Overexpression Of Follistatin Research Articles

Follistatin drives neuropathic pain in mice through IGF1R signaling in nociceptive neurons.

Neuropathic pain is a debilitating chronic condition that lacks effective treatment. The role of cytokine- and chemokine-mediated neuroinflammation in its pathogenesis has been well documented. Follistatin (FST) is a secreted protein known to antagonize the biological activity of cytokines in the transforming growth factor-β (TGF-β) superfamily. The involvement of FST in neuropathic pain and the underlying mechanism remain largely unknown. Here, we report that FST was up-regulated in A-fiber sensory neurons after spinal nerve ligation (SNL) in mice. Inhibition or deletion of FST alleviated neuropathic pain and reduced the nociceptive neuron hyperexcitability induced by SNL. Conversely, intrathecal or intraplantar injection of recombinant FST, or overexpression of FST in the dorsal root ganglion (DRG) neurons, induced pain hypersensitivity. Furthermore, exogenous FST increased neuronal excitability in nociceptive neurons. The biolayer interferometry (BLI) assay and coimmunoprecipitation (co-IP) demonstrated direct binding of FST to the insulin-like growth factor-1 receptor (IGF1R), and IGF1R inhibition reduced FST-induced activation of extracellular signal-regulated kinase (ERK) and protein kinase B (AKT), as well as neuronal hyperexcitability. Further co-IP analysis revealed that the N-terminal domain of FST exhibits the highest affinity for IGF1R, and blocking this interaction with a peptide derived from FST attenuated Nav1.7-mediated neuronal hyperexcitability and neuropathic pain after SNL. In addition, FST enhanced neuronal excitability in human DRG neurons through IGF1R. Collectively, our findings suggest that FST, released from A-fiber neurons, enhances Nav1.7-mediated hyperexcitability of nociceptive neurons by binding to IGF1R, making it a potential target for neuropathic pain treatment.

Follistatin Alleviates Hepatic Steatosis in NAFLD via the mTOR Dependent Pathway.

In this study, we aimed to investigate the effect of follistatin (FST) on hepatic steatosis in NAFLD and the underlying mechanism, which has rarely been reported before. Liver samples from NAFLD patients and normal liver samples (from liver donors) were collected to investigate hepatic FST expression in humans. Additionally, human liver cells (LO2) were treated with free fatty acid (FFA) to induce lipid accumulation. Furthermore, lentivirus with FST overexpression or knockdown vectors were used to generate stable cell lines, which were subsequently treated with FFA or FFA and rapamycin. In the animal experiments, male C57BL/6J mice were fed with a high-fat diet (HFD) to induce NAFLD, after which the adeno-associated virus (AAV) gene vectors for FST overexpression were administered. In both cell culture and mice, we evaluated morphological changes and the protein expression of sterol regulatory element-binding protein1 (SREBP1), acetyl-CoA carboxylase1 (ACC1), carbohydrate-responsive element-binding protein (ChREBP), fatty acid synthase (FASN), and Akt/mTOR signaling. The body weight and serum parameters of the mice were also measured. Hepatic FST expression level was higher in NAFLD patients compared to normal samples. In LO2 cells, FST overexpression alleviated lipid accumulation and lipogenesis, whereas FST knockdown aggravated hepatic steatosis. FST could regulate Akt/mTOR signaling, and the mTOR inhibitor rapamycin abolished the effect of FST knockdown on hepatic de novo lipogenesis (DNL). Furthermore, FST expression was increased in HFD mice compared to the corresponding controls. FST overexpression in mice reduced body weight gain, hyperlipidemia, hepatic DNL, and suppressed Akt/mTOR signaling. Hepatic FST expression increases in NAFLD and plays a protective role in hepatic steatosis. FST overexpression gene therapy alleviates hepatic steatosis via the mTOR pathway.Therefore, gene therapy for FST is a promising treatment in NAFLD.

Follistatin Attenuates Myocardial Fibrosis in Diabetic Cardiomyopathy via the TGF-β-Smad3 Pathway.

Follistatin (FST) is an endogenous protein that irreversibly inhibits TGF-β superfamily members and plays an anti-fibrotic role in other diseases. However, the role of FST in diabetic cardiomyopathy remains unclear. In this study, we investigated the effects of FST on diabetic cardiomyopathy. The expression of FST was downregulated in the hearts of db/db mice. Remarkably, overexpressing FST efficiently protected against cardiac dysfunction. In addition, overexpression of FST promoted cardiac hypertrophy with an unchanged expression of atrial natriuretic peptide (ANP) and the ratio of myosin heavy chain-β/myosin heavy chain-α (MYH7/MYH6). Furthermore, FST reduced cardiac fibrosis and the production of reactive oxygen species (ROS), and enhanced matrix metallopeptidase 9 (MMP9) activities in db/db mouse hearts. We also observed that overexpressing FST decreased the level of transforming growth factor beta (TGF-β) superfamily members and the phosphorylation of Smad3; consistently, in vitro experiments also verified the above results. Our findings revealed the cardioprotective role of FST in attenuating diabetic cardiomyopathy through its anti-fibrotic effects through the TGF-β–Smad3 pathway and provided a promising therapeutic strategy for diabetic cardiomyopathy.

Dysregulation of Follistatin Promotes Leukemogenesis in Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is characterized by an abnormal increase of myeloblasts in bone marrow (BM) and peripheral blood (PB). Chemotherapy and allogeneic hematopoietic stem cell transplantation (HSCT) are the mainstays of treatment. However, these approaches have reached an impasse and despite an initial remission, leukemia relapses are frequent, with an overall cure rate of only 30-40% (Rowe and Tallman 2010). Internal tandem duplication (ITD) of the fms-like tyrosine kinase 3 (FLT3) gene is the commonest mutation in AML, occurring in nearly 30% cases with inferior disease-free and overall survivals (Boissel et al. 2002; Meshinchi et al. 2001). FLT3-ITD as a therapeutic target has been tested in phase I/II clinical trials (Man et al. 2012; Fischer et al. 2010; Levis et al. 2011; Cortes et al. 2011). In most circumstances, there was an initial response to FLT3 inhibitors with reduction or clearance of myeloblasts in BM and PB. However, resistance invariably developed despite continuous treatment (Kindler, Lipka, and Fischer 2010). Therefore, the identification of novel therapeutic targets for FLT3-ITD+ AML becomes urgent.In our previous study (He et al. 2014), we showed that over-expression of human leukemic gene FLT3-ITD resulted in significant myeloid cells expansion which could be effectively rescued by FLT3-ITD inhibitor AC220 treatment. Interestingly, a proportion of zebrafish embryos expressing FLT3-ITD showed double-headed and axis-duplication phenotype as shown by the whole mount in situ hybridization (WISH) of a notochord-specific marker col9a2 . Mechanistically, FLT3-ITD-induced axis-duplication was correlated with the expansion of Spemann's Organizer as shown by the ectopic expression of Spemann's Organizer-specific marker goosecoid (gsc) by WISH as well as by the -2,067gsc-GFP reporter assay. It has been reported that some key signaling pathways are conserved between embryogenesis and tumorigenesis. We hypothesized that certain Spemann's Organizers were dysregulated in FLT3-ITD+ AML.To test this hypothesis, we performed an in silico analysis to quantify the gene expression of Spemann's Organizers in normal HSC and AML by the public database BloodSpot. Intriguingly, the Activin antagonist Follistatin (FST) was significantly increased in hematological malignancies, comparing to the normal HSC. In human malignancies, FST has been shown to express in prostate (Sepporta et al. 2013; Tumminello et al. 2010), ovarian (Ren et al. 2012; Karve et al. 2012; Di Simone et al. 1996), and liver cancers (Patella et al. 2006; Rossmanith et al. 2002) promote cancer cell growth (Gao et al. 2010; Rossmanith et al. 2002). However, the role of FST in human AML and other hematological malignancies is currently unknown. Firstly, we confirmed the upregulation of FST in AML by real-time quantitative PCR, Western Blotting, and immunofluorescence imaging. Mechanistically, FLT3-ITD up-regulated FST via the phosphorylation of CREB as demonstrated by ChIP-PCR. Moreover, the upregulation of FST by FLT3-ITD was effectively blocked by the pharmacological inhibition of CREB in a dosage-dependent manner. FST gene knock-down (by shRNA) or knock-out (by CRISPR/Cas9) in FLT3-ITD+ MOLM-13 AML cell line significantly reduced leukemic growth in vitro, reduced leukemic burden, and prolonged survival of mice in the xenotransplantation experiment. Over-expression of FST in MOLM-13 and ML-2 AML cell lines promoted leukemic growth in vitro and in vivo xenograft mouse model via AKT/p70S6K/mTOR signaling. Furthermore, proteins (RPS19, RPS6, RPS13, RPL6, RPS21, RPSA, RPS18, RPLP2, RPL10A, WIBG, and PPP2R1A) associated with nonsense-mediated decay of mRNA were decreased after FST over-expression in ML-2 AML cells, while the chemoresistant and anti-apoptotic protein CD44 was consistently increased by a label-free proteomics analysis. Notably, the serum FST levels were significantly increased in a patient-derived xenograft AML mouse model. Consistently, plasma FST levels from FLT3-ITD+ AML patients were significantly reduced after an FLT3-ITD inhibitor quizartinib treatment in a phase I/II clinical trial.In conclusion, the CREB targeted gene FST promotes AML cell growth via AKT/p70S6K/mTOR signaling and CD44-mediated anti-apoptosis. Therefore, FST may be a promising therapeutic target for AML treatment. DisclosuresNo relevant conflicts of interest to declare.

Follistatin-induced muscle hypertrophy in aged mice improves neuromuscular junction innervation and function

Sarcopenia, or age-related loss of muscle mass and strength, is an important contributor to loss of physical function in older adults. The pathogenesis of sarcopenia is likely multifactorial, but recently the role of neurological degeneration, such as motor unit loss, has received increased attention. Here, we investigated the longitudinal effects of muscle hypertrophy (via overexpression of human follistatin, a myostatin antagonist) on neuromuscular integrity in C57BL/6J mice between the ages of 24 and 27 months. Following follistatin overexpression (delivered via self-complementary adeno-associated virus subtype 9 injection), muscle weight and torque production were significantly improved. Follistatin treatment resulted in improvements of neuromuscular junction innervation and transmission but had no impact on age-related losses of motor units. These studies demonstrate that follistatin overexpression-induced muscle hypertrophy not only increased muscle weight and torque production but also countered age-related degeneration at the neuromuscular junction in mice.

Voluntary wheel running with and without follistatin overexpression improves NMJ transmission but not motor unit loss in late life of C57BL/6J mice

Sarcopenia, or pathological loss of muscle mass and strength during aging, is an important contributor to loss of physical function in older adults. Sarcopenia is a multifactorial syndrome associated with intrinsic muscle and upstream neurological dysfunction. Exercise is well-established as an effective intervention for sarcopenia, but less is known about the long-term neurobiological impact of exercise. The goals of this study were to investigate the effects of exercise, alone or in combination with follistatin (FST) overexpression (antagonist of myostatin), on neuromuscular junction transmission and motor unit numbers in mice between the age of 22 and 27 months, ages at which prior studies have demonstrated that some motor unit loss is already evident. C57BL/6J mice underwent baseline assessment and were randomized to housing with or without voluntary running wheels and injection with adeno-associated virus to overexpress FST or vehicle. Groups for comparison included sedentary and running with and without FST. Longitudinal assessments showed significantly increased muscle mass and contractility in the ‘running plus FST’ group, but running, with and without FST, showed no effect on motor unit degeneration. In contrast, running, with and without FST, demonstrated marked improvement of neuromuscular junction transmission stability.

Myostatin inhibition promotes fast fibre hypertrophy but causes loss of AMP-activated protein kinase signalling and poor exercise tolerance in a model of limb-girdle muscular dystrophy R1/2A.

Limb-girdle muscular dystrophy R1 (LGMD R1) is caused by mutations in the CAPN3 gene and is characterized by progressive muscle loss, impaired mitochondrial function and reductions in the slow oxidative gene expression programme. Myostatin is a negative regulator of muscle growth, and its inhibition improves the phenotype in several muscle wasting disorders. The effect of genetic and pharmacological inhibition of myostatin signalling on the disease phenotype in a mouse model of LGMD R1 (CAPN3 knockout mouse-C3KO) was studied. Inhibition of myostatin signalling in C3KO muscles resulted in significant muscle hypertrophy; however, there were no improvements in muscle strength and exacerbation of exercise intolerance concomitant with further reduction of muscle oxidative capacity was observed. Inhibition of myostatin signalling is unlikely to be a valid therapeutic strategy for LGMD R1. Limb-girdle muscular dystrophy R1 (LGMD R1) is caused by mutations in the CAPN3 gene and is characterized by progressive muscle loss, impaired mitochondrial function and reductions in the slow oxidative gene expression programme. There are currently no therapies available to patients. We sought to determine if induction of muscle growth, through myostatin inhibition, represents a viable therapeutic strategy for this disease. Myostatin is a negative regulator of muscle growth, and its inhibition improves the phenotype in several muscle wasting disorders. However, the effect of myostatin depends on the genetic and pathophysiological context and may not be efficacious in all contexts. We found that genetic inhibition of myostatin through overexpression of follistatin (an endogenous inhibitor of myostatin) in our LGMD R1 model (C3KO) resulted in 1.5- to 2-fold increase of muscle mass for the majority of limb muscles. However, muscle strength was not improved and exercise intolerance was exacerbated. Pharmacological inhibition of myostatin, using an anti-myostatin antibody, resulted in statistically significant increases in muscle mass; however, functional testing did not reveal changes in muscle strength nor endurance in treated C3KO mice. Histochemical and biochemical evaluation of follistatin overexpressing mice revealed a reduction in the percentage of oxidative fibres and decreased activation of AMP-activated protein kinase signalling in transgenics compared to C3KO muscles. Our data suggest that muscle hypertrophy, induced by myostatin inhibition, leads to loss of oxidative capacity, which further compromises metabolically impaired C3KO muscles and thus is unlikely to be a valid strategy for treatment of LGMD R1.

Metabolic profiling of follistatin overexpression: a novel therapeutic strategy for metabolic diseases.

BackgroundFollistatin (Fst) promotes brown adipocyte characteristics in adipose tissues.MethodsAbdominal fat volume (CT scan), glucose clearance (GTT test), and metabolomics analysis (mass spectrometry) of adipose tissues from Fst transgenic (Fst-Tg) and wild type (WT) control mice were analyzed. Oxygen consumption (Seahorse Analyzer) and lipidomics (gas chromatography) was analyzed in 3T3-L1 cells.ResultsFst-Tg mice show significant decrease in abdominal fat content, increased glucose clearance, improved plasma lipid profiles and significant changes in several conventional metabolites compared to the WT mice. Furthermore, overexpression of Fst in 3T3-L1 cells resulted in up regulation of key brown/beige markers and changes in lipidomics profiles.ConclusionFst modulates key factors involved in promoting metabolic syndrome and could be used for therapeutic intervention.

Increased Expression of Follistatin in Breast Cancer Reduces Invasiveness and Clinically Correlates with Better Survival.

Activin and its antagonist follistatin (FST) have been implicated in several solid tumours. This study investigated the role of FST in breast cancer. FST expression was examined using reverse transcription polymerase chain reaction (RT-PCR), real-time quantitative polymerase chain reaction (qPCR) and immunohistochemistry in a cohort of breast cancer samples. Expression was correlated to pathological and prognostic parameters in our patient cohort. FST was overexpressed in MCF-7 cells and assays for growth and invasion were performed. FST is expressed in breast tissue, in the cytoplasm of mammary epithelial cells. Expression was decreased in breast cancer tissue in comparison to normal mammary tissue. Over-expression of FST in vitro led to significantly increased growth rate and reduced invasion. Higher FST associates with lower-grade tumours and better survival. Our results suggest a role for FST as a suppressor of invasion and metastasis in breast cancer.

Gene therapy for follistatin decreases systemic inflammation and pain sensitivity following knee injury in high-fat diet induced obese mice

Purpose: Both obesity and joint trauma are risk factors for osteoarthritis (OA) and OA-related pain. In addition, muscle may have a role in knee OA development as changes in muscle strength could affect kinematics of joint loading. Furthermore, because of its key function in glucose storage and utilization, muscle strength and physiology are particularly vulnerable to obesity-associated inflammation. However, the mechanical, inflammatory, and metabolic links among obesity, muscle, and OA remain unclear. Follistatin (FST), an activin-binding protein, has been used as an effective treatment for muscle degenerative diseases by increasing muscle growth due to its ability to inhibit myostatin, a negative regulator of muscle mass. Moreover, FST has been reported to reduce the infiltration of inflammatory cells in the synovial membrane of the knee joint. Therefore, we hypothesized that overexpression of FST using a gene therapy approach will mitigate obesity-associated inflammation and pain sensitization in a high-fat diet (HFD) induced obesity model of mouse OA. Methods: Six-week old male C57BL/6 mice were fed either a chow control-diet or HFD (60% fat by kcal, n = 16/dietary group). At 7-weeks of age, either an AAV9-mediated FST 314 gene vector or GFP-labeled control vector (n = 10 for each dietary groups) was delivered via tail vein at a final dose of 6 × 1011 vg/ mouse. At 16-weeks of age, mice in each group (n = 10) underwent surgery to destabilize the medial meniscus (DMM) on the left knee. The remaining mice served as non-surgical controls. At time of surgery, Dual Energy X-ray Absorptiometry was conducted to measure body composition and bone mineral density (BMD). Six-weeks post-surgery (22-weeks of age), blood was collected and analyzed using a 31-plex Luminex® assay. Peripheral thermal sensitivity was determined using a hot/cold plate, while mechanical pain algesia at the knee was evaluated by a pressure-pain test. A two-way ANOVA between groups or a paired t-test within each group was performed to determine statistical significance, as appropriate (α = 0.05). Results: Prior to surgery, HFD resulted in increased body weight for both GFP- and FST-treated animals compared to chow (p < 0.05). However, overexpression of FST significantly decreased percentage of body fat in both chow and HFD treated mice as compared to their own corresponding GFP-controls. There was a main effect of both diet (p = 0.028) and FST-treatment (p = 0.007) on bone mineral density, such that HFD decreased BMD (HFD-GFP: 0.46 ± 0.23g/cm2 vs. chow-GFP: 0.51 ± 0.15g/cm2) while FST increased BMD (HFD-FST: 0.52g/cm2 ± 0.17, chow-FST: 0.56g/cm2 ± 0.17). Six weeks post-surgery, serum inflammatory profiles for HFD-GFP animals demonstrated increased systemic inflammation compared to chow diet, which was attenuated in the HFD-FST animals across fourteen markers (Table 1). HFD increased hot-plate paw-withdrawal latency time in GFP-treated animals compared to all other groups (p < 0.05), and the phenotype was rescued by FST-treatment in the HFD animals. Similarly, reduced cold plate responsiveness was also observed in HFD animals, which was rescued by FST-treatment. No differences in thermal peripheral pain were found with surgery. Additionally, FST treatment protected HFD animals from mechanical algesia at the knee post-DMM surgery (Fig. 1). Conclusions: Overexpression of FST decreased body fat, pain sensitivity, and obesity-associated systemic inflammation, while increasing whole body BMD in mice with HFD-induced obesity. These findings further support the important role of muscle in obesity and inflammation, which are well-known risk factors associated with OA onset and progression. We are currently evaluating the relationship among these factors in these mice, and whether FST treatment will mitigate OA development due to obesity and joint injury.Tabled 1Serum Markers Decreased with FST Treatment in HFD Animals compared to HFD animals with GFP.MarkerHFD-GFP Mean ± SEM (ng/mL)HFD-FST Mean ± SEM (ng/mL)p-valueCXCL1/KC0.25 ± 0.030.16 ± 0.020.050CXCL10/IP-100.09 ± 0.010.06 ± 0.040.005, #IL-1α1.03 ± 0.170.53 ± 0.080.022*IL-1β0.06 ± 0.020.03 ± 0.010.028*Leptin137.93 ± 17.4317.69 ± 4.640.001*Insulin25.22 ± 4.884.30 ± 0.650.001** indicates p < 0.05; # indicates p < 0.05 between DMM animals and control surgery animals. Open table in a new tab * indicates p < 0.05; # indicates p < 0.05 between DMM animals and control surgery animals.

PP75. DROSOPHILA MELANOGASTER AS A MODEL ORGANISM TO STUDY GLIOMA: LOCATION AND IDENTIFICATION OF FOLLISTATIN

Gliomas are the most common, malignant brain tumour found in the central nervous system. The main characteristics of gliomas is their rapid proliferation, resistance to current chemotherapies and their ability to infiltrate the brain. Brain tumours have a low survival rate due to the lack of available treatments. In the UK between 2010 and 2011, the survival rate was 14% for 10 or more year. The mortality rates for malignant brain and CNS tumours have increased by up to 24% in the UK between 1970 and 2012. As there is difficulty obtaining glioma samples, it is important to identify a suitable model organism. The main signalling pathways seen to be activated in human glioma development are the epithelial growth factor receptor (EGFR) and phosphatidylinositol-3 kinase (PI3K) pathways. The homologous EFGR and PI3K pathways have been studied greatly in drosophila, and show that there must be co-activation of the EGFR and PI3K pathways for cancer development to occur; leading to an increase in glial cell numbers by 50-100x fold. Follistatin is a protein which has been identified as a biomarker in malignant gliomas; by comparing normal serum to glioma serum it has shown that there is an overexpression of follistatin. Gliomas can be replicated in drosophila as there has been a high level of homology identified in human diseased states and in neural development. Follistatin studies have shown it has important roles in development and differentiation of tissues and organs. It acts as an inhibitor, binding to activin, bone morphogenetic proteins and other proteins which can be found in vertebrates. Follistatin has been identified within a number of different cancer types (such as prostate and ovarian), and plays an important role in cancer metastasis and angiogenesis. Serum studies have revealed follistatin is present in the early stages of cancer development; however the location and role of follistatin in cancer development has not been identified. Drosophila contain a homolog of follistatin, which will allow studies of follistatin in cancer within drosophila. By using drosophila as a model organism it will allow the location of follistatin and its involvement in glioma development to be identified. Immunohistochemistry is used to identify the location and distribution of a protein within a tissue or an organ. It is commonly used to identify which proteins are present in specific types of cancer. Follistatin has been identified to play a role in the early stages of glioma development. Therefore by using immunohistochemistry follistatin can be identified throughout the different stages of glioma development and possibly identify its role in this development. The aim of the research is to identify and locate the protein follistatin in drosophila by using immunohistochemistry. Using different larval instar stages, this will identify the distribution of follistatin in different stages of neural development. The hypothesis would be that follistatin is involved in the early stages of glial development and the distribution is important in glioma development compared to normal neural development.

Transgenic overexpression of follistatin 2 in blunt snout bream results in increased muscle mass caused by hypertrophy

As a competitive binding protein, follistatin (FST) antagonizes myostatin (MSTN) function and promotes muscle growth. Overexpression of FST in transgenic mice results in dramatically increased muscle mass caused by hyperplasia and/or hypertrophy. Previously, we identified two functional divergent fst genes (gcfst1 and gcfst2) in grass carp (Ctenopharyngodon idella). The objectives of this study were to determine if gcfst genes have similar roles on promoting muscle growth in fish. We generated a heterozygous gcfst1- and gcfst2-transgenic F0 generation in blunt snout bream (Megalobrama amblycephala). We then produced a homozygous gynogenetic F1 generation, and eventually a homozygous transgenic F2 generation. Driven by the zebrafish skeletal muscle-specific gene Mylz2 promoter, high levels of gcfst1 or gcfst2 mRNAs were expressed in embryonic somites and adult muscles in homozygous gcfst1- or gcfst2-transgenic F2 blunt snout bream, respectively. In contrast to gcfst1-transgenic fish or wild-type controls, adult gcfst2-transgenic F2 blunt snout bream exhibited a double-muscling phenotype and marked increases in body height and thickness. Overexpression of gcfst2 caused significant fiber size enlargement in skeletal muscle in gcfst2-transgenic F2 blunt snout bream. Our findings suggest that enhanced muscle growth induced by gcfst2 overexpression in transgenic blunt snout bream is caused by hypertrophic muscle growth. This approach may be used to promote directional breeding in important aquaculture species such as blunt snout bream in the future. Statement of relevanceTo promote directional breeding in important aquaculture species blunt snout bream, our research papers presents novel data on fish breeding. Our article complies with the Policy Statement for submission of manuscripts to the Genetics Section.

Transcriptional activation of follistatin by Nrf2 protects pulmonary epithelial cells against silica nanoparticle-induced oxidative stress.

Silica nanoparticles (SiO2 NPs) cause oxidative stress in respiratory system. Meanwhile, human cells launch adaptive responses to overcome SiO2 NP toxicity. However, besides a few examples, the regulation of SiO2 NP-responsive proteins and their functions in SiO2 NP response remain largely unknown. In this study, we demonstrated that SiO2 NP induced the expression of follistatin (FST), a stress responsive gene, in mouse lung tissue as well as in human lung epithelial cells (A549). The levels of Ac-H3(K9/18) and H3K4me2, two active gene markers, at FST promoter region were significantly increased during SiO2 NP treatment. The induction of FST transcription was mediated by the nuclear factor erythroid 2-related factor 2 (Nrf2), as evidenced by the decreased FST expression in Nrf2-deficient cells and the direct binding of Nrf2 to FST promoter region. Down-regulation of FST promoted SiO2 NP-induced apoptosis both in cultured cells and in mouse lung tissue. Furthermore, knockdown of FST increased while overexpression of FST decreased the expression level of NADPH oxidase 1 (NOX1) and NOX5 as well as the production of cellular reactive oxygen species (ROS). Taken together, these findings demonstrated a protective role of FST in SiO2 NP-induced oxidative stress and shed light on the interaction between SiO2 NPs and biological systems.

Human umbilical cord matrix mesenchymal stem cells suppress the growth of breast cancer by expression of tumor suppressor genes.

Human and rat umbilical cord matrix mesenchymal stem cells (UCMSC) possess the ability to control the growth of breast carcinoma cells. Comparative analyses of two types of UCMSC suggest that rat UCMSC-dependent growth regulation is significantly stronger than that of human UCMSC. Their different tumoricidal abilities were clarified by analyzing gene expression profiles in the two types of UCMSC. Microarray analysis revealed differential gene expression between untreated naïve UCMSC and those co-cultured with species-matched breast carcinoma cells. The analyses screened 17 differentially expressed genes that are commonly detected in both human and rat UCMSC. The comparison between the two sets of gene expression profiles identified two tumor suppressor genes, adipose-differentiation related protein (ADRP) and follistatin (FST), that were specifically up-regulated in rat UCMSC, but down-regulated in human UCMSC when they were co-cultured with the corresponding species’ breast carcinoma cells. Over-expression of FST, but not ADRP, in human UCMSC enhanced their ability to suppress the growth of MDA-231 cells. The growth of MDA-231 cells was also significantly lower when they were cultured in medium conditioned with FST, but not ADRP over-expressing human UCMSC. In the breast carcinoma lung metastasis model generated with MDA-231 cells, systemic treatment with FST-over-expressing human UCMSC significantly attenuated the tumor burden. These results suggest that FST may play an important role in exhibiting stronger tumoricidal ability in rat UCMSC than human UCMSC and also implies that human UCMSC can be transformed into stronger tumoricidal cells by enhancing tumor suppressor gene expression.

Overcoming Insulin Insufficiency by Forced Follistatin Expression in β-cells of db/db Mice

Diabetes poses a substantial burden to society as it can lead to serious complications and premature death. The number of cases continues to increase worldwide. Two major causes of diabetes are insulin resistance and insulin insufficiency. Currently, there are few antidiabetic drugs available that can preserve or protect β-cell function to overcome insulin insufficiency in diabetes. We describe a therapeutic strategy to preserve β-cell function by overexpression of follistatin (FST) using an AAV vector (AAV8-Ins-FST) in diabetic mouse model. Overexpression of FST in the pancreas of db/db mouse increased β-cell islet mass, decreased fasting glucose level, alleviated diabetic symptoms, and essentially doubled lifespan of the treated mice. The observed islet enlargement was attributed to β-cell proliferation as a result of bioneutralization of myostatin and activin by FST. Overall, our study indicates overexpression of FST in the diabetic pancreas preserves β-cell function by promoting β-cell proliferation, opening up a new therapeutic avenue for the treatment of diabetes.

AAV2-mediated follistatin overexpression induces ovine primary myoblasts proliferation.

BackgroundFollistatin (FST) has been shown to bind to some TGF-β family members and can function as a potent myostatin (MSTN) antagonist. Recent studies have revealed that over-expression of FST by adeno-associated viruses increases muscle growth in mice, humans and nonhuman primates. In the present study, to determine the effect of FST on ovine primary myoblast (OPM) proliferation, FST was over-expressed using an adeno-associated virus serotype 2 (AAV 2) vector.ResultsWestern blot results showed that AAV induced the expression of FST protein in transduced OPM cells. Real-time quantitative PCR results indicated that over-expression of FST resulted in a dramatic increase in Akt I and CDK2 expression and a decrease in p21 expression. Moreover, cell cycle analysis confirmed that FST down-regulated p21, a CDK inhibitor, and increased the level of CDK2 expression in OPM cells. Hence, follistatin positively regulated the G1 to S progression. Our results showed that FST induced proliferation through a down-regulation of p21, as only the p21 expression level was down-regulated as a result of FST over-expression in myoblasts, whereas no change was observed in the level of p57 expression.ConclusionsThese results expanded our understanding of the regulation mechanism of FST in ovine primary myoblasts. Our results provide the first evidence that the AAV viral system can be used for gene transfer in ovine myoblast cells. Moreover, the results showed that an AAV vector can successfully induce the expression of FST in OPM cells in vitro. These findings demonstrated that FST over-expression induces proliferation through a down-regulation of the p21 gene under proliferating conditions.

Follistatin could promote the proliferation of duck primary myoblasts by activating PI3K/Akt/mTOR signalling.

FST (follistatin) is essential for skeletal muscle development, but the intracellular signalling networks that regulate FST-induced effects are not well defined. We sought to investigate whether FST promotes the proliferation of myoblasts through the PI3K (phosphoinositide 3-kinase)/Akt (protein kinase B)/mTOR (mammalian target of rapamycin) signalling. In the present study, we transfected the pEGFP-duFST plasmid and added PI3K and mTOR inhibitors to the medium of duck primary myoblasts. Then, we analysed the cellular phenotypic changes that occurred and analysed the expression of target genes. The results showed that FST promoted myoblast proliferation, induced the mRNA expression of PI3K, Akt, mTOR, 70-kDa ribosomal protein S6K (S6 kinase) and the protein expression of phospho-Akt (Thr308), mTOR, phospho-mTOR (serine 2448), phospho-S6K (Ser417), inhibited the mRNA expression of FoxO1, MuRF1 (muscle RING finger-1) and the protein expression of phospho-FoxO1 (Ser256). Moreover, we found that the overexpression of FST could alleviate the inhibitory effect of myoblast proliferation caused by the addition of LY294002, a PI3K inhibitor. Additionally, the overexpression of duck FST also relieved the inhibition of myoblast proliferation caused by the addition of rapamycin (an mTOR inhibitor) through PI3K/Akt/mTOR signalling. In light of the present results, we hypothesize that duck FST could promote myoblast proliferation, which is dependent on PI3K/Akt/mTOR signalling.

Construction of a eukaryotic expression vector for pEGFP-FST and its biological activity in duck myoblasts

article i nfo Background: Follistatin (FST), a secreted glycoprotein, is intrinsically linked to muscle hypertrophy. To explore the function of duck FST in myoblast proliferation and differentiation, the pEGFP-FST eukaryotic expression vector was constructed and identified. The biological activities of this vector were analyzed by transfecting pEGFP-FST into cultured duck myoblasts using Lipofectamine™ 2000 and subsequently determining the mRNA expression profiles of FST and myostatin (MSTN). Results: The duck pEGFP-FST vector was successfully constructed and was confirmed to have high liposome-mediated transfection efficiency in duck myoblasts. Additionally, myoblasts transfected with pEGFP-FST had a higher biological activity. Significantly, the overexpression of FST in these cells significantly inhibited the mRNA expression of MSTN (a target gene that is negatively regulated by FST). Conclusions: The duck pEGFP-FST vector has been constructed successfully and exhibits biological activity by promoting myoblast proliferation and differentiation in vitro.

ERRβ signalling through FST and BCAS2 inhibits cellular proliferation in breast cancer cells

Background:The overexpression of oestrogen-related receptor-β (ERRβ) in breast cancer patients is correlated with improved prognosis and longer relapse-free survival, and the level of ERRβ mRNA is inversely correlated with the S-phase fraction of cells from breast cancer patients.Methods:Chromatin immunoprecipitation (ChIP) cloning of ERRβ transcriptional targets and gel supershift assays identified breast cancer amplified sequence 2 (BCAS2) and Follistatin (FST) as two important downstream genes that help to regulate tumourigenesis. Confocal microscopy, co-immunoprecipitation (CoIP), western blotting and quantitative real-time PCR confirmed the involvement of ERRβ in oestrogen signalling.Results:Overexpressed ERRβ induced FST-mediated apoptosis in breast cancer cells, and E-cadherin expression was also enhanced through upregulation of FST. However, this anti-proliferative signalling function was challenged by ERRβ-mediated BCAS2 upregulation, which inhibited FST transcription through the downregulation of β-catenin/TCF4 recruitment to the FST promoter. Interestingly, ERRβ-mediated upregulation of BCAS2 downregulated the major G1-S transition marker cyclin D1, despite the predictable oncogenic properties of BCAS2.Interpretation:Our study provides the first evidence that ERRβ, which is a coregulator of ERα also acts as a potential tumour-suppressor molecule in breast cancer. Our current report also provides novel insights into the entire cascade of ERRβ signalling events, which may lead to BCAS2-mediated blockage of the G1/S transition and inhibition of the epithelial to mesenchymal transition through FST-mediated regulation of E-cadherin. Importantly, matrix metalloprotease 7, which is a classical mediator of metastasis and E-cadherin cleavage, was also restricted as a result of ERRβ-mediated FST overexpression.

Abstract 220: Human umbilical cord matrix-derived stem cells control tumor growth by tumor suppressor gene expression.

Abstract Umbilical cord matrix derived stem cells (UCMSC) have the potential to treat various diseases including cancer. We have shown that naïve human and rat UCMSC significantly attenuate proliferation of multiple cancer cells (Ganta et al., Cancer Res., 2009. Ayuzawa et al., Cancer Lett., 2009). However, the growth attenuation ability of rat UCMSC is stronger than that of human UCMSC. To clarify their different tumoricidal abilities, differential gene expression profiles of these two cells were studied by micro-array analysis using Illumina HumanRef-8 V2 for human and RatRef-12 BeadChip for rat UCMSC. The gene expression profiles were compared between UCMSC untreated and co-cultured with either human (MDA-231) or rat breast carcinoma cells (Mat B III). Selection criteria used for the screening of candidate genes associated with UCMSC-dependent tumoricidal ability are as follows; 1) gene expression difference should be at least a 1.5 fold between naive UCMSC and those co-cultured with mammary tumor cells; 2) they must encode secretory proteins; and 3) they must encode cell growth regulation-related proteins. These analyses screened 16 common genes from both human and rat UCMSC. The comparison between two sets of gene expression profiles identified that two genes, adipose-differentiation related protein (ADRP) and follistatin (FST), were specifically up-regulated in rat UCMSC, whereas they were down-regulated in human UCMSC when they were co-cultured with the corresponding species’ breast cancer cells. The suppression of either protein by adding specific neutralizing antibody in culture of rat UCMSC significantly abrogated their ability to attenuate cell growth. Over-expression of both genes by adenovirus vector in human UCMSC enhanced their ability to suppress the growth of MDA-231 cells. FST over-expression in MDA 231 cells also decreased their growth significantly. In the experimental lung metastasis model with MDA 231 cells in immunodeficient mice, the treatment with FST-over-expressing human UCMSC was administered three times, at one week intervals, via tail vein injection, starting 6 days after tumor cell inoculation. Examination 4 weeks after tumor inoculation showed a significant decrease in the number of tumor nodules in the lungs. Both ADRP and FST are considered to be tumor suppressor genes. These results suggest that both ADRP and FST may play important roles in exhibiting a stronger tumoricidal ability in rat UCMSC than human UCMSC. These results imply that human UCMSC can be transformed into stronger tumoricidal cells by enhancing tumor suppressor gene expression. This work was supported by the Kansas State University (KSU) Terry C. Johnson Center for Basic Cancer Research, KSU College of Veterinary Medicine Dean's Fund, NIH RR017686, RR15563, CA135599, Kansas Bioscience Authority research grant. Citation Format: Naomi Ohta, Susumu Ishiguro, Atsushi Kawabata, Deepthi Uppalapati, Marla Pyle, Deryl Troyer, Supriyo De, Yongqing Zhang, Kevin G. Becker, Masaaki Tamura. Human umbilical cord matrix-derived stem cells control tumor growth by tumor suppressor gene expression. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 220. doi:10.1158/1538-7445.AM2013-220