Human INHBB Gene Variant (c.1079T>C:p.Met360Thr) Alters Testis Germ Cell Content, but Does Not Impact Fertility in Mice.

Testicular-derived inhibin B (α/ß B dimers) acts in an endocrine manner to suppress pituitary production of follicle-stimulating hormone (FSH), by blocking the actions of activins (ß A/B/ß A/B dimers). Previously, we identified a homozygous genetic variant (c.1079T>C:p.Met360Thr) arising from uniparental disomy of chromosome 2 in the INHBB gene (ß B-subunit of inhibin B and activin B) in a man suffering from infertility (azoospermia). In this study, we aimed to test the causality of the p.Met360Thr variant in INHBB and testis function. Here, we used CRISPR/Cas9 technology to generate InhbbM364T/M364T mice, where mouse INHBB p.Met364 corresponds with human p.Met360. Surprisingly, we found that the testes of male InhbbM364T/M364T mutant mice were significantly larger compared with those of aged-matched wildtype littermates at 12 and 24 weeks of age. This was attributed to a significant increase in Sertoli cell and round spermatid number and, consequently, seminiferous tubule area in InhbbM364T/M364T males compared to wildtype males. Despite this testis phenotype, male InhbbM364T/M364T mutant mice retained normal fertility. Serum hormone analyses, however, indicated that the InhbbM364T variant resulted in reduced circulating levels of activin B but did not affect FSH production. We also examined the effect of this p.Met360Thr and an additional INHBB variant (c.314C>T: p.Thr105Met) found in another infertile man on inhibin B and activin B in vitro biosynthesis. We found that both INHBB variants resulted in a significant disruption to activin B in vitro biosynthesis. Together, this analysis supports that INHBB variants that limit activin B production have consequences for testis composition in males.

Oncogene-Induced Senescence Limits the Progression of Pancreatic Neoplasia through Production of Activin A.

Pancreatic ductal adenocarcinoma (PDAC) is a deadly and aggressive cancer. Understanding mechanisms that drive preneoplastic pancreatic lesions is necessary to improve early diagnostic and therapeutic strategies. Mutations and inactivation of activin-like kinase (ALK4) have been demonstrated to favor PDAC onset. Surprisingly, little is known regarding the ligands that drive ALK4 signaling in pancreatic cancer or how this signaling pathway limits the initiation of neoplastic lesions. In this study, data mining and histologic analyses performed on human and mouse tumor tissues revealed that activin A is the major ALK4 ligand that drives PDAC initiation. Activin A, which is absent in normal acinar cells, was strongly induced during acinar-to-ductal metaplasia (ADM), which was promoted by pancreatitis or the activation of KrasG12D in mice. Activin A expression during ADM was associated with the cellular senescence program that is induced in precursor lesions. Blocking activin A signaling through the use of a soluble form of activin receptor IIB (sActRIIB-Fc) and ALK4 knockout in mice expressing KrasG12D resulted in reduced senescence associated with decreased expression of p21, reduced phosphorylation of H2A histone family member X (H2AX), and increased proliferation. Thus, this study indicates that activin A acts as a protective senescence-associated secretory phenotype factor produced by Kras-induced senescent cells during ADM, which limits the expansion and proliferation of pancreatic neoplastic lesions. SIGNIFICANCE: This study identifies activin A to be a beneficial, senescence-secreted factor induced in pancreatic preneoplastic lesions, which limits their proliferation and ultimately slows progression into pancreatic cancers.

The activin-follistatin anti-inflammatory cycle is deregulated in synovial fibroblasts.

BACKGROUND: Activin A and follistatin exhibit immunomodulatory functions, thus affecting autoinflammatory processes as found in rheumatoid arthritis (RA). The impact of both proteins on the behavior of synovial fibroblasts (SF) in RA as well as in osteoarthritis (OA) is unknown. METHODS: Immunohistochemical analyses of synovial tissue for expression of activin A and follistatin were performed. The influence of RASF overexpressing activin A on cartilage invasion in a SCID mouse model was examined. RASF and OASF were stimulated with either IL-1ß or TNFα in combination with or solely with activin A, activin AB, or follistatin. Protein secretion was measured by ELISA and mRNA expression by RT-PCR. Smad signaling was confirmed by western blot. RESULTS: In human RA synovial tissue, the number of activin A-positive cells as well as its extracellular presence was higher than in the OA synovium. Single cells within the tissue expressed follistatin in RA and OA synovial tissue. In the SCID mouse model, activin A overexpression reduced RASF invasion. In human RASF, activin A was induced by IL-1ß and TNFα. Activin A slightly increased IL-6 release by unstimulated RASF, but decreased protein and mRNA levels of follistatin. CONCLUSION: The observed decrease of cartilage invasion by RASF overexpressing activin A in the SCID mouse model appears to be mediated by an interaction between activin/follistatin and other local cells indirectly affecting RASF because activin A displayed certain pro-inflammatory effects on RASF. Activin A even inhibits production and release of follistatin in RASF and therefore prevents itself from being blocked by its inhibitory binding protein follistatin in the local inflammatory joint environment.

FlexiBAC: a versatile, open-source baculovirus vector system for protein expression, secretion, and proteolytic processing.

BACKGROUND: Baculovirus-mediated expression in insect cells is a powerful approach for protein production. However, many existing methods are time-consuming, offer limited options for protein tagging, and are unsuitable for secreted proteins requiring proteolytic maturation, such as TGF-ß family growth factors. RESULTS: To overcome the limitations of traditional baculovirus expression systems, we engineered "FlexiBAC". This system allows recombinant baculovirus formation inside insect cells and reduces the time between initial cloning and protein production to 13 days. FlexiBAC includes 143 shuttle vectors that append combinations of purification tags, fluorescent markers, proteolytic cleavage sites, trafficking signals, and chemical conjugation tags to the termini of the target protein. This system also overexpresses recombinant furin convertase to allow efficient proteolytic processing of secreted proteins. We demonstrate that FlexiBAC can be used to produce high levels of mature, active forms of TGF-ß family growth factors, such as Activin A, as well as other proteins that are typically difficult to reconstitute, such as proteins rich in coiled-coil, low complexity, and disordered domains. CONCLUSIONS: FlexiBAC is a protein expression system for production of both cytosolic proteins and secreted proteins that require proteolytic maturation. The design of FlexiBAC and its expansive complementary shuttle vector system reduces cloning steps and simplifies baculovirus production.

Human Epicardial Adipose Tissue Activin A Expression Predicts Occurrence of Postoperative Atrial Fibrillation in Patients Receiving Cardiac Surgery.

BACKGROUND: Activin A secreted by epicardial adipose tissue (EAT) plays a major role in the progress of atrial fibrosis. However, the potential of Activin A in predicting the occurrence of postoperative atrial fibrillation (POAF) has yet to be elucidated. We aimed to investigate the predicable value of Activin A expression in EAT on POAF. METHODS: A total of 89 patients receiving cardiac surgery without atrial fibrillation (AF) history were enrolled in this study, including 49 patients with valvular heart disease (VHD) and 40 patients with non-valvular heart disease (NVHD). Activin A expression in EAT was determined by quantitative polymerase chain reaction (qPCR), while the thickness of EAT (EATT) was estimated by echocardiography. New onset POAF before discharge was documented. RESULTS: Eventually 32 patients (36.0%) developed POAF, including 20 patients with VHD (40.8%) and 12 patients with NVHD (30.0%). Activin A expression was higher in POAF than sinus rhythm (SR) patients, whether for VHD or NVHD group (All p<0.001). In general, Activin A expression predicted the occurrence of POAF with a sensitivity of 65.6% and specificity of 91.2% (AUC: 0.795; 95%CI: 0.693-0.897, p<0.001). Subgroup analysis showed that EATT was not significant for the VHD group in predicting POAF (p=0.07), while Activin A expression demonstrated a sensitivity of 60.0% and specificity of 89.7% (AUC: 0.745; 95%CI: 0.601-0.889, p<0.001). Multivariate regression analysis showed that Activin A expression in EAT was an independent risk factor for POAF (OR: OR=1.067, 95%CI:1.002-1.136, p=0.042). CONCLUSIONS: Activin A expression in EAT is an independent risk factor for POAF, which can be used for prediction of POAF, especially for patients with VHD.

Downstream mRNA Target Analysis in Neonatal Hypoxic-Ischaemic Encephalopathy Identifies Novel Marker of Severe Injury: a Proof of Concept Paper.

Human microRNA miR-374a is downregulated in the umbilical cord blood (UCB) of infants with hypoxic-ischaemic encephalopathy (HIE). The downstream targets of this microRNA (miRNA) are unclear, but one putative target is the activin-A receptor type IIb (ACVR2B). ACVR2B is required for activin-A function and previous reports have shown alterations of activin-A levels in neonatal HIE. Our aim was to investigate the expression of the potential downstream targets of miR-374a, activin-A and ACVR2B, at birth in a cohort of full-term infants with perinatal asphyxia (PA) only, and those with PA who developed clinical and electrographic HIE. UCB was drawn and processed immediately after delivery. Levels of serum activin-A were measured using ELISA. mRNA levels of ACVR2B in whole blood were quantified using qRT-PCR. Outcome was assessed at 3 years of age using standardised developmental assessment. In total, 171 infants were enrolled: 88 healthy controls, 56 PA and 27 HIE. A statistically significant elevation of median (IQR) ACVR2B was detected in infants with severe HIE compared to moderate/mild HIE, PA and control groups (3.3 (2.94-3.67) vs. 0.91 (0.55-1.21) vs. 0.88 (0.57-1.38) vs. 0.84 (0.74-1.24), p values = 0.04, 0.027 and 0.025, respectively). Although serum activin-A levels were elevated in infants with severe HIE, this elevation did not reach significance. ACVR2B may be a potential novel marker of HIE severity. This is the first study to examine the relationship between activin-A, its receptor AVCR2B and potentially upstream miRNA miR-374a in a cohort of carefully categorised and phenotyped infants. We have shown that miRNA analysis, combined with downstream target exploration, may yield novel biomarkers for the prediction of HIE severity.

Atypical Activin A and IL-10 Production Impairs Human CD16+ Monocyte Differentiation into Anti-Inflammatory Macrophages.

Human CD14(++)CD16(-) and CD14(+/lo)CD16(+) monocyte subsets comprise 85 and 15% of blood monocytes, respectively, and are thought to represent distinct stages in the monocyte differentiation pathway. However, the differentiation fates of both monocyte subsets along the macrophage (Mϕ) lineage have not yet been elucidated. We have now evaluated the potential of CD14(++) CD16(-) and CD16(+) monocytes to differentiate and to be primed toward pro- or anti-inflammatory Mϕs upon culture with GM-CSF or M-CSF, respectively (subsequently referred to as GM14, M14, GM16, or M16). Whereas GM16 and GM14 were phenotypic and functionally analogous, M16 displayed a more proinflammatory profile than did M14. Transcriptomic analyses evidenced that genes associated with M-CSF-driven Mϕ differentiation (including FOLR2, IL10, IGF1, and SERPINB2) are underrepresented in M16 with respect to M14. The preferential proinflammatory skewing of M16 relative to M14 was found to be mediated by the secretion of activin A and the low levels of IL-10 produced by M16. In fact, activin A receptor blockade during the M-CSF-driven differentiation of CD16(+) monocytes, or addition of IL-10-containing M14-conditioned medium, significantly enhanced their expression of anti-inflammatory-associated molecules while impairing their acquisition of proinflammatory-related markers. Thus, we propose that M-CSF drives CD14(++)CD16- monocyte differentiation into bona fide anti-inflammatory Mϕs in a self-autonomous manner, whereas M-CSF-treated CD16(+) monocytes generate Mϕs with a skewed proinflammatory profile by virtue of their high activin A expression unless additional anti-inflammatory stimuli such as IL-10 are provided.

Low miR-143/miR-145 Cluster Levels Induce Activin A Overexpression in Oral Squamous Cell Carcinomas, Which Contributes to Poor Prognosis.

Deregulated expression of activin A is reported in several tumors, but its biological functions in oral squamous cell carcinoma (OSCC) are unknown. Here, we investigate whether activin A can play a causal role in OSCCs. Activin A expression was assessed by qPCR and immunohistochemistry in OSCC tissues. Low activin A-expressing cells were treated with recombinant activin A and assessed for apoptosis, proliferation, adhesion, migration, invasion and epithelial-mesenchymal transition (EMT). Those phenotypes were also evaluated in high activin A-expressing cells treated with follistatin (an activin A antagonist) or stably expressing shRNA targeting activin A. Transfections of microRNA mimics were performed to determine whether the overexpression of activin A is regulated by miR-143/miR-145 cluster. Activin A was overexpressed in OSCCs in comparison with normal oral mucosa, and high activin A levels were significantly associated with lymph node metastasis, tumor differentiation and poor survival. High activin A levels promoted multiple properties associated with malignant transformation, including decreased apoptosis and increased proliferation, migration, invasion and EMT. Both miR-143 and miR-145 were markedly downregulated in OSCC cell lines and in clinical specimens, and inversely correlated to activin A levels. Forced expression of miR-143 and miR-145 in OSCC cells significantly decreased the expression of activin A. Overexpression of activin A in OSCCs, which is controlled by downregulation of miR-143/miR-145 cluster, regulates apoptosis, proliferation and invasiveness, and it is clinically correlated with lymph node metastasis and poor survival.

Adipose Stromal Cell Contact with Endothelial Cells Results in Loss of Complementary Vasculogenic Activity Mediated by Induction of Activin A.

Adipose stem/stromal cells (ASCs) after isolation produce numerous angiogenic growth factors. This justifies their use to promote angiogenesis per transplantation. In parallel, local coimplantation of ASC with endothelial cells (ECs) leading to formation of functional vessels by the donor cells suggests the existence of a mechanism responsible for fine-tuning ASC paracrine activity essential for vasculogenesis. As expected, conditioned media (CM) from ASC promoted ECs survival, proliferation, migration, and vasculogenesis. In contrast, media from EC-ASC cocultures had neutral effects upon EC responses. Media from cocultures exhibited lower levels of vascular endothelial growth factor (VEGF), hepatic growth factor, angiopoietin-1, and stromal cell-derived factor-1 compared with those in ASC CM. Activin A was induced in ASC in response to EC exposure and was responsible for overall antivasculogenic activity of EC-ASC CM. Except for VEGF, activin A diminished secretion of all tested factors by ASC. Activin A mediated induction of VEGF expression in ASC, but also upregulated expression of VEGF scavenger receptor FLT-1 in EC in EC-ASC cocultures. Blocking the FLT-1 expression in EC led to an increase in VEGF concentration in CM. In vitro pre-exposure of ASC to low number of EC before subcutaneous coimplantation with EC resulted in decrease in vessel density in the implants. In vitro tests suggested that activin A was partially responsible for this diminished ASC activity. This study shows that neovessel formation is associated with induction of activin A expression in ASC; this factor, by affecting the bioactivity of both ASC and EC, directs the crosstalk between these complementary cell types to establish stable vessels.

Elimination of BMP7 from the developing limb mesenchyme leads to articular cartilage degeneration and synovial inflammation with increased age.

While osteo- and chondro-inductive activities of recombinant human bone morphogenetic protein 7 are well established, evaluation of the role of endogenous BMP7 in skeletal homeostasis has been hampered by perinatal lethality in BMP7 knockout mice. Here, we examined physiological roles of endogenous BMP7 in joint homeostasis and showed that proteoglycan contents in articular cartilage were significantly reduced in the absence of BMP7. Loss of BMP7 did not affect survival of articular cartilage cells, but resulted in reduced expression of aggrecan and enhanced expression of matrix metalloproteinase 13. We also found extensive synovial hyperplasia and enhanced expression of Activin A. These findings suggest that locally produced BMP7 is prerequisite for postnatal synovial joint homeostasis and may be involved in osteoarthritic changes in adults.

Upregulation of alveolar levels of activin B, but not activin A, in lungs of west highland white terriers with idiopathic pulmonary fibrosis and diffuse alveolar damage.

Activins, cytokines belonging to the transforming growth factor-ß superfamily, have an important role in inflammation and fibrosis. Activin A has been suggested to participate in the pathophysiology of human idiopathic pulmonary fibrosis (IPF), but studies on the role of activin B are sparse. Canine IPF (CIPF) is an incurable interstitial lung disease occurring particularly in West Highland white terriers (WHWTs). During the disease course there are acute exacerbations (AEs) and the condition has a poor prognosis. Microscopically, AEs of CIPF are characterized by diffuse alveolar damage, which is also a key feature of acute respiratory distress syndrome (ARDS). The aim of this study was to study expression of activin A and B in lung tissue of WHWTs with CIPF and WHWTs with CIPF and concurrent AE, and dogs of various breeds with ARDS and to compare these findings with those of healthy WHWTs. In addition, western blot analysis of activin B from bronchoalveolar lavage fluid (BALF) from WHWTs with CIPF and healthy WHWTs was conducted. Activin B, but not activin A, was strongly expressed in the altered alveolar epithelium in the lungs of WHWTs with CIPF as well as in the lungs of dogs with ARDS. Activin B was detected in the BALF of WHWTs with CIPF, most notably in samples from dogs with AE, but activin B was not detected in BALF from healthy WHWTs. These findings suggest that activin B may be part of the pathophysiology of CIPF and might act as a marker of alveolar epithelial damage.

Contribution of corneal neovascularization to dendritic cell migration into the central area during human corneal infection.

Compared with the peripheral corneal limbus, the human central cornea lacks blood vessels, which is responsible for its immunologically privileged status and high transparency. Dendritic cells (DCs) are present in the central avascular area of inflamed corneas, but the mechanisms of their migration to this location are poorly understood. Here, we investigated the contribution of vessel formation to DC migration into the central cornea, and analyzed the DC chemotactic factors produced by human corneal epithelial (HCE) cells. Using human eyes obtained from surgical procedures, we then assessed vessel formation, DC distribution, and activin A expression immunohistochemically. The results demonstrated increased numbers of vessels and DCs in the central area of inflamed corneas, and a positive correlation between the number of vessels and DCs. Activin A was expressed in the subepithelial space and the endothelium of newly formed blood vessels in the inflamed cornea. In infected corneas, DCs were present in the central area but no vascularization was observed, suggesting the presence of chemotactic factors that induced DC migration from the limbal vessels. To test this hypothesis, we assessed the migration of monocyte-derived DCs toward HCE cell supernatants with or without lipopolysaccharide (LPS) stimulation of HCE cells and inflammatory cytokines (released by HCE cells). DCs migrated toward tumor necrosis factor alpha (TNF-α), interleukin (IL)-6, and activin A, as well as LPS-stimulated HCE cell supernatants. The supernatant contained elevated TNF-α, IL-6, and activin A levels, suggesting that they were produced by HCE cells after LPS stimulation. Therefore, vessels in the central cornea might constitute a DC migration route, and activin A expressed in the endothelium of newly formed vessels might contribute to corneal vascularization. Activin A also functions as a chemotactic factor, similar to HCE-produced TNF-α and IL-6. These findings enhance our understanding of the pathophysiology of corneal inflammation during infection.

TGFß and Wnt in cardiac outflow tract defects in offspring of diabetic pregnancies.

BACKGROUND: Diabetes mellitus in pregnancy causes defects in infant heart, including the outflow tracts (OFTs). Development of the aorta and pulmonary artery, which are derived from the common OFT in the embryo, is regulated by the transforming growth factor ß (TGFß) and Wnt families, and can be perturbed by hyperglycemia-generated intracellular stress conditions. However, the underlying cellular and molecular mechanisms remain to be delineated. METHODS: Female mice were induced diabetic with streptozotocin. Embryonic and fetal OFTs were examined morphologically and histologically. Cell proliferation was assessed using 5'-bromo-2'-deoxyuridine incorporation assay. Oxidative and endoplasmic reticulum (ER) stress markers and TGFß factors were detected using immunohistochemistry. The expression of genes in the Wnt-signaling system was assessed using real-time reverse transcription polymerase chain reaction array. The role of activin-A in cell proliferation was addressed by treating embryos cultured in high glucose with activin-A. RESULTS: Maternal diabetes caused complex abnormalities in the OFTs, including aortic and pulmonary stenosis and persistent truncus arteriosus. The development of the endocardial cushions was suppressed, manifested with insufficient cellularization of the tissues. Cell proliferation was significantly decreased under oxidative and ER stress conditions. The expression of genes in the Wnt signaling was significantly altered. Activin-A and Smad3 were found to be expressed in the OFT. Treatment with activin-A rescued cell proliferation in the endocardial cushions. CONCLUSIONS: Maternal diabetes generates oxidative and ER stress conditions, suppresses TGFß and Wnt signaling, inhibits cell proliferation and cellularization of the endocardial cushions, leading to OFT septal defects. Activin-A plays a role in hyperglycemia-suppressed proliferation of the endocardial cells.

Ulipristal acetate modulates the expression and functions of activin a in leiomyoma cells.

Uterine leiomyoma is the most common benign gynecological tumor in women of reproductive age and represents the single most common indication for hysterectomy. A development of new treatments is necessary for a medical management, and in this direction, several hormonal drugs are under investigation. Ulipristal acetate (UPA; a selective progesterone receptor modulator) is considered as one of the most promising because progesterone has a critical role in development and growth of uterine leiomyoma. The effect of steroids is partly mediated by growth factors like activin A which increases extracellular matrix expression contributing to the growth of leiomyoma. The present study aimed to test whether UPA acts on leiomyoma cells affecting expression and functions of activin A system. Cultured myometrial and leiomyoma cells were treated with UPA, and messenger RNA (mRNA) expression levels of activin A (inhibin ßA [INHBA] subunits), its binding proteins (follistatin [FST] and FST-related gene), and its receptors (activin receptor-like kinase 4 [ALK4], activin receptor type [ActR] II, and ActRIIB) were evaluated. The effect of UPA on activin A modulation of fibronectin and vascular endothelial growth factor A (VEGF-A) mRNA expression in cultured myometrial and leiomyoma cells was also studied. Ulipristal acetate decreased INHBA, FST, ActRIIB, and Alk4 mRNA expressions in leiomyoma cultured cells. In addition, UPA was able to block the activin A-induced increase in fibronectin or VEGF-A mRNA expression in myometrial and in leiomyoma cultured cells. The present data show that UPA inhibits activin A expression and functions in leiomyoma cells, and this may represent a possible mechanism of action of the drug on uterine leiomyoma.

PMA induces SnoN proteolysis and CD61 expression through an autocrine mechanism.

Phorbol-12-myristate-13-acetate, also called PMA, is a small molecule that activates protein kinase C and functions to differentiate hematologic lineage cells. However, the mechanism of PMA-induced cellular differentiation is not fully understood. We found that PMA triggers global enhancement of protein ubiquitination in K562, a myelogenous leukemia cell line and one of the enhanced-ubiquitination targets is SnoN, an inhibitor of the Smad signaling pathway. Our data indicated that PMA stimulated the production of Activin A, a cytokine of the TGF-ß family. Activin A then activated the phosphorylation of both Smad2 and Smad3. In consequence, SnoN is ubiquitinated by the APC(Cdh1) ubiquitin ligase with the help of phosphorylated Smad2. Furthermore, we found that SnoN proteolysis is important for the expression of CD61, a marker of megakaryocyte. These results indicate that protein ubiquitination promotes megakaryopoiesis via degrading SnoN, an inhibitor of CD61 expression, strengths the roles of ubiquitination in cellular differentiation.

Activin A as a mediator of NK-dendritic cell functional interactions.

The interaction of NK cells with dendritic cells (DCs) results in reciprocal cell activation through the interaction of membrane proteins and the release of soluble factors. In this article, we report that in NK-DC cocultures, among a set of 84 cytokines investigated, activin A was the second highest induced gene, with CXCL8 being the most upregulated one. Activin A is a member of the TGF-ß superfamily and was previously shown to possess both proinflammatory and anti-inflammatory activities. In NK-DC cocultures, the induction of activin A required cell contact and was dependent on the presence of proinflammatory cytokines (i.e., IFN-γ, TNF-α, and GM-CSF), as well as on NK cell-mediated DC killing. CD1(+) DCs were the main activin A producer cells among myeloid blood DC subsets. In NK-DC cocultures, inhibition of activin A by follistatin, a natural inhibitory protein, or by a specific blocking Ab, resulted in the upregulation of proinflammatory cytokine release (i.e., IL-6, IL-8, TNF-α) by DCs and in the increase of DC maturation. In conclusion, our study reports that activin A, produced during NK-DC interactions, represents a relevant negative feedback mechanism that might function to prevent excessive immune activation by DCs.

Activin A induction of erythroid differentiation sensitizes K562 chronic myeloid leukemia cells to a subtoxic concentration of imatinib.

Chronic myeloid leukemia (CML) is a hematopoietic stem/progenitor cell disorder in which Bcr-Abl oncoprotein inhibits cell differentiation. Differentiation induction is considered an alternative strategy for treating CML. Activin A, a member of the transforming growth factor-ß superfamily, induces erythroid differentiation of CML cells through the p38 MAPK pathway. In this study, treatment of the K562 CML stem/progenitor cell line with activin A followed by a subtoxic concentration of the Bcr-Abl inhibitor imatinib strongly induced growth inhibition and apoptosis compared with simultaneous treatment with activin A and imatinib. Imatinib-induced growth inhibition and apoptosis following activin A pretreatment were dose- and time-dependent. Imatinib-induced growth inhibition and apoptosis were also dependent on the pretreatment dose of activin A. More than 90% of the activin A-induced increases in glycophorin A-positive cells were sensitive to imatinib. However, only some of original glycophorin A-positive cells in the activin A treatment group were sensitive to imatinib. Sequential treatment with activin A and imatinib decreased Bcr-Abl, procaspase-3, Mcl-1, and Bcl-xL and also induced cleavage of procaspase-3/poly(ADP-ribose)polymerase. The reduction of erythroid differentiation in p38 MAPK dominant-negative mutants or by short hairpin RNA knockdown of p38 MAPK decreased the growth inhibition and apoptosis mediated by sequential treatment with activin A and imatinib. Furthermore, the same inhibition level of multidrug resistance 1 expression was observed in cells treated with activin A alone, treated sequentially with activin A and imatinib, or treated simultaneously with activin A and imatinib. The p38 MAPK inhibitor SB-203580 can restore activin A-inhibited multidrug resistance 1 expression. Taken together, our results suggest that a subtoxic concentration of imatinib could exhibit strong cytotoxicity against erythroid-differentiated K562 CML cells.

Germline mutations of inhibins in early-onset ovarian epithelial tumors.

To identify novel genetic bases of early-onset epithelial ovarian tumors, we used the trio exome sequencing strategy in a patient without familial history of cancer who presented metastatic serous ovarian adenocarcinomas at 21 years of age. We identified a single de novo mutation (c.1157A>G/p.Asn386Ser) within the INHBA gene encoding the ßA-subunit of inhibins/activins, which play a key role in ovarian development. In vitro, this mutation alters the ratio of secreted activins and inhibins. In a second patient with early-onset serous borderline papillary cystadenoma, we identified an unreported germline mutation (c.179G>T/p.Arg60Leu) of the INHA gene encoding the α-subunit, the partner of the ßA-subunit. This mutation also alters the secreted activin/inhibin ratio, by disrupting both inhibin A and inhibin B biosynthesis. In a cohort of 62 cases, we detected an additional unreported germline mutation of the INHBA gene (c.839G>A/p.Gly280Glu). Our results strongly suggest that inhibin mutations contribute to the genetic determinism of epithelial ovarian tumors.

Activin A: a potential therapeutic target for characterizing and stopping joint pain early in rheumatoid arthritis patients.

It is common for rheumatoid arthritis patients to suffer from persistent joint pain for decades during their lifetime. Activin A, a member of the transforming growth factor ß superfamily, which plays a role in the very early phase of inflammatory processes, can also induce pain behaviors. In view of the contribution of synovial inflammation to the progression of rheumatoid arthritis, here we present the hypothesis that activin A may be a potential therapeutic target, serving as a marker for joint pain in which inflammatory processes are involved and as a target for early intervention.

Effects of activin A on survival, function and gene expression of pancreatic islets from non-diabetic and diabetic human donors.

Emerging evidence suggests that activin with its associated receptors, second messengers, and antagonists would be excellent targets for therapeutic drug development in the treatment of diabetes. We undertook the current study to investigate the ability to extrapolate findings from rodent studies to human islets in which data thus far has been scarce. We tested the hypothesis that human islets synthesize activin and that activin participates in the regulation of islet ß-cells. Human islets from 33 separate isolations were categorized based on functional status, culture status and diabetic status. Statistical comparisons were made by ANOVA with Tukey post-hoc adjustment for multiple comparisons. Experiments investigating activin utilized qPCR, FACS cell sorting, immunofluorescent antibody staining, functionality assays, viability assays and protein secretion assays. We have defined the transcript expression patterns of activin and the TGFß superfamily in human islets. We found INHBA (the gene encoding activin A) to be the most highly expressed of the superfamily in normal, cultured islets. We elucidated a link between the islet microenvironment and activin A. We found differential ligand expression based on diabetic, culture and functional status. Further, this is also the first report that links direct effects of activin A with the ability to restore glucose-stimulated insulin secretion in human islets from type 2 diabetic donors thereby establishing the relevance of targeting activin for therapeutic drug development.