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Growth differentiation factor 11 (GDF11) is a transforming factor-β superfamily member that functions as a negative regulator of neurogenesis during embryonic development. However, when recombinant GDF11 (rGDF11) is administered systemically in aged mice, it promotes neurogenesis, the opposite of its role during development. The goal of the present study was to reconcile this apparent discrepancy by performing the first detailed investigation into the expression of endogenous GDF11 in the adult brain and its effects on neurogenesis. Using quantitative histological analysis, we observed that Gdf11 is most highly expressed in adult neurogenic niches and non-neurogenic regions within the hippocampus, choroid plexus, thalamus, habenula, and cerebellum. To investigate the role of endogenous GDF11 during adult hippocampal neurogenesis, we generated a tamoxifen inducible mouse that allowed us to reduce GDF11 levels. Depletion of Gdf11 during adulthood increased proliferation of neural progenitors and decreased the number of newborn neurons in the hippocampus, suggesting that endogenous GDF11 remains a negative regulator of hippocampal neurogenesis in adult mice. These findings further support the idea that circulating systemic GDF11 and endogenously expressed GDF11 in the adult brain have different target cells or mechanisms of action. Our data describe a role for GDF11-dependent signaling in adult neurogenesis that has implications for how GDF11 may be used to treat CNS disease., (© 2021. The Author(s).)

We previously reported bidirectional gene expression regulation of the Bone Morphogenetic Proteins (BMP2, 4, and 7) in chick retinal pigment epithelium (RPE) in response to imposed optical defocus and form-deprivation (FD). This study investigated whether there are local (regional) differences in these effects. 19-day old White-Leghorn chicks wore monocular +10 or - 10 D lenses, or diffusers (FD) for 2 or 48 hr, after which RPE samples were collected from both eyes, from a central circular zone (3 mm radius), and 3 mm wide annular mid-peripheral and peripheral zones in all cases. BMP2, 4, and 7 gene expression levels in RPE from treated and fellow control eyes were compared as well as differences across zones. With the +10 D lens, increased expression of both BMP2 and BMP4 genes was observed in central and mid-peripheral zones but not the peripheral zone after 2 and 48 hr. In contrast, with the -10 D lens BMP2 gene expression was significantly decreased in all three zones after 2 and 48 hr. Similar patterns of BMP2 gene expression were observed in all three zones after 48 hr of FD. Smaller changes were recorded for BMP4 and BMP7 gene expression for both myopia-inducing treatments. That optical defocus- and FD-induced changes in BMP gene expression in chick RPE show treatment-dependent local (regional) differences suggest important differences in the nature and contributions of local retinal and underlying RPE regions to eye growth regulation., (© 2020 Wiley Periodicals LLC.)

Understanding limb development not only gives insights into the outgrowth and differentiation of the limb, but also has clinical relevance. Limb development begins with two paired limb buds (forelimb and hindlimb buds), which are initially undifferentiated mesenchymal cells tipped with a thickening of the ectoderm, termed the apical ectodermal ridge (AER). As a transitional embryonic structure, the AER undergoes four stages and contributes to multiple axes of limb development through the coordination of signalling centres, feedback loops, and other cell activities by secretory signalling and the activation of gene expression. Within the scope of proximodistal patterning, it is understood that while fibroblast growth factors (FGFs) function sequentially over time as primary components of the AER signalling process, there is still no consensus on models that would explain proximodistal patterning itself. In anteroposterior patterning, the AER has a dual-direction regulation by which it promotes the sonic hedgehog (Shh) gene expression in the zone of polarizing activity (ZPA) for proliferation, and inhibits Shh expression in the anterior mesenchyme. In dorsoventral patterning, the AER activates Engrailed-1 (En1) expression, and thus represses Wnt family member 7a (Wnt7a) expression in the ventral ectoderm by the expression of Fgfs, Sp6/8, and bone morphogenetic protein (Bmp) genes. The AER also plays a vital role in shaping the individual digits, since levels of Fgf4/8 and Bmps expressed in the AER affect digit patterning by controlling apoptosis. In summary, the knowledge of crosstalk within AER among the three main axes is essential to understand limb growth and pattern formation, as the development of its areas proceeds simultaneously.

In this study, we aimed to determine the origin of the difference, in terms of anti-Müllerian hormone production, existing between the bovine and porcine ovaries. We first confirmed by quantitative real-time-Polymerase-Chain Reaction, ELISA assay and immunohistochemistry that anti-Müllerian hormone mRNA and protein production are very low in porcine ovarian growing follicles compared to bovine ones. We then have transfected porcine and bovine granulosa cells with vectors containing the luciferase gene driven by the porcine or the bovine anti-Müllerian hormone promoter. These transfection experiments showed that the porcine anti-Müllerian hormone promoter is less active and less responsive to bone morphogenetic protein stimulations than the bovine promoter in both porcine and bovine cells. Moreover, bovine but not porcine granulosa cells were responsive to bone morphogenetic protein stimulation after transfection of a plasmidic construction including a strong response element to the bone morphogenetic proteins (12 repetitions of the GCCG sequence) upstream of the luciferase reporter gene. We also showed that SMAD6, an inhibitor of the SMAD1-5-8 pathway, is strongly expressed in porcine compared to the bovine granulosa cells. Overall, these results suggest that the low expression of anti-Müllerian hormone in porcine growing follicles is due to both a lack of activity/sensitivity of the porcine anti-Müllerian hormone promoter, and to the lack of responsiveness of porcine granulosa cells to bone morphogenetic protein signaling, potentially due to an overexpression of SMAD6 compared to bovine granulosa cells. We propose that the low levels of anti-Müllerian hormone in the pig would explain the poly-ovulatory phenotype in this species., (© The Author(s) 2020. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Hepcidin (HAMP) synthesis is suppressed by erythropoiesis to increase iron availability for red blood cell production. This effect is thought to result from factors secreted by erythroid precursors. Growth differentiation factor 11 (GDF11) expression was recently shown to increase in erythroid cells of β-thalassaemia, and decrease with improvement in anaemia. Whether GDF11 regulates hepatic HAMP production has never been experimentally studied. Here, we explore GDF11 function during erythropoiesis-triggered HAMP suppression. Our results confirm that exogenous erythropoietin significantly increases Gdf11 as well as Erfe (erythroferrone) expression, and Gdf11 is also increased, albeit at a lower degree than Erfe, in phlebotomized wild type and β-thalassaemic mice. GDF11 is expressed predominantly in erythroid burst forming unit- and erythroid colony-forming unit- cells during erythropoiesis. Exogeneous GDF11 administration results in HAMP suppression in vivo and in vitro. Furthermore, exogenous GDF11 decreases BMP-SMAD signalling, enhances SMAD ubiquitin regulatory factor 1 (SMURF1) expression and induces ERK1/2 (MAPK3/1) signalling. ERK1/2 signalling activation is required for GDF11 or SMURF1-mediated suppression in BMP-SMAD signalling and HAMP expression. This research newly characterizes GDF11 in erythropoiesis-mediated HAMP suppression, in addition to ERFE., (© 2019 British Society for Haematology and John Wiley & Sons Ltd.)

New Findings: What is the central question of this study? What is the effect and mechanism of interval running training on age-related muscle wasting and bone loss in an ovariectomized rat model? What is the main finding and its importance? Interval running training improved muscle growth and osteogenic differentiation by enhancing the expression of bone morphogenic proteins and sirtuins in ageing-induced ovariectomized rats. Therefore, the repetition of low and high intensities within a single exercise bout, such as interval running training, may be recommended as a practical intervention to prevent skeletal muscle wasting and bone loss in the elderly., Abstract: Effective prophylactic strategies are needed for the suppression of age-related muscle wasting and bone loss after menopause. Exercise training is attractive due to its potential for improving energy metabolism, as well as age-related muscle wasting and bone loss. In particular, interval running (IR) training involves a repetition of low and high intensities within a single exercise bout. Therefore, this study elucidated the effect of interval training on muscle and bone health, as well as anti-ageing, in ovariectomized (OVX) rats. The anti-ageing effect of IR on muscle and bone was tested using western blotting and micro-computed tomography analysis, tartrate-resistant acid phosphatase and immunohistochemical staining. IR significantly inhibited the expression of inflammatory molecules, and improved antioxidant activity via down-regulation of mitogen-activated protein kinases (MAPKs) in the ageing-induced OVX rats skeletal muscle. IR compared with continuous running (CR) improved muscle mass and growth in OVX rats by the promotion of muscle growth-related factors including MyoD, myogenin, phospho-mechanistic target of rapamycin (p-mTOR), sirtuins (SIRTs), and bone morphogenic proteins (BMPs). IR also effectively recovered OVX-induced bone loss via the down-regulation of bone resorption and osteoclast formation in receptor activator of nuclear factor κB ligand (RANKL)-treated bone marrowmacrophages (BMMs). In particular, IR led to high expression of SIRT1 and 6, which promoted osteogenic differentiation and bone formation via modulating the BMP signalling pathway compared with CR training. The in vivo effect of IR was confirmed by immunohistochemical staining with the improvement of bone formation molecules such as BMPs and SIRTs. These results suggested that IR training affected myogenic and osteogenic formation. So, IR training may be considered for prevention of muscle wasting and bone loss for the elderly., (© 2019 The Authors. Experimental Physiology © 2019 The Physiological Society.)

Chronic kidney disease-mineral bone disorder (CKD-MBD), comprising mineral, hormonal, and bone metabolic imbalance, is a major CKD-related issue; it causes osteoporosis prevalence in CKD patients. Osteocyte-derived sclerostin inhibits the osteogenic Wnt/β-catenin signaling pathway; its levels rise when kidney function declines. Exercise modulates the physiological functions of osteocytes, potentially altering sclerostin production. It may aid bone and mineral electrolyte homeostasis in CKD. Mild CKD was induced in rats by partial nephrectomy. They were divided into: sham (no CKD), CKD, and CKD + exercise (8 weeks of treadmill running) groups. Micro-CT scanning demonstrated that the CKD + exercise-group rats had a higher bone mineral density (BMD) of the spine and femoral metaphysis and higher femoral trabecular bone volume than the CKD-group rats. Bone formation rates were not significantly different. The CKD + exercise-group rats had lower serum sclerostin (157.1 ± 21.1 vs 309 ± 38.1 pg/mL, p < 0.05) and CTX-1 (bone resorption marker) levels. Immunohistochemistry revealed higher tibial β-catenin concentrations in the CKD + exercise-group rats. Serum FGF-23, intact parathyroid hormone (iPTH), alkaline phosphatase (ALP), calcium, and phosphate levels showed no significant differences between these groups. Thus, exercise improves BMD and bone microstructure in mild CKD by inhibiting sclerostin production, but does not alter serum minerals.

The two-stage induced-membrane (IM) technique is increasingly used for treatment of large bone defects. In stage one, the bone defect is filled with polymethylmethacrylate (PMMA), which induces a membrane around the implant. In stage two, PMMA is replaced with bone graft. Bioactive glasses (BAGs) are bone substitutes with bone-stimulating and angiogenic properties. We have previously shown that a certain type of BAG can also induce a foreign-body membrane similar to PMMA. The aim of this study was to evaluate the bone-forming capacity of sintered BAG-S53P4 and poly(lactide-co-glycolide) (PLGA)-coated BAG-S53P4 scaffolds for potential use as bone substitutes in a single-stage IM technique. Sintered porous rods of BAG-S53P4, BAG-S53P4-PLGA, or PMMA were implanted in rabbit femurs for 2, 4, or 8 weeks. The expression of bone morphogenic protein (BMP)-2, -4, and -7 in the IMs of implanted materials were analyzed with real-time quantitative polymerase chain reaction. Micro-computed tomography imaging was used to evaluate bone growth and further verified with scanning electron microscopy. BAG-S53P4 and BAG-S53P4-PGLA scaffold IMs show similar or superior expression of BMP-2, -4, and -7 compared with PMMA IM. Bone ingrowth into BAG scaffolds increased over time. Active bone formation occurred inside the BAG scaffolds and the respective BMP expressions were similar or superior for the BAG IMs compared with PMMA, thus making BAGs a promising device for single-stage treatment of bone defects. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res B Part B: Appl Biomater, 2018. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B: 847-857, 2019., (© 2018 Wiley Periodicals, Inc.)

Growth differentiation factor 11 (GDF11), is a member of the transforming growth factor-beta (TGF-β) superfamily and bone morphogenetic protein (BMP) subfamily. In this study, we aimed to assess the expression profile of GDF11, its prognostic value in terms of OS, as well as the potential mechanisms leading to its dysregulation in uveal melanoma. A retrospective study was conducted using our primary data and genetic, clinicopathological and overall survival (OS) data from the Cancer Genome Atlas-Uveal Melanoma (TCGA-UVM). Results showed that GDF11 expression was significantly higher in tumor tissues compared with that in adjacent normal tissues. High GDF11 expression was associated with uveal melanoma in advanced stages (IV), epithelioid cell dominant subtype, as well as extrascleral extension. Univariate analysis showed that older age, epithelioid cell dominant, with extrascleral extension and increased GDF11 expression were associated with unfavorable OS. Multivariate analysis confirmed that GDF11 expression was an independent prognostic indicator of unfavorable OS (HR: 1.704, 95%CI: 1.143-2.540, p = 0.009), after adjustment of age, histological subtypes and extrascleral extension. Among the 80 cases of uveal melanoma, only 3 cases had low-level copy gain (+1) and 2 cases had heterozygous loss (-1). No somatic mutations, including SNPs and small INDELs were observed in GDF11 DNA. The methylation of these four CpG sites had weakly (cg22950598 and cg23689080), moderately (cg09890930), or strongly (cg05511733) negative correlation with GDF11 expression. In addition, the patients with high methylation of these four sites had significantly better OS compared to the group with low methylation. Based on these findings, we infer that methylation modulated GDF11 expression might be a valuable prognostic biomarker regarding OS in uveal melanoma., Competing Interests: The authors have declared that no competing interests exist.

Bone morphogenetic proteins (BMPs) are secreted proteins that belong to the transforming growth factor-β superfamily. In the adult brain, they modulate neurogenesis, favor astrogliogenesis, and inhibit oligodendrogenesis. Because BMPs may be involved in the failure of remyelination in multiple sclerosis (MS), we characterized the expression of BMP-2, BMP-4, BMP-5, and BMP-7; BMP type II receptor (BMPRII); and phosphorylated SMAD (pSMAD) 1/5/8 in lesions of MS and other demyelinating diseases. A total of 42 MS lesions, 12 acute ischemic lesions, 8 progressive multifocal leukoencephalopathy lesions, and 10 central nervous system areas from four nonneuropathological patients were included. Lesions were histologically classified according to the inflammatory activity. The expression of BMP-2, BMP-4, BMP-5, BMP-7, BMPRII, and pSMAD1/5/8 was quantified by immunostaining, and colocalization studies were performed. In MS lesions, astrocytes, microglia/macrophages, and neurons expressed BMP-2, BMP-4, BMP-5, and BMP-7; BMPRII; and pSMAD1/5/8. Oligodendrocytes expressed BMP-2 and BMP-7 and pSMAD1/5/8. The percentage of cells that expressed BMPs, BMPRII, and pSMAD1/5/8 correlated with the inflammatory activity of MS lesions, and changes in the percentage of positive cells were more relevant in MS than in other white matter-damaging diseases. These data indicate that BMPs are increased in active MS lesions, suggesting a possible role in MS pathogenesis., (Copyright © 2019 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

This study was done with aim to assess the serum sclerostin and dickkopf-1 (DKK-1) level in patients of rheumatoid arthritis (RA) and to correlate their level with disease activity and bone mineral density. Fifty patients of RA and equal age and sex matched healthy controls were included in the study. Patients were evaluated clinically and investigated with routine blood tests along with rheumatoid factor (RF), anti-citrullinated protein antibody (anti-CCP 2 ), radiographs and bone mineral density (BMD). Serum sclerostin and DKK-1 levels of both cases and controls was assayed by using enzyme-linked immunosorbent assay (ELISA) assay [RayBio ® , Georgia, USA with coefficient of variation percent (CV %), < 10%] and compared with disease activity and bone mineral density. Disease activity was measured by Disease Activity Score 28 (DAS28) along with Modified Health Assessment Questionnaire (MHAQ) score. Mean serum sclerostin and DKK-1 was significantly higher in study group as compared to control group. Serum sclerostin showed significant correlation with disease activity scores (DAS score and MHAQ score), erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP) level. Serum sclerostin at level of 394 pg/mL was found to have diagnostic significance with sensitivity of 100% and specificity of 90%. DKK-1 level shows significantly positive correlation with larson score which denotes radiological progression (r value 0.468; p value 0.001). More studies with larger sample size of RA patients are needed for better determination of the role of sclerostin and DKK-1 in RA. Also, the correlation of these and other bone turn over markers will help decipher their role with disease progression in RA patients.

We have previously shown myofibroblasts subjacent to the squamous epithelium in the normal human esophagus and an increase in esophagitis. Myofibroblast contribution to bone morphogenetic protein (BMP) signaling and to paracrine mediated epithelial-mesenchymal interactions in the human esophagus remains incompletely defined. We investigated BMP4 and BMP inhibitor GREM1 gene expression and protein levels in previously characterized human esophageal myofibroblast primary cultures and in a human esophageal myofibroblast cell line. We adapted human esophageal myofibroblast conditioned media into a 3D organotypic model to investigate the effect of myofibroblast secreted factors on squamous epithelial morphology, proliferation, differentiation and BMP signaling. Human esophageal myofibroblasts constitutively secrete GREM1 and increase BMP4 expression and BMP4 secretion in response to epithelial Hedgehog ligand SHH. Detection of secreted BMP4 is decreased in the presence of GREM1. Myofibroblast conditioned media increases epithelial proliferation and expression of basal markers p63 and CK14 leading to an overall increase in epithelial thickness. Epithelial BMP signaling increases with myofibroblast conditioned media. These findings were partially reversed with GREM1 inhibition. Our results demonstrate that myofibroblasts are potential sources of GREM1 and of BMP4 in the human esophagus and that human esophageal myofibroblast-epithelial paracrine interactions contribute in part to the regulation of epithelial growth.

A considerable amount of research has focused on improving regenerative therapy strategies for repairing defects in load-bearing bones. The enhancement of tissue regeneration with microRNAs (miRNAs) is being developed because miRNAs can simultaneously regulate multiple signaling pathways in an endogenous manner. In this study, we developed a miR-210-based bone repair strategy. We identified a miRNA (miR-210-3p) that can simultaneously up-regulate the expression of multiple key osteogenic genes in vitro. This process resulted in enhanced bone formation in a subcutaneous mouse model with a miR-210-3p/poly-l-lactic acid (PLLA)/bone marrow-derived stem cell (BMSC) construct. Furthermore, we constructed a model of critical-sized load-bearing bone defects and implanted a miR-210-3p/β-tricalcium phosphate (β-TCP)/bone mesenchymal stem cell (BMSC) construct into the defect. We found that the load-bearing defect was almost fully repaired using the miR-210-3p construct. We also identified a new mechanism by which miR-210-3p regulates Sclerostin protein levels. This miRNA-based strategy may yield novel therapeutic methods for the treatment of regenerative defects in vital load-bearing bones by utilizing miRNA therapy for tissue engineering., Statement of Significance: The destroyed maxillofacial bone reconstruction is still a real challenge for maxillofacial surgeon, due to that functional bone reconstruction involved load-bearing. Base on the above problem, this paper developed a novel miR-210-3p/β-tricalcium phosphate (TCP)/bone marrow-derived stem cell (BMSC) construct (miR-210-3p/β-TCP/BMSCs), which lead to functional reconstruction of critical-size mandible bone defect. We found that the load-bearing defect was almost fully repaired using the miR-210-3p construct. In addition, we also found the mechanism of how the delivered microRNA activated the signaling pathways of endogenous stem cells, leading to the defect regeneration. This miRNA-based strategy can be used to regenerate defects in vital load-bearing bones, thus addressing a critical challenge in regenerative medicine by utilizing miRNA therapy for tissue engineering., (Copyright © 2018 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Chronic obstructive pulmonary disease (COPD) is a chronic inflammatory lung disease, which is associated with significant mortality and costs. The molecular mechanisms underlying the roles of cigarette smoke (an accepted risk factor for COPD) and growth differentiation factor 11 (GDF11), which is reduced in patients with COPD, in the occurrence of COPD are unclear. The aim of the present study was to explore the function of GDF11 in the progression of COPD. Western blotting analysis was used to determine the expression levels of GDF11 in serum and primary lung mesenchymal cells from patients with COPD and the healthy people, and the effect of cigarette smoke extract (CSE) on the expression of AKT, p-AKT (Ser473), p-AKT (Thr308) and GDF11 was examined. The correlations between the expression level of GDF11 and the ratio of forced expiratory volume in one second (FEV1) and forced vital capacity (FVC), as well as GDF11 and p-AKT (Ser473 and Thr308) in vivo and in vitro were examined. GDF11 expression was decreased in COPD patients' serum and cells when compared with that from the healthy people, and it was positively correlated with the FEV1/FVC ratio. Exposure to CSE reduced the expression of GDF11 but increased the expression of p-AKT (Ser473 and Thr308). Together, the results suggested that CSE promoted the progression of COPD by downregulating the expression of GDF11, which then activated the AKT signaling pathway. This study suggests that GDF11 may be a novel target for the diagnosis and treatment of COPD., (Copyright © 2018 Elsevier Masson SAS. All rights reserved.)

Background: Several hypotheses have been proposed regarding the mechanisms underlying meningioma-related hyperostosis. In this study, we investigated the role of osteoprotegerin (OPG), insulin-like growth factor 1 (IGF-1), endothelin 1 (ET-1), and bone morphogenetic protein (BMP) 2 and 4., Methods: A total of 149 patients (39 males and 110 females; mean age, 62 years) who underwent surgery were included. Depending on the relationship with the bone, meningiomas were classified as hyperostotic, osteolytic, infiltrative, or unrelated. Expression of OPG, and IGF-1, ET-1, BMP-2, and BMP-4 was evaluated by tissue microarray analysis of surgical samples., Results: Our series comprised 132 cases of grade I, 14 cases of grade II, and 3 cases of grade III meningiomas, according to the World Health Organization classification. Based on preoperative computed tomography scan, the cases were classified as follows: hyperostotic, n = 11; osteolytic, n = 11; infiltrative, n = 15; unrelated to the bone, n = 108. Four cases were excluded from the statistical analysis. Using receiver operating characteristic curve analysis, we identified a 2% cutoff for the mean value of IGF-1 that discriminated between osteolytic and osteoblastic lesions; cases with a mean IGF-1 expression of <2% were classified as osteolytic (P = 0.0046), whereas those with a mean OPG expression of <10% were classified as osteolytic (P = 0.048). No other significant relationships were found., Conclusions: Expression of OPG and expression of IGF-1 were found to be associated with the development of hyperostosis. Preliminary findings suggest that hyperostosis can be caused by an overexpression of osteogenic molecules that influence osteoblast/osteoclast activity. Based on our results, further studies on hyperostotic bony tissue in meningiomas are needed to better understand how meningiomas influence bone overproduction., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Cerebellar granule cell precursors (GCPs) and granule cells (GCs) represent good models to study neuronal development. Here, we report that the transcription factor myeloid ectopic viral integration site 1 homolog (Meis1) plays pivotal roles in the regulation of mouse GC development. We found that Meis1 is expressed in GC lineage cells and astrocytes in the cerebellum during development. Targeted disruption of the Meis1 gene specifically in the GC lineage resulted in smaller cerebella with disorganized lobules. Knock-down/knock-out (KO) experiments for Meis1 and in vitro assays showed that Meis1 binds to an upstream sequence of Pax6 to enhance its transcription in GCPs/GCs and also suggested that the Meis1-Pax6 cascade regulates morphology of GCPs/GCs during development. In the conditional KO (cKO) cerebella, many Atoh1-positive GCPs were observed ectopically in the inner external granule layer (EGL) and a similar phenomenon was observed in cultured cerebellar slices treated with a bone morphogenic protein (BMP) inhibitor. Furthermore, expression of Smad proteins and Smad phosphorylation were severely reduced in the cKO cerebella and Meis1-knock-down GCPs cerebella. Reduction of phosphorylated Smad was also observed in cerebellar slices electroporated with a Pax6 knock-down vector. Because it is known that BMP signaling induces Atoh1 degradation in GCPs, these findings suggest that the Meis1-Pax6 pathway increases the expression of Smad proteins to upregulate BMP signaling, leading to degradation of Atoh1 in the inner EGL, which contributes to differentiation from GCPs to GCs. Therefore, this work reveals crucial functions of Meis1 in GC development and gives insights into the general understanding of the molecular machinery underlying neural differentiation from neural progenitors. SIGNIFICANCE STATEMENT We report that myeloid ectopic viral integration site 1 homolog (Meis1) plays pivotal roles in the regulation of mouse granule cell (GC) development. Here, we show Meis1 is expressed in GC precursors (GCPs) and GCs during development. Our knock-down and conditional knock-out (cKO) experiments and in vitro assays revealed that Meis1 is required for proper cerebellar structure formation and for Pax6 transcription in GCPs and GCs. The Meis1-Pax6 cascade regulates the morphology of GCs. In the cKO cerebella, Smad proteins and bone morphogenic protein (BMP) signaling are severely reduced and Atoh1-expressing GCPs are ectopically detected in the inner external granule layer. These findings suggest that Meis1 regulates degradation of Atoh1 via BMP signaling, contributing to GC differentiation in the inner EGL, and should provide understanding into GC development., (Copyright © 2018 the authors 0270-6474/18/381278-18$15.00/0.)

Bone loss is a serious clinical issue in patients with cerebral palsy (CP). Sclerostin has garnered interest as a key mechanosensor in osteocytes, leading to considerations of the therapeutic utilization of anti-sclerostin medications. This study was undertaken to determine associations among mechanical unloading, sclerostin levels, and bone imbalance in patients with CP. A total of 28 patients with CP participated in this cross-sectional study. The following measurements were taken: anthropometrics, clinical diagnosis of CP subtype and ambulatory status, bone mineral density (BMD) z-scores at the lumbar spine and hip, and blood biochemical markers, including sclerostin, parathyroid hormone (PTH), osteocalcin, C-terminal telopeptide, 25-hydroxyvitamin D, 1,25-dihydroxyvitamin D, creatinine, calcium, and phosphorus. In analysis according to CP subtype, patients with spastic CP showed significantly lower BMD z-scores at the lumbar spine and femur neck regions than patients with dyskinetic CP. In analysis according to ambulatory status, patients with non-ambulatory CP showed significantly lower BMD z-scores at all lumbar spine and femoral sites, lower PTH and creatinine levels, and higher plasma sclerostin levels than patients with ambulatory CP. In regression analysis, ambulatory status was a significant determinant of plasma sclerostin levels. This study is the first to report on sclerostin levels and BMD in patients with CP, based on the hypothesis that patients who lack sufficient weight-bearing activities would show increased sclerostin levels and decreased BMD scores, compared with patients who sustain relatively sufficient physical activity. Therefore, this report may provide clinical insights for clinicians considering ambulatory status, sclerostin levels, and bone loss in patients with CP., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

Growth differentiation factor 11 (GDF11), a member of the transforming growth factor β (TGF-β) family, plays diverse roles in mammalian development. It is synthesized as a large, inactive precursor protein containing a prodomain, pro-GDF11, and exists as a homodimer. Activation requires two proteolytic processing steps that release the prodomains and transform latent pro-GDF11 into active mature GDF11. In studying proteolytic activation in vitro, we discovered that a 6-kDa prodomain peptide containing residues 60-114, PDP 60-114 , remained associated with the mature growth factor. Whereas the full-length prodomain of GDF11 is a functional antagonist, PDP 60-114 had no impact on activity. The specific activity of the GDF11/PDP 60-114 complex (EC 50 = 1 nM) in a SMAD2/3 reporter assay was identical to that of mature GDF11 alone. PDP 60-114 improved the solubility of mature GDF11 at neutral pH. As the growth factor normally aggregates/precipitates at neutral pH, PDP 60-114 can be used as a solubility-enhancing formulation. Expression of two engineered constructs with PDP 60-114 genetically fused to the mature domain of GDF11 through a 2x or 3x G4S linker produced soluble monomeric products that could be dimerized through redox reactions. The construct with a 3x G4S linker retained 10% activity (EC 50 = 10 nM), whereas the construct connected with a 2x G4S linker could only be activated (EC 50 = 2 nM) by protease treatment. Complex formation with PDP 60-114 represents a new strategy for stabilizing GDF11 in an active state that may translate to other members of the TGF-β family that form latent pro/mature domain complexes.

Osseointegration is the key to implant stability and occlusal support. Biomechanical response and remodeling of peri-implant bone occurs under impact loading. Sclerostin participates in bone formation and resorption through Wnt and RANKL pathways. However the mechanism of microdamage and expression of sclerostin in peri-implant bone under impact load is still unclear. In present study, specific impact forces were applied to the implants with favorable osseointegration in rabbits. The microdamage of peri-implant bone and the expression of sclerostin, β-catenin and RANKL during the process of bone damage and remodeling were investigated by micro-CT, histology, immunofluorescence and RT-qPCR analysis. Interface separation and trabecular fracture were found histologically, which were consistent with micro-CT analyses. Throughout remodeling, bone resorption was observed during the first 14 days after impact, and osseointegration and normal trabecular structure were found by 28 d. The expression of sclerostin and RANKL increased after impact and reached a maximum by 14 d, then decreased gradually to normal levels by 28 d. And β-catenin expression was opposite. Results indicated that sclerostin may involve in the peri-implant bone damage caused by impact and remodeling through Wnt/β-catenin and RANKL/RANK pathways. It will provide a new insight in the diagnosis and treatment for patients suffering impact.

Multiple signaling pathways guide the behavior and differentiation of both germline stem cells (GSCs) and somatic follicle stem cells (FSCs) in the Drosophila germarium, necessitating careful control of signal generation, range and responses. Signal integration involves escort cells (ECs), which promote differentiation of the GSC derivatives they envelop, provide niche signals for FSCs and derive directly from FSCs in adults. Hedgehog (Hh) signaling induces the Hippo pathway effector Yorkie (Yki) to promote proliferation and maintenance of FSCs, but Hh also signals to ECs, which are quiescent. Here, we show that in ECs both Hh and Yki limit production of BMP ligands to allow germline differentiation. Loss of Yki produced a more severe germarial phenotype than loss of Hh signaling and principally induced a different BMP ligand. Moreover, Yki activity reporters and epistasis tests showed that Yki does not mediate the key actions of Hh signaling in ECs. Thus, both the coupling and output of the Hh and Yki signaling pathways differ between FSCs and ECs despite their proximity and the fact that FSCs give rise directly to ECs., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2017. Published by The Company of Biologists Ltd.)

Purpose: Bariatric surgery has been associated with bone remodeling changes. The action of adipokines on the expression of receptor activator of nuclear factor kappa β ligand (RANKL) and osteoprotegerin (OPG) and on an increase in sclerostin could be related to these changes., Materials and Methods: This study aimed to assess the repercussions of weight loss, fat mass (FM), and fat-free mass (FFM) loss and biochemical and hormonal changes on bone remodeling markers after Roux-en-Y gastric bypass (RYGB). Anthropometric data, parathyroid hormone (PTH), bone-specific alkaline phosphatase (BSAP), collagen type 1 C-telopeptide (CTX), 25-hydroxy vitamin D (25-OH-VitD), leptin, adiponectin, RANKL, OPG, and sclerostin of 30 menstruating women were measured preoperatively (Pre), and 3, 12, and 24 months (m) after RYGB., Results: Leptin (34.4 (14.7; 51.9) vs. 22.5 (1.9; 52.7) ng/mL) and OPG (3.6 (1.1; 11.5) vs. 3.4 (1.5; 6) pmol/L) decreased, and adiponectin (7.4 (1.7; 18.4) vs. 13.8 (3.0; 34.6) μg/mL), CTX (0.2 (0.1; 2.2) vs. 0.6 (0.4; 6.0) ng/mL), RANKL (0.1 (0.0; 0.5) vs. 0.3 (0.0; 2.0) pmol/L), and sclerostin (21.7 (3.2; 75.1) vs. 34.8 (6.4; 80.5) pmol/L) increased after 3 m. BSAP increased after 12 m (10.1 (5.4; 18.9) vs. 13.9 (6.9; 30.2) μg/mL) (p < 0.005). CTX correlated positively with adiponectin at 24 m and inversely with leptin Pre; OPG at 3 m; weight, FM, FFM, and leptin at 24 m. RANKL correlated directly with weight at 3 m. Sclerostin correlated inversely with weight Pre and FM at 3 m. BSAP correlated negatively with 25-OH-VitD at 12 m, and positively with PTH at 24 m., Conclusions: RYGB induced weight loss, and biochemical, hormonal, and body composition changes are associated with higher bone remodeling.

Growth and differentiation factor (GDF) 11 is a member of the transforming growth factor β superfamily recently identified as a potential therapeutic for age-related cardiac and skeletal muscle decrements, despite high homology to myostatin (Mstn), a potent negative regulator of muscle mass. Though several reports have refuted these data, the in vivo effects of GDF11 on skeletal muscle mass have not been addressed. Using in vitro myoblast culture assays, we first demonstrate that GDF11 and Mstn have similar activities/potencies on activating p-SMAD2/3 and induce comparable levels of differentiated myotube atrophy. We further demonstrate that adeno-associated virus-mediated systemic overexpression of GDF11 in C57BL/6 mice results in substantial atrophy of skeletal and cardiac muscle, inducing a cachexic phenotype not seen in mice expressing similar levels of Mstn. Greater cardiac expression of Tgfbr1 may explain this GDF11-specific cardiac phenotype. These data indicate that bioactive GDF11 at supraphysiological levels cause wasting of both skeletal and cardiac muscle. Rather than a therapeutic agent, GDF11 should be viewed as a potential deleterious biomarker in muscle wasting diseases., (© 2017 The Authors. Published under the terms of the CC BY 4.0 license.)

To define a complete catalog of the genes that are activated during mouse sclerotome formation, we sequenced RNA from embryonic mouse tissue directed to form sclerotome in culture. In addition to well-known early markers of sclerotome, such as Pax1, Pax9, and the Bapx2/Nkx3-2 homolog Nkx3-1, the long-noncoding RNA PEAT (Pax1 enhancer antisense transcript) was induced in sclerotome-directed samples. Strikingly, PEAT is located just upstream of the Pax1 gene. Using CRISPR/Cas9, we generated a mouse line bearing a complete deletion of the PEAT-transcribed unit. RNA-seq on PEAT mutant embryos showed that loss of PEAT modestly increases bone morphogenetic protein target gene expression and also elevates the expression of a large subset of ribosomal protein mRNAs., Competing Interests: The authors declare no conflict of interest.

Background: Abusive alcohol utilization of pregnant woman may cause congenital heart disease (CHD) of fetus, where alcohol ignites histone H3 hyperacetylation leading to abnormal development of heart morphogenesis and associated genes. Knowledge about the regularized upstream genes is little, but bone morphogenetic protein (BMP) signaling may actively and prominently take part in alteration in acetylation of histone H3. The supreme objective of this study was to unearth the involvement of BMP signaling pathway in alcohol-driven hyperacetylation of histone H3 in cardiomyoblast cells., Methods: Cardiomyoblast cells (H9c2 cells) were addicted with alcohol (100 mM) for 24 hours. Dorsomorphin (5 μM) was used for the inhibition of BMP signaling pathway. We detected the phosphorylation activity of SMAD1/5/8, mRNA expression, histone acetyltransferases (HAT)/histone deacetylase (HDAC) activity, and acetylation of histone H3., Results: Following alcohol exposure, phosphorylation of SMAD1/5/8 and HAT activities was increased to a significant extent, while histone H3 acetylation and expression of heart development-related genes were also increased. The said phenomenon influenced by alcohol was reverted upon dorsomorphin treatment to the cells without effecting HDAC activity., Conclusions: The data clearly identified that BMP-mediated histone H3 acetylation of heart development-related genes might be one of the possible cellular mechanisms to control alcohol-induced expression of heart development-related genes. Dorsomorphin, on the other hand, may modulate alcohol-induced hyperacetylation of histone H3 through BMP targeting, which could be a potential way to block CHD., (Copyright © 2016 by the Research Society on Alcoholism.)

Objective. Current studies of serum sclerostin levels in AS and RA patients are inconsistent. This meta-analysis was performed to identify the association of serum sclerostin level with AS and RA patients. Methods. Embase, PubMed, MEDLINE, and Cochrane Library databases (up to 25 January 2017) were used to collect all relevant published articles. Studies were pooled and standard mean difference (SMD) with 95% confidence interval (CI) was calculated. All data analyses were performed using RevMan 5.3. Results. Totally eight studies of AS including 420 AS patients and 317 healthy controls (HC) and three studies of RA including 145 RA patients and 127 HC were finally included in this meta-analysis. The results revealed that the serum sclerostin levels in both AS patients (SMD = -0.14; 95% CI = [-0.39,0.11]; P = 0.28) and RA patients (SMD = -0.10; 95% CI = [-0.34,0.15]; P = 0.43) were not significantly different from those in HC. Conclusion. The difference of serum sclerostin levels in AS and RA patients was not significantly different from HC, indicating that the sclerostin may not associate with the development of AS and RA.

Chondrogenesis can regulate bone formation. Fibroblast growth factor receptor 3, highly expressed in chondrocytes, is a negative regulator of bone growth. To investigate whether chondrocyte FGFR3 regulates osteogenesis, thereby contributing to postnatal bone formation and bone remodeling, mice with conditional knock-out of Fgfr3 in chondrocytes (mutant (MUT)) were generated. MUT mice displayed overgrowth of bone with lengthened growth plates. Bone mass of MUT mice was significantly increased at both 1 month and 4 months of age. Histological analysis showed that osteoblast number and bone formation were remarkably enhanced after deletion of Fgfr3 in chondrocytes. Chondrocyte-osteoblast co-culture assay further revealed that Fgfr3 deficiency in chondrocytes promoted differentiation and mineralization of osteoblasts by up-regulating the expressions of Ihh, Bmp2, Bmp4, Bmp7, Wnt4, and Tgf-β1, as well as down-regulating Nog expression. In addition, osteoclastogenesis was also impaired in MUT mice with decreased number of osteoclasts lining trabecular bone, which may be related to the reduced ratio of Rankl to Opg in Fgfr3-deficient chondrocytes. This study reveals that chondrocyte FGFR3 is involved in the regulation of bone formation and bone remodeling by a paracrine mechanism., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

SOST encodes sclerostin, an inhibitor of bone formation that antagonizes canonical Wnt signaling. Variations of SOST expression have an impact on bone mineral density (BMD) and bone strength. We hypothesized that genetic and epigenetic DNA modifications have an impact on SOST gene expression. By analyzing 43 bone samples from women, we found that rs851054 (G/A) is associated with SOST mRNA expression, donors with one or two G allele(s) displaying higher SOST expression (p<0.05). Beside this polymorphism, we also investigated the role of CpG methylation in SOST mRNA expression, and analyzed methylation variation at 13 CpG sites on the 1st exon of SOST in 14 human bone samples. Principal component analysis identified three groups of CpG sites that explained most of the methylation variation. We calculated the percentage of methylation in the main group of CpGs, and showed that higher rates of methylated CpGs are associated with higher SOST mRNA expression. To demonstrate that change in SOST expression might be related to human bone disease, we analyzed 131 patients with osteogenesis imperfecta (OI), a rare disease characterized by low BMD, bone fragility, and marked intra-familial variability of bone phenotypes. We found an association between rs851054 of the SOST promoter and the fracture rate only during childhood (p<0.01). In conclusion, genetic and epigenetic changes contribute to variation in SOST expression in human bone. Our data also indicate that these variations may be related to the severity of OI., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Aims: To assess the immunophenotypic and mRNA expression of sclerostin in human skeletal tissues and in a wide range of benign and malignant bone tumours and tumour-like lesions., Methods and Results: Sclerostin expression was evaluated by immunohistochemistry and quantitative polymerase chain reaction (PCR). In lamellar and woven bone, there was strong sclerostin expression by osteocytes. Osteoblasts and other cell types in bone were negative. Hypertrophic chondrocytes in the growth plate and mineralized cartilage cells in zone 4 of hyaline articular cartilage strongly expressed sclerostin, but most chondrocytes in hyaline cartilage were negative. In primary bone-forming tumours, including osteosarcomas, there was patchy expression of sclerostin in mineralized osteoid and bone. Sclerostin staining was seen in woven bone in fibrous dysplasia, in osteofibrous dysplasia, and in reactive bone formed in fracture callus, in myositis ossificans, and in the wall of solitary bone cysts and aneurysmal bone cysts. Sclerostin was expressed by hypertrophic chondrocytes in osteochondroma and chondroblasts in chondroblastoma, but not by tumour cells in other bone tumours, including myeloma and metastatic carcinoma. mRNA expression of sclerostin was identified by quantitative PCR in osteosarcoma specimens and cell lines., Conclusions: Sclerostin is an osteocyte marker that is strongly expressed in human woven and lamellar bone and mineralizing chondrocytes. This makes it a useful marker with which to identify benign and malignant osteogenic tumours and mineralizing cartilage tumours, such as chondroblastomas and other lesions in which there is bone formation., (© 2016 John Wiley & Sons Ltd.)

The BMP signaling pathway has a conserved role in dorsal-ventral axis patterning during embryonic development. In Drosophila, graded BMP signaling is transduced by the Mad transcription factor and opposed by the Brinker repressor. In this study, using the Drosophila embryo as a model, we combine RNA-seq with Mad and Brinker ChIP-seq to decipher the BMP-responsive transcriptional network underpinning differentiation of the dorsal ectoderm during dorsal-ventral axis patterning. We identify multiple new BMP target genes, including positive and negative regulators of EGF signaling. Manipulation of EGF signaling levels by loss- and gain-of-function studies reveals that EGF signaling negatively regulates embryonic BMP-responsive transcription. Therefore, the BMP gene network has a self-regulating property in that it establishes a balance between its activity and that of the antagonistic EGF signaling pathway to facilitate correct patterning. In terms of BMP-dependent transcription, we identify key roles for the Zelda and Zerknüllt transcription factors in establishing the resulting expression domain, and find widespread binding of insulator proteins to the Mad and Brinker-bound genomic regions. Analysis of embryos lacking the BEAF-32 insulator protein shows reduced transcription of a peak BMP target gene and a reduction in the number of amnioserosa cells, the fate specified by peak BMP signaling. We incorporate our findings into a model for Mad-dependent activation, and discuss its relevance to BMP signal interpretation in vertebrates.

Altered expression of the E3 ubiquitin ligase UBE3A, which is involved in protein degradation through the proteasome-mediated pathway, is associated with neurodevelopmental and behavioral defects observed in Angelman syndrome (AS) and autism. However, little is known about the neuronal function of UBE3A and the pathogenesis of UBE3A-associated disorders. To understand the in vivo function of UBE3A in the nervous system, we generated multiple mutations of ube3a, the Drosophila ortholog of UBE3A. We found a significantly increased number of total boutons and satellite boutons in conjunction with compromised endocytosis in the neuromuscular junctions (NMJs) of ube3a mutants compared to the wild type. Genetic and biochemical analysis showed upregulation of bone morphogenetic protein (BMP) signaling in the nervous system of ube3a mutants. An immunochemical study revealed a specific increase in the protein level of Thickveins (Tkv), a type I BMP receptor, but not other BMP receptors Wishful thinking (Wit) and Saxophone (Sax), in ube3a mutants. Ube3a was associated with and specifically ubiquitinated lysine 227 within the cytoplasmic tail of Tkv, and promoted its proteasomal degradation in Schneider 2 cells. Negative regulation of Tkv by Ube3a was conserved in mammalian cells. These results reveal a critical role for Ube3a in regulating NMJ synapse development by repressing BMP signaling. This study sheds new light onto the neuronal functions of UBE3A and provides novel perspectives for understanding the pathogenesis of UBE3A-associated disorders.

Objective: During orthodontic tooth movement, bone resorption and inhibition of bone formation occur on the compressed side, thereby preventing ankylosis. Periodontal ligament (PDL) cells control bone metabolism and inhibition of bone formation on the compressed side by secreting bone-formation inhibitory factors such as asporin (ASPN) or sclerostin (encoded by SOST). The aim of this study was to identify the inhibitory factors of bone formation in PDL cells., Design: In vitro, the changes in expression of ASPN and SOST and subsequent protein release in human PDL (hPDL) cells were assessed by semi-quantitative polymerase chain reaction (PCR), real-time PCR, and immunofluorescence in hPDL cells subjected to centrifugal force using a centrifuge (45, 90, 135, and 160 × g). In vivo, we applied a compressive force using the Waldo method in rats, and examined the distribution of ASPN or sclerostin by immunohistochemistry., Results: In vitro, hPDL cells subjected to 90 × g for 24h demonstrated upregulated ASPN and downregulated SOST expressions, which were confirmed by immunofluorescent staining. In addition, the formation of mineralized tissue by human osteoblasts was significantly inhibited by the addition of medium from hPDL cells cultured during compressive force as well as the addition of equivalent amounts of ASPN peptide. In vivo, asporin-positive immunoreactive PDL cells and osteoclasts were found on the compressed side, whereas few sclerostin-positive PDL cells were observed., Conclusions: PDL cells subjected to an optimal compressive force induce the expression and release of ASPN, which inhibits bone formation during orthodontic tooth movement on the compressed side., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Both bone morphogenetic protein (BMP) and Wnt signaling have significant roles in osteoblast differentiation and the interaction between BMP and Wnt signaling is well known. Sclerostin is an important inhibitor of bone formation, inhibiting Wnt signaling and downstream effects of BMP such as alkaline phosphatase activity and matrix mineralization in vitro. However, little is known about the effect of BMP and Wnt signaling interaction on the regulation of SOST, the gene encoding sclerostin. Possibly, uncoupling of osteoblast differentiation regulators and SOST expression could increase osteoblast differentiation. Therefore, we investigated the effect of BMP and Wnt signaling interaction on the expression of SOST and the subsequent effect on osteoblast differentiation. Human osteosarcoma cells (SaOS-2) and murine pre-osteoblast cells (KS483) were treated with different concentrations of Wnt3a, a specific GSK3β inhibitor (GIN) and BMP4. Both Wnt3a and GIN increased BMP4-induced BMP signaling and BMP4 increased Wnt3a and GIN-induced Wnt signaling. However, the effect of GIN was much stronger. Quantitative RT-PCR analysis showed that SOST expression dose-dependently decreased with increasing Wnt signaling, while BMP4 induced SOST expression. GIN significantly decreased the BMP4-induced SOST expression. This resulted in an increased osteoblast differentiation as measured by ALP activity in the medium and matrix mineralization. We conclude that GSK3β inhibition by GIN caused an uncoupling of BMP signaling and SOST expression, resulting in an increased BMP4-induced osteoblast differentiation. This effect can possibly be used in clinical practice to induce local bone formation, for example, fracture healing or osseointegration of implants., (© 2015 Wiley Periodicals, Inc.)

To explore how limbal niche cells (LNCs) may control quiescence, self-renewal, and corneal epithelial lineage commitment/differentiation of limbal epithelial progenitor/stem cells (LEPCs), we have established an in vitro sphere assay by reunion between the two cell types in three-dimensional Matrigel. The resultant sphere exhibits inhibition of corneal epithelial lineage commitment/differentiation and marked clonal growth of LEPCs, of which the latter is correlated with activation of canonical Wnt signaling. Herein, we have created a similar reunion assay in immobilized heavy chain-hyaluronic acid/pentraxin 3 (HC-HA/PTX3), which is purified from amniotic membrane (AM) and consists of a complex formed by hyaluronic covalently linked to heavy chain 1 of inter-α-inhibitor and noncovalently linked to pentraxin 3. The resultant spheres exhibited similar suppression of corneal epithelial lineage commitment/differentiation but upregulation of quiescence markers including nuclear translocation of Bmi-1, and negligible clonal growth of LEPCs. This outcome was correlated with the suppression of canonical Wnt but activation of noncanonical (Planar cell polarity) Wnt signaling as well as BMP signaling in both LEPCs and LNCs. The activation of BMP signaling in LNCs was pivotal because nuclear translocation of pSmad1/5/8 was prohibited in hLEPCs when reunioned with mLNCs of conditionally deleted Bmpr1a;Acvr1(DCKO) mice. Furthermore, ablation of BMP signaling in LEPCs led to upregulation of cell cycle genes, downregulation of Bmi-1, nuclear exclusion of phosphorylated Bmi-1, and marked promotion of the clonal growth of LEPCs. Hence, HC-HA/PTX3 uniquely upregulates BMP signaling in LNCs which leads to BMP signaling in LEPCs to achieve quiescence, helping explain how AM transplantation is clinically useful to be used as a matrix for ex vivo expansion of LEPCs and to treat corneal blindness caused by limbal stem cells deficiency., (© 2015 AlphaMed Press.)

Background: Aortic valve calcification is characterized as the active process of aortic valve interstitial cells (AVICs), and considered as an inflammatory disease. As an antioxidant, the anti-inflammatory activity of Lazaroid has been exhibited in various models. We hypothesized that Lazaroid U-74389G would inhibit the osteoblastic differentiation of AVICs induced by IL-1β., Methods: Normal tricuspid aortic valve leaflets were collected from patients with acute aortic dissection (Type A) undergoing the Bentall procedure. AVICs were isolated and stimulated with IL-1β in presence or absence of U-74389G in culture. Cell lysates were analyzed for osteogenic markers and nuclear factor-κB using real-time PCR and Immunoblotting. Culture media was analyzed for IL-6 and IL-8 with enzyme-linked immunosorbent assay. Alizarin Red Staining was adopted to demonstrate the calcium deposition., Results: The expression of alkaline phosphatase and bone morphogenetic protein, accompanied by the production of IL-6 and IL-8, was up-regulated in response to IL-1β and was inhibited by the addition of U-74389G. The NF-κB pathway was activated by IL-1β and involved in the suppression of U-74389G on osteoblastic differentiation in AVICs. The negative effects of U-74389G on ostengenic gene expression and mineralization of AVICs were blocked by glucocorticoid receptor antagonist mifepristone and the NF-κB inhibitor Bay 11-7082., Conclusions: U-74389G inhibits the pro-osteogenic response to IL-1β stimulation in AVICs. The osteoblastic differentiation and mineralization of AVICs were inhabited by U-74389G though the modulation of NF-κB activation, and this pathway could be potential therapeutic targets for medical treatment of calcified aortic valve disease., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Introduction: Bone morphogenetic proteins (BMPs) are multifunctional secreted growth factors regulating a broad spectrum of functions in numerous systems. An increased expression and production of specific BMPs have been described in the rheumatoid arthritis (RA) synovium. The aim of this study was to analyze the involvement of the BMP signaling pathway in RA synoviocytes in response to interleukin-17 (IL-17) and tumor necrosis factor-alpha (TNF-α)., Methods: The expression of components of the BMP signaling pathway (BMP receptors, BMP ligands, BMP signal transducers, and BMP antagonists) was analyzed by quantitative polymerase chain reaction before and after treatment of RA synoviocytes with TNF-α or IL-17 or both. Regulation was studied in the presence of the specific BMP inhibitor DMH1 (dorsomorphin homologue 1) or an exogenous BMP ligand, BMP6. Expression and production of pro-inflammatory cytokines (IL-6 and granulocyte-macrophage colony-stimulating factor), chemokines (IL-8, CCL2, CCL5, and CXCL10), and matrix metalloproteinases (MMP-1, -2, -3, -9, and -13) were analyzed., Results: RA synoviocytes express BMP receptors (mainly BMPRIA, ACTRIA, and BMPRII), signal transducers of the Smad family (Smad1 and 5 and co-Smad4), and different BMP antagonists. The modulation of the expression of the BMP target genes-Id (inhibitor of DNA-binding/differentiation) proteins and Runx (Runt-related transcription factor) transcription factors-after the addition of exogenous BMP shows that the BMP signaling pathway is active. RA synoviocytes also express BMP ligands (BMP2, BMP6, and BMP7) which are highly upregulated after activation with TNF-α and IL-17. Autocrine BMP signaling pathway can be blocked by treatment with the inhibitor DMH1, leading to an increase in the upregulated expression of pro-inflammatory cytokines, chemokines, and MMPs induced by the activation of RA synoviocytes with TNF-α and IL-17. Conversely, the additional stimulation of the BMP pathway with the exogenous addition of the BMP6 ligand decreases the expression of those pro-inflammatory and pro-destructive factors., Conclusion: The results indicate that the canonical BMP pathway is functionally active in human RA synoviocytes and that the inhibition of autocrine BMP signaling exacerbates the pro-inflammatory phenotype induced in RA synoviocytes by the stimulation with IL-17 and TNF-α.

Introduction: Sclerostin (SOST), a soluble antagonist of Wnt signaling, is expressed in chondrocytes and contributes to chondrocytes' hypertrophic differentiation; however its role in osteoarthritis (OA) pathogenesis is not well known. Based on our previous findings on the interaction between Wnt/β-catenin pathway and BMP-2 in OA, we aimed to investigate the role of DNA methylation and BMP-2 on SOST's expression in OA chondrocytes., Methods: SOST mRNA and protein expression levels were investigated using real-time polymerase chain reaction (PCR) and Western blot, respectively. The methylation status of SOST promoter was analysed using methylation-specific PCR (MSP), quantitative methylation-specific PCR (qMSP) and bisulfite sequencing analysis. The effect of BMP-2 and 5'-Aza-2-deoxycytidine (5-AzadC) on SOST's expression levels were investigated and Smad1/5/8 binding to SOST promoter was assessed by Chromatin Immunoprecipitation (ChΙP)., Results: We observed that SOST's expression was upregulated in OA chondrocytes compared to normal. Moreover, we found that the CpG region of SOST promoter was hypomethylated in OA chondrocytes and 5-AzadC treatment in normal chondrocytes resulted in decreased SOST methylation, whereas its expression was upregulated. BMP-2 treatment in 5-AzadC-treated normal chondrocytes resulted in SOST upregulation, which was mediated through Smad 1/5/8 binding on the CpG region of SOST promoter., Conclusions: We report novel findings that DNA methylation regulates SOST's expression in OA, by changing Smad 1/5/8 binding affinity to SOST promoter, providing evidence that changes in DNA methylation pattern could underlie changes in genes' expression observed in OA.

The skeleton responds to mechanical stimulation. We wished to ascertain the magnitude and speed of the growing skeleton's response to a standardised form of mechanical stimulation, vibration. 36 prepubertal boys stood for 10 minutes in total on one of two vibrating platforms (high (>2 g) or low (<1 g) magnitude vibration) on either 1, 3 or 5 successive days (n=12 for each duration); 15 control subjects stood on an inactive platform. Blood samples were taken at intervals before and after vibration to measure bone formation (P1NP, osteocalcin) and resorption (CTx) markers as well as osteoprotegerin and sclerostin. There were no significant differences between platform and control groups in bone turnover markers immediately after vibration on days 1, 3 and 5. Combining platform groups, at day 8 P1NP increased by 25.1% (CI 12.3 to 38.0; paired t-test p=0.005) and bone resorption increased by 10.9% (CI 3.6 to 18.2; paired t-test p=0.009) compared to baseline. Osteocalcin, osteoprotogerin and sclerostin did not change significantly. The growing skeleton can respond quickly to vibration of either high or low magnitude. Further work is needed to determine the utility of such "stimulation-testing" in clinical practice., Competing Interests: The authors have no conflict of interest.

Uterus circadian rhythms have been implicated in the gestation processes of mammals through entraining of the clock proteins to numerous downstream genes. Bone morphogenetic proteins (BMPs), having clock-controlled regulatory sites in their gene promoters, are expressed in the uterus during decidualization, but the regulation of the Bmp gene expression is poorly understood. The present study was designed to dissect the physiological roles of the uterus oscillators in the Bmp expression using the uterus endometrial stromal cells (UESCs) isolated from Per2-dLuc transgenic rats on day 4.5 of gestation. The in vitro decidualization of UESCs was induced by medroxyprogesterone acetate and 2-O-dibutyryl cAMP. A significant decline of Per2-dLuc bioluminescence activity was induced in decidual cells, and concomitantly, the expression of canonical clock genes was downregulated. Conversely, the expression of the core Bmp genes Bmp2, Bmp4, Bmp6, and Bmp7 was upregulated. In UESCs transfected with Bmal1-specific siRNA, in which Rev-erbα expression was downregulated, Bmp genes, such as Bmp2, Bmp4, and Bmp6 were upregulated. However, Bmp1, Bmp7, and Bmp8a were not significantly affected by Bmal1 silencing. The expression of all Bmp genes was enhanced after treatment with the REV-ERBα antagonist (SR8278), although their rhythmic profiles were differed from each other. The binding of REV-ERBα to the proximal regions of the Bmp2 and Bmp4 promoters was revealed by chromatin immunoprecipitation-PCR analysis. Collectively, these results indicate that the Bmp genes are upregulated by the attenuation of the cellular circadian clock; in particular, its core component REV-ERBα functions as a transcriptional silencer in the Bmp gene family., (Copyright © 2015 the American Physiological Society.)

Tissue homeostasis requires balanced self-renewal and differentiation of stem/progenitor cells, especially in tissues that are constantly replenished like the esophagus. Disruption of this balance is associated with pathological conditions, including eosinophilic esophagitis (EoE), in which basal progenitor cells become hyperplastic upon proinflammatory stimulation. However, how basal cells respond to the inflammatory environment at the molecular level remains undetermined. We previously reported that the bone morphogenetic protein (BMP) signaling pathway is critical for epithelial morphogenesis in the embryonic esophagus. Here, we address how this pathway regulates tissue homeostasis and EoE development in the adult esophagus. BMP signaling was specifically activated in differentiated squamous epithelium, but not in basal progenitor cells, which express the BMP antagonist follistatin. Previous reports indicate that increased BMP activity promotes Barrett's intestinal differentiation; however, in mice, basal progenitor cell-specific expression of constitutively active BMP promoted squamous differentiation. Moreover, BMP activation increased intracellular ROS levels, initiating an NRF2-mediated oxidative response during basal progenitor cell differentiation. In both a mouse EoE model and human biopsies, reduced squamous differentiation was associated with high levels of follistatin and disrupted BMP/NRF2 pathways. We therefore propose a model in which normal squamous differentiation of basal progenitor cells is mediated by BMP-driven NRF2 activation and basal cell hyperplasia is promoted by disruption of BMP signaling in EoE.

Peroxisome proliferator activated receptor gamma (PPARγ) has been known for their anti-inflammatory effects. But the application of this molecule in implant-induced inflammation has not been clearly studied yet. Here, we determined in vivo anti-inflammatory and osteogenic effects of PPARγ coated dental implant in the rat mandible. We used chitosan gold nanoparticles (Ch-GNPs) as a non viral vector to carry PPARγ plasmid DNA. Ch-GNPs were conjugated with PPARγ plasmid DNA through a coacervation process. Conjugation was cast over titanium (Ti) implants (4.5 × 0.8 mm) by dipping, and implants were installed in rat mandibles. One, 2, 3, and 6 weeks post-implantation, mandibles were examined by microcomputed tomography (µCT), immunohistochemistry, hematoxylin & eosin, and tartrate resistance acid phosphatase (TRAP) staining. In vivo Ch-GNPs/PPARγcoated implants were associated with inhibition of implant induced inflammatory molecules interleukin-1β and receptor activator of nuclear factor kappa-B ligand and enhanced expression of osteogenic molecules like bone morphogenetic protein 2 and 7 (BMP-2/-7) by up-regulating anti-oxidant molecules heme oxygenase-1. µCT demonstrated that PPARγ overexpression increased the density and volume of newly formed bone surrounding the implants compared to control (n = 4; p < 0.05). Also, PPARγ reduced the number of TRAP positive cells. These results support the view that PPARγ overexpression diminishes inflammation and enhances osteogenesis around the dental implants. Thus, implant coated with anti-inflammatory molecules could have a significant utilization for the preparation of new biomaterials and may serve as prosthetic materials in patients suffering from inflammatory bone disease., (© 2014 Wiley Periodicals, Inc.)

Introduction: Pathological bone changes differ considerably between inflammatory arthritic diseases and most studies have focused on bone erosion. Collagen-induced arthritis (CIA) is a model for rheumatoid arthritis, which, in addition to bone erosion, demonstrates bone formation at the time of clinical manifestations. The objective of this study was to use this model to characterise the histological and molecular changes in bone remodelling, and relate these to the clinical disease development., Methods: A histological and gene expression profiling time-course study on bone remodelling in CIA was linked to onset of clinical symptoms. Global gene expression was studied with a gene chip array system., Results: The main histopathological changes in bone structure and inflammation occurred during the first two weeks following the onset of clinical symptoms in the joint. Hereafter, the inflammation declined and remodelling of formed bone dominated. Global gene expression profiling showed simultaneous upregulation of genes related to bone changes and inflammation in week 0 to 2 after onset of clinical disease. Furthermore, we observed time-dependent expression of genes involved in early and late osteoblast differentiation and function, which mirrored the histopathological bone changes. The differentially expressed genes belong to the bone morphogenetic pathway (BMP) and, in addition, include the osteoblast markers integrin-binding sialoprotein (Ibsp), bone gamma-carboxyglutamate protein (Bglap1), and secreted phosphoprotein 1 (Spp1). Pregnancy-associated protein A (Pappa) and periostin (Postn), differentially expressed in the early disease phase, are proposed to participate in bone formation, and we suggest that they play a role in early bone formation in the CIA model. Comparison to human genome-wide association studies (GWAS) revealed differential expression of several genes associated with human arthritis., Conclusions: In the CIA model, bone formation in the joint starts shortly after onset of clinical symptoms, which results in bony fusion within one to two weeks. This makes it a candidate model for investigating the relationship between inflammation and bone formation in inflammatory arthritis.

Objective: To explore the effects of Elemene, the essential oil of Curcuma wenyujin, on Bone morphogenetic protein/drosophila mothers against decapentaplegic proteins (BMP/SMADs) signal pathway in ankylosing spondylitis (AS) fibroblasts., Methods: Hip joint capsules were obtained from AS patients (n=10) receiving total hip replacement. Healthy hip joint capsules from patients with hip fracture (n=10) receiving surgery were included as a control. Primary fibroblast cell lines were established from these tissue samples. Fibroblasts were incubated with Elemene for 48 hours. The protein expression was detected by Western blot. The mRNA expression was detected by real-time fluorescent quantitative polymerase chain reaction (PCR)., Results: The results showed that the expression of proteins including SMAD1, pSMAD1, SMAD4 and Runt-related transcription factor 2 (RUNX2), and mRNA of RUNX2, which were over-expressed in AS fibroblasts were decreased in the AS fibroblasts cultured in medium with Elemene., Conclusions: Ele could have a hand in anti-osteogenic differentiation of AS fibroblasts by inhibiting the BMP/SMADs signal pathway and subsequently blocking expression of ossification marker genes RUNX2 that initiate the osteogenic differentiation., (Copyright © 2014 Société française de rhumatologie. Published by Elsevier SAS. All rights reserved.)

Background: Sclerostin, encoded by the SOST gene, has been implicated in the response to mechanical loading in bone. Some studies demonstrated that unloading leads to up-regulated SOST expression, which may induce bone loss., Purpose: Most reported studies regarding the changes caused by mechanical unloading were only based on a single site. Considering that the longitudinal bone growth leads to cells of different age with different sensitivity to unloading, we hypothesized that bone turnover in response to unloading is site specific., Methods: We established a disuse rat model by sciatic neurectomy in tibia. In various regions at two time-points, we evaluated the bone mass and microarchitecture in surgically-operated rats and control rats by micro-Computed Tomography (micro-CT) and histology, sclerostin/SOST by immunohistochemistry, enzyme-linked immunosorbent assay (ELISA), and quantitative reverse transcription polymerase chain reaction (qPCR), tartrate resistant acid phosphatase 5b (TRAP 5b) by ELISA and TRAP staining, and other bone markers by ELISA., Results: Micro-CT and histological analysis confirmed bone volume in the disuse rats was significantly decreased compared with those in the time-matched control rats, and microarchitecture also changed 2 and 8 weeks after surgery. Compared with the control groups, SOST mRNA expression in the diaphysis was down-regulated at both week 2 and 8. On the contrary, the percentage of sclerostin-positive osteocytes showed an up-regulated response in the 5 - 6 mm region away from the growth plate, while in the 2.5 - 3.5 mm region, the percentage was no significant difference. Nevertheless, in 0.5 - 1.5 mm region, the percentage of sclerostin-positive osteocytes decreased after 8 weeks, consistent with serum SOST level. Besides, the results of TRAP also suggested that the expression in response to unloading may be opposite in different sites or system., Conclusion: Our data indicated that unloading-induced changes in bone turnover are probably site specific. This implies a more complex response pattern to unloading and unpredictable therapeutics which target SOST or TRAP 5b.

Unlabelled: The study is about the regulatory effects of estrogen and parathyroid hormone (PTH) on sclerostin, a protein that inhibits the Wnt/β-catenin pathway. The results indicate that estrogen may down-regulate sclerostin expression and that estrogen displays synergistic action with PTH. These results provide a new perspective on the relationship between estrogen and bone., Purpose: To investigate whether estrogen can down-regulate SOST and MEF2 (myocyte enhancer factor 2) expression and whether co-treatment with estrogen and PTH has a stronger effect on suppressing SOST than PTH applied alone in ovariectomized rats., Methods: Forty-three-month-old virgin female Sprague-Dawley (SD) rats were ovariectomized and divided into four groups (n = 10). Another ten age-matched rats received sham operations as controls. After allowing 8 weeks for the development of vertebral osteopenia, the rats were administered the drug intervention. For this intervention, the estrogen group was subcutaneously injected with 17β-estradiol at 25 μg/kg body weight, the PTH group was injected with 80 μg/kg synthetic human PTH (1-34), and the co-treatment group was concurrently treated with PTH and estrogen at the above dosage. The OVX group and sham group were treated with vehicle. The drug treatment was conducted for 12 weeks. After the lumbar spine bone mineral density (BMD) was measured, the rats were sacrificed, and the lumbar spine and blood were collected for qPCR, Western blot, immunohistochemistry and other tests., Results: Estrogen can down-regulate MEF2 and sclerostin expression, and co-treatment with estrogen and PTH has a stronger effect on suppressing MEF2 and SOST mRNA than PTH alone. The co-treatment group displayed slightly higher bone mass and biomechanical properties than the PTH group, but the differences were not significant., Conclusions: Estrogen appears to be a regulator of sclerostin, and the effect may involve suppressing MEF2s. Combined treatment with PTH and estrogen is not more beneficial for vertebral bone mass and strength than treatment with PTH alone in ovariectomized rats.

Astrocytes constitute a major cell population in the brain with a myriad of essential functions, yet we know remarkably little about the signaling pathways and mechanisms that direct astrocyte maturation. To explore the signals regulating astrocyte development, we prospectively purified and cultured immature postnatal rodent astrocytes. We identified fibroblast growth factors (FGFs) and bone morphogenetic proteins (BMPs) as robust trophic factors for immature astrocytes. We showed that astrocytes respond directly to BMPs via phosphorylation of the smad1/5/8 pathway. In vitro, BMP signaling promoted immature astrocytes to adopt multiple characteristics of mature astrocytes, including a more process-bearing morphology, aquaporin-4 (AQP4) and S100β immunoreactivity, limited proliferation, and strong downregulation of epidermal growth factor receptor (EGFR). In vivo, activation of the smad1/5/8 pathway in astrocytes was seen during early postnatal development, but inhibition of astrocyte proliferation was not observed. These insights can aid in the further dissection of the mechanisms and pathways controlling astrocyte biology and development.

Natural killer (NK) cells are critical for innate tumor immunity due to their specialized ability to recognize and kill neoplastically transformed cells. However, NK cells require a specific set of cytokine-mediated signals to achieve optimal effector function. Th1-associated cytokines promote effector functions that are inhibited by the prototypic Th2 cytokine IL4 and the TGFβ superfamily members TGFβ1 and activin-A. Interestingly, the largest subgroup of the TGFβ superfamily are the bone morphogenetic proteins (BMP), but the effects of BMP signaling on NK cell effector functions have not been evaluated. Here, we demonstrate that blood-circulating NK cells express type I and II BMP receptors, BMP-2 and BMP-6 ligands, and phosphorylated isoforms of Smad-1/-5/-8, which mediate BMP family member signaling. In opposition to the inhibitory effects of TGFβ1 or activin-A, autocrine BMP signaling was supportive to NK cell function. Mechanistic investigations in cytokine and TLR-L-activated NK cells revealed that BMP signaling optimized IFNγ and global cytokine and chemokine production, phenotypic activation and proliferation, and autologous dendritic cell activation and target cytotoxicity. Collectively, our findings identify a novel auto-activatory pathway that is essential for optimal NK cell effector function, one that might be therapeutically manipulated to help eradicate tumors. Cancer Res; 74(18); 5019-31. ©2014 AACR., (©2014 American Association for Cancer Research.)

Unlabelled: Spinal cord injury (SCI) causes rapid and marked bone loss. The present study demonstrates that low-intensity vibration (LIV) improves selected biomarkers of bone turnover and gene expression and reduces osteoclastogenesis, suggesting that LIV may be expected to benefit to bone mass, resorption, and formation after SCI., Introduction: Sublesional bone is rapidly and extensively lost following spinal cord injury (SCI). Low-intensity vibration (LIV) has been suggested to reduce loss of bone in children with disabilities and osteoporotic women, but its efficacy in SCI-related bone loss has not been tested. The purpose of this study was to characterize effects of LIV on bone and bone cells in an animal model of SCI., Methods: The effects of LIV initiated 28 days after SCI and provided for 15 min twice daily 5 days each week for 35 days were examined in female rats with moderate severity contusion injury of the mid-thoracic spinal cord., Results: Bone mineral density (BMD) of the distal femur and proximal tibia declined by 5 % and was not altered by LIV. Serum osteocalcin was reduced after SCI by 20 % and was increased by LIV to a level similar to that of control animals. The osteoclastogenic potential of bone marrow precursors was increased after SCI by twofold and associated with 30 % elevation in serum CTX. LIV reduced the osteoclastogenic potential of marrow precursors by 70 % but did not alter serum CTX. LIV completely reversed the twofold elevation in messenger RNA (mRNA) levels for SOST and the 40 % reduction in Runx2 mRNA in bone marrow stromal cells resulting from SCI., Conclusion: The findings demonstrate an ability of LIV to improve selected biomarkers of bone turnover and gene expression and to reduce osteoclastogenesis. The study indicates a possibility that LIV initiated earlier after SCI and/or continued for a longer duration would increase bone mass.

Sclerostin, the product of the SOST gene, is a key regulator of bone homeostasis. Sclerostin interferes with the Wnt signalling pathway and, therefore, has a negative effect on bone formation. Although the importance of sclerostin in bone homeostasis is well established, many aspects of its biology are still unknown. Due to its restricted pattern of expression, in vitro studies of SOST gene regulation are technically challenging. Furthermore, a more profound investigation of the molecular mechanism controlling sclerostin expression has been hampered by the lack of a good human in vitro model. Here, we describe two cell lines derived from the human osteosarcoma cell line SaOS-2 that produce elevated levels of sclerostin. Analysis of the super-producer cell lines showed that sclerostin levels were still reduced in response to parathyroid hormone treatment or in response to mechanical loading, indicating that these regulatory mechanisms were not affected in the presented cell lines. In addition, we did not find differences between the promoter or ECR5 sequences of our clones and the SaOS-2 parental line. However, the methylation of the proximal CpG island located at the SOST promoter was lower in the super-producer clones, in agreement with a higher level of SOST transcription. Although the underlying biological causes of the elevated levels of sclerostin production in this cell line are not yet clear, we believe that it could be an extremely useful tool to study the molecular mechanisms driving sclerostin expression in humans.

The growth and differentiation factor-11 (GDF-11) gene is thought to code for a single protein that plays a crucial role in regulating the development of multiple tissues. In this study, we aimed to investigate if the GDF-11 gene has another transcript and, if so, to characterise this transcript and determine its tissue-specific and developmental expression. We have identified a novel transcript of GDF-11 in mouse muscle, which contains the 3' region of intron 1, exon 2, exon 3 and 3'UTR, and has two transcription initiation sites and a single termination site. We named the novel transcript GDF-11ΔEx1 because it does not contain exon 1 of canonical GDF-11. The GDF-11ΔEx1 transcript was expressed in the skeletal muscles, heart, brain and kidney, but was undetectable in the liver and gut. The concentration of the GDF-11ΔEx1 transcript was increased in gastrocnemius muscles from three to 6 weeks of age, a period of accelerated muscle growth, steadily declined thereafter and was higher in male than female mice (P < 0.001 for age and sex). GDF-11ΔEx1 cDNA was predicted to code for a putative N-terminal-truncated propeptide and the canonical ligand for GDF-11. However, propeptide-specific antibodies could not identify proteins of the expected size in skeletal muscle. Interestingly, in silico analysis of the GDF-11ΔEx1 RNA predicted a secondary structure with the potential to coordinate multiple protein interactions as a molecular scaffold. Therefore, we postulate that GDF-11ΔEx1 may act as a long non-coding RNA to regulate the transcription of canonical GDF-11 and/or other genes in skeletal muscle and other tissues.