cells of the ovarian clear cell adenocarcinoma tissue by immunohistochemistry. In the present study, in order to investigate the role of activin concerning cell growth in ovarian clear cell adenocarcinoma cells, we determined the production of activin A and inhibin A, and the expression of activin receptors and Smads using the human ovarian clear cell adenocarcinoma cell line JHOC-5. Moreover, we examined the effects of activin A on the activation of activin signaling pathway and on the proliferation in JHOC-5 cells. We detected a measurable amount of activin A in the culture medium of JHOC-5 cells, although inhibin A was not detected. The expression of activin receptor type IA, IB, IIA, IIB, Smad2, Smad3 and Smad4 was observed in JHOC-5 cells. Activin A induced a significant increase in proliferation of JHOC-5 cells compared with the untreated control. On the other hand, activin A did not affect the growth of JHOC-5 cells and no statistically significant difference was observed in the presence of follistatin which is a specific binding protein of activin. Phosphorylated Smad2, an activated form of Smad2, was detected both in treated JHOC-5 cells and in untreated cells by activin A. Activin A significantly increased the expression of phosphorylated Smad2 in JHOC-5 cells. Therefore, it is possible that activin has autocrine roles in tumor growth of ovarian clear cell adenocarcinoma cells.

The activin-follistatin system in the neonatal ovine uterus.

Uterine gland development or adenogenesis in the neonatal ovine uterus involves budding and tubulogenesis followed by coiling and branching morphogenesis of the glandular epithelium (GE) from the luminal epithelium (LE) between birth (Postnatal Day [PND] 0) and PND 56. Activins, which are members of the transforming growth factor beta superfamily, and follistatin, an inhibitor of activins, regulate epithelial branching morphogenesis in other organs. The objective of the present study was to determine effects of postnatal age on expression of follistatin, inhibin alpha subunit, betaA subunit, betaB subunit, activin receptor (ActR) type IA, ActRIB, and ActRII in the developing ovine uterus. Ewes were ovariohysterectomized on PND 0, 7, 14, 21, 28, 35, 42, 49, or 56. The uterus was analyzed by in situ hybridization and immunohistochemistry. Neither inhibin alpha subunit mRNA or protein was detected in the neonatal uterus. Expression of betaA and betaB subunits was detected predominantly in the endometrial LE and GE and myometrium between PND 0 and PND 56. In all uterine cell types, ActRIA, ActRIB, and ActRII were expressed, with the highest levels observed in the endometrial LE and GE and myometrium. Between PND 0 and PND 14, follistatin was detected in all uterine cell types. However, between PND 21 and PND 56, follistatin was only detected in the stroma and myometrium and not in the developing GE. Collectively, the present results indicate that components of the activin-follistatin system are expressed in the developing neonatal ovine uterus and are potential regulators of endometrial gland morphogenesis.

Possible role of hepatocyte growth factor/scatter factor and activin A produced by the target organ in liver metastasis

The molecular mechanism of organ-specific metastasis to the liver remains largely unknown. However, it is conceivable that paracrine growth factors produced by a target organ induce migration and proliferation of malignant cells to that organ, and this is the cause of organ-specific metastasis. In this study, we investigated the effect of hepatocyte growth factor/scatter factor (HGF/SF) and activin A, which are known to be produced by the liver, on the motility and growth of liver-metastatic cell line FBJ-LL. HGF/SF and activin A induced motility synergistically, but they did not affect the proliferation of FBJ-LL cells. Expression of the HGF/SF receptor, the c-met gene, and the activin-receptor type IA, type IB, and type IIA genes in FBJ-LL cells was detected by reverse transcription polymerase chain reaction. These findings suggest that both HGF/SF and activin A promote organ-specific metastasis to the liver by induction of migration through their specific receptors on liver-metastatic cells.

Expression of serine/threonine kinase receptors in traumatic brain injury.

The aim of this study was to investigate the expression of serine/threonine kinase receptors in the brain following traumatic brain injury. We report here that, the recently cloned and characterized bone morphogenetic protein (BMP) receptor type II (BMPR-II) and the activin receptor type IA (ActR-1) Act: mRNAs were simultaneously up-regulated in neurones in the dentate gyrus 6 h after a mild cerebral contusion injury. This findings was specific for these receptors since other investigated genes (i.e. ActR-II, ActR-IB, trkB and c-fos) showed other temporal patterns. These data suggest that type I and type II receptors act together in signal transduction in vivo and that BMPs may be involved in neuronal plasticity after traumatic brain injury.