BMP Signaling Research Articles

Microarray meta-analysis reveals comprehensive effects of 3,4,5-tricaffeolyquinic acid in cell differentiation and signaling

Caffeoylquinic acids (CQA) are polyphenolic compounds found in fruits, vegetables, coffee, and spices that have exhibited several beneficial activities, including antioxidant, antibacterial, neuroprotective, anti-inflammatory, anticancer, antiviral, antidiabetic, and cardiovascular effects. A derivative, TCQA (3,4,5-Tri-O-caffeoylquinic acid), has also shown both neurogenic and pigment differentiation potential. A transcriptomic-based meta-analysis was conducted to explore potential biochemical processes and molecular targets of TCQA. This approach involved integrating data from various cell and tissue types, including human amniotic stem cells, human neural stem cells, human dermal papilla cells, and the brain cortex of aging model mice. It offered a comprehensive perspective on the significant gene regulations in response to TCQA treatment. The objective was to uncover the mechanism and novel targets of TCQA, facilitating a further understanding of its functions. New areas of interest found were TCQA's effect on adipogenesis, heart, and muscle tissue development. In addition, significantly enhanced biological activities found through meta-analysis included cell cycle, VEGFA-VEGFR2 pathway, and BMP signaling. Overall, a comprehensive functional and visual analysis using available biological databases uncovered the multi-target potential of this natural compound.

FSHD muscle shows perturbation in fibroadipogenic progenitor cells, mitochondrial function and alternative splicing independently of inflammation.

Facioscapulohumeral muscular dystrophy (FSHD) is a prevalent, incurable myopathy. FSHD is highly heterogeneous, with patients following a variety of clinical trajectories, complicating clinical trials. Skeletal muscle in FSHD undergoes fibrosis and fatty replacement that can be accelerated by inflammation, adding to heterogeneity. Well controlled molecular studies are thus essential to both categorize FSHD patients into distinct subtypes and understand pathomechanisms. Here, we further analyzed RNA-sequencing data from 24 FSHD patients, each of whom donated a biopsy from both a non-inflamed (TIRM-) and inflamed (TIRM+) muscle, and 15 FSHD patients who donated peripheral blood mononucleated cells (PBMCs), alongside non-affected control individuals. Differential gene expression analysis identified suppression of mitochondrial biogenesis and up-regulation of fibroadipogenic progenitor (FAP) gene expression in FSHD muscle, which was particularly marked on inflamed samples. PBMCs demonstrated suppression of antigen presentation in FSHD. Gene expression deconvolution revealed FAP expansion as a consistent feature of FSHD muscle, via meta-analysis of 7 independent transcriptomic datasets. Clustering of muscle biopsies separated patients in an unbiased manner into clinically mild and severe subtypes, independently of known disease modifiers (age, sex, D4Z4 repeat length). Lastly, the first genome-wide analysis of alternative splicing in FSHD muscle revealed perturbation of autophagy, BMP2 and HMGB1 signalling. Overall, our findings reveal molecular subtypes of FSHD with clinical relevance and identify novel pathomechanisms for this highly heterogeneous condition.

Capturing heart valve development with Gene Ontology.

Introduction: The normal development of all heart valves requires highly coordinated signaling pathways and downstream mediators. While genomic variants can be responsible for congenital valve disease, environmental factors can also play a role. Later in life valve calcification is a leading cause of aortic valve stenosis, a progressive disease that may lead to heart failure. Current research into the causes of both congenital valve diseases and valve calcification is using a variety of high-throughput methodologies, including transcriptomics, proteomics and genomics. High quality genetic data from biological knowledge bases are essential to facilitate analyses and interpretation of these high-throughput datasets. The Gene Ontology (GO, http://geneontology.org/) is a major bioinformatics resource used to interpret these datasets, as it provides structured, computable knowledge describing the role of gene products across all organisms. The UCL Functional Gene Annotation team focuses on GO annotation of human gene products. Having identified that the GO annotations included in transcriptomic, proteomic and genomic data did not provide sufficient descriptive information about heart valve development, we initiated a focused project to address this issue. Methods: This project prioritized 138 proteins for GO annotation, which led to the curation of 100 peer-reviewed articles and the creation of 400 heart valve development-relevant GO annotations. Results: While the focus of this project was heart valve development, around 600 of the 1000 annotations created described the broader cellular role of these proteins, including those describing aortic valve morphogenesis, BMP signaling and endocardial cushion development. Our functional enrichment analysis of the 28 proteins known to have a role in bicuspid aortic valve disease confirmed that this annotation project has led to an improved interpretation of a heart valve genetic dataset. Discussion: To address the needs of the heart valve research community this project has provided GO annotations to describe the specific roles of key proteins involved in heart valve development. The breadth of GO annotations created by this project will benefit many of those seeking to interpret a wide range of cardiovascular genomic, transcriptomic, proteomic and metabolomic datasets.

Selenium nanoparticles functionalized by mushroom polysaccharide-protein complex: A novel nano-mineral for managing postmenopausal osteoporosis

With an increase in aging population, osteoporosis has become one of the major public health issues nowadays, affecting over 200 million people worldwide. Since current medications are associated with various side effects, there is a clear clinical need to develop alternative therapeutics for managing osteoporosis. Selenium is an essential trace mineral, which has been proved to play important role in bone health. However, the narrow therapeutic window of selenium has greatly hindered its further development. Recently, selenium nanoparticles (SeNPs) have become a new selenium source due to its promising bioactivity and low toxicity. Nevertheless, scientific research concerning their effects on bone health is still very limited. By using the mushroom polysaccharide-protein complex isolated from Cordyceps sinensis, we have successfully prepared novel SeNPs (“Cs4-SeNPs”) with well-characterized structure and high stability. Interestingly, Cs4-SeNPs (10 μM) were found to markedly induce proliferation, differentiation, and mineralization of the pre-osteoblast murine MC3T3-E1 cells. Further mechanistic study discovered that after cellular internalization via endocytosis, Cs4-SeNPs would trigger Nox4-derived intracellular ROS generation in the MC3T3-E1 cells followed by promoting downstream BMP-2 gene transcription and activating BMP signaling via both Smad dependent and Smad independent p38 MAPK pathways. More importantly, Cs4-SeNPs (25–500 μg/kg BW/d) exhibitedpromising in vivo bone protective efficacy against OVX-induced osteoporosis by promoting bone formation, inhibiting bone resorption, and improving bone microarchitecture after oral gavage for 6 weeks. Findings of this study collectively suggested that Cs4-SeNPs is of great potential to be further developed into a safe and evidence-based nano-mineral for managing postmenopausal osteoporosis.

OR27-02 The Bone Anabolic Effect Of An Antibody Blocking Oxidized Phospholipids Is Associated With An Increase In Wnt10b In Osteoblasts.

Abstract Disclosure: M. Palmieri: None. I. Nookaew: None. M.J. Schuller-Almeida: None. H. Kim: None. T.E. Jospeh: None. S.C. Manolagas: None. C.A. O'Brien: None. E. Ambrogini: None. Oxidized phospholipids containing phosphatidylcholine (PC-OxPLs) are toxic molecules present on oxidized low-density lipoproteins (OxLDL) and apoptotic cells; and are pathogenic in many diseases, including osteoporosis. OxPLs decrease Wnt and BMP2 signaling, induce ferroptosis and stimulate pro-inflammatory cytokine production, in atherosclerosis and nonalcoholic steatohepatitis. The natural antibody E06 blocks PC-OxPLs. Transgenic expression of a single chain (scFv) form of the antigen-binding domain of E06 IgM (E06-scFv) increases bone mass in 6-month-old mice and attenuates high fat diet- and age-induced bone loss by increasing osteoblast number and bone formation rate and reducing osteoblast apoptosis. To elucidate the mechanism(s) mediating the bone anabolic effect of E06-scFv we performed bulk-RNA sequencing (bulk-RNA-seq) of vertebral bone and single cell RNA-sequencing (scRNA-seq) of mesenchymal and myeloid cells from long bones of WT and E06-scFv transgenic mice. Earlier bulk-RNA-seq performed in vertebral bone of 22- month-old females had shown that E06-scFv upregulates Wnt10b, but not other Wnt ligands, and the Wnt signaling pathway. We report now that scRNA-seq of mesenchymal cells isolated from 7-month-old female mice revealed that Wnt10b expression is increased by 3-fold (adj-P=3.04E-11) in pre-osteoblasts and osteoblasts from E06-scFv transgenic mice as compared to cells from WT mice. Wnt target genes were also upregulated. An integrated analysis of 9 published scRNA-seq datasets from bone resident mesenchymal and hematopoietic cell types revealed that the majority of Wnt10b in the bone microenvironment originates from cells of the osteoblast lineage. Further, OxLDL decreased Wnt10b and Wnt target genes in calvaria- or bone marrow-derived osteoblastic cell cultures. In contrast, OxLDL did not cause the ferroptosis associated upregulation of Chac1, Ptgs2, Slc7A11, and Acsl4, and the downregulation of Rgs4 in calvaria cells. In addition, no changes in the expression of these genes were detected in the scRNA-seq of mesenchymal cells from E06-scFv compared to WT mice. BMP signaling pathways were also unchanged in this scRNA-seq analysis. Finally, using bulk-RNA-seq of vertebral bone and scRNA-seq of myeloid cells, we could not detect changes in the expression of pro-inflammatory cytokines, such as Tnf-α, Il-6, Il1-α, Il-β, Il-4, Il-12, Lif, Il-23, interferon-α, and interferon-β, between WT and E06-scFv mice. Together, these findings indicate the PC-OxPLs decrease Wnt10b and E06-scFv increases Wnt10b in bone but has no effect on other pathways shown to be affected by OxPLs in other tissues. Thus, Wnt10b may be a critical mediator of the bone anabolic effects of E06-scFv. Presentation: Saturday, June 17, 2023

FRI670 Osteoblast Rna Sequencing In T1d Mice Reveals Genetic Signature Of Maladaptation To Hypoxia And Compromised Collagen Synthesis

Abstract Disclosure: S. Kim: None. J. Hong: None. F. Sultana: None. E. Shelly: None. F. Korkmaz: None. J. Gimenez: None. T. Yuen: None. I. Daehn: None. J. Choi: None. M. Zaidi: None. The main feature of skeletal abnormality in type 1 diabetes (T1D) is low bone turnover with decreased osteoblastic bone formation [1, 2]. However, the mechanism of osteoblast dysfunction remains unclear. We generated T1D mice using low dose streptozotocin (STZ). T1D mice showed lower bone mass (g/cm2) at the femur (0.07 vs. 0.09*) and trends at lumbar spine (0.05 vs. 0.06, p=0.08) compared with control group at 8 weeks after inducing T1D. Osteoblasts were differentiated using bone marrow mesenchymal stem cells, and RNAseq was performed to analyze the transcriptome of osteoblasts. In gene set enrichment analysis, several pathways including immune, hypoxia, and glycolytic pathway were enriched. The osteogenic genes panel showed decreased Bglap and Col1a1 expression, together with lower expression of Bmp2 and Wnt5a, suggesting decreased bone formation might be related to the suppression of BMP and WNT signaling. Particularly, P4ha2, encoding prolyl 4-hydroxylase subunit alpha–2, which are responsible for posttranslational modification of collagen were downregulated in T1D osteoblasts (log2FC = – 1.7**). Among genes in hypoxia pathway, Egln3 encoding prolyl hydroxylase domain-containing protein 2 and Vhl encoding von Hippel-Lindau tumor suppressor, both of which stabilize HIF–α were downregulated (log2FC = – 2.8** and – 0.31*, respectively) likely for promoting HIF–α dependent gene expression in hypoxia. However, genes of the growth signaling pathway namely Pgf, Vegfa, and Igf1r were downregulated (log2FC = – 3.1**, – 0.4* and – 0.5**, respectively). Altogether, our findings suggest that maladaptation to hypoxic condition in T1D with compromised growth signaling pathway and impaired collagen synthesis might be related to osteoblasts dysfunction in T1D bone. (* p < 0.05 and ** p < 0.001) [1] Shanbhogue VV et al, JBMR. 2015[2] Rubin MR et al, JCEM. 2022 Presentation: Friday, June 16, 2023

Concurrent impact of de novo mutations on cranial and cortical development in nonsyndromic craniosynostosis.

Nonsyndromic craniosynostosis (nsCS), characterized by premature cranial suture fusion, is considered a primary skull disorder in which impact on neurodevelopment, if present, results from the mechanical hindrance of brain growth. Despite surgical repair of the cranial defect, neurocognitive deficits persist in nearly half of affected children. Therefore, the authors performed a functional genomics analysis of nsCS to determine when, where, and in what cell types nsCS-associated genes converge during development. The authors integrated whole-exome sequencing data from 291 nsCS proband-parent trios with 29,803 single-cell transcriptomes of the prenatal and postnatal neurocranial complex to inform when, where, and in what cell types nsCS-mutated genes might exert their pathophysiological effects. The authors found that nsCS-mutated genes converged in cranial osteoprogenitors and pial fibroblasts and their transcriptional networks that regulate both skull ossification and cerebral neurogenesis. Nonsyndromic CS-mutated genes also converged in inhibitory neurons and gene coexpression modules that overlapped with autism and other developmental disorders. Ligand-receptor cell-cell communication analysis uncovered crosstalk between suture osteoblasts and neurons via the nsCS-associated BMP, FGF, and noncanonical WNT signaling pathways. These data implicate a concurrent impact of nsCS-associated de novo mutations on cranial morphogenesis and cortical development via cell- and non-cell-autonomous mechanisms in a developmental nexus of fetal osteoblasts, pial fibroblasts, and neurons. These results suggest that neurodevelopmental outcomes in nsCS patients may be driven more by mutational status than surgical technique.

Bee Pollen Stimulated BMP2 and RANK Signaling Pathway in Osteoprogenitors and Improved Bone Mineralization in Rat Model of Femur Fracture

Objectives Bee pollen (BP) contains isoflavonoids and minerals that can be effective in bone repair, stimulation of osteoprogenitors, differentiation of osteoblasts, and inhibition of osteoclasts. For this purpose, in the present study, BP was used to stimulate osteogenesis in an animal model of a femur fracture. Materials and Methods After induction of the femur fracture model, 80 Wistar rats in eight groups ( n = 10) were studied as follows. Normal control group (0.5 cc of distilled water, DW/gavage/90 days), BP group (200 mg/kg BP/daily/gavage/90 days), fracture (FX-0.5 cc of distilled water/gavage/90 days), FX group treated with 100 and 200 mg/kg BP (100 and 200 mg/kg BP/daily/gavage/90 days), FX group treated with osteocare (OC) syrup (1 mL/day/gavage/90 days) and combinatorial FX groups treated with BP and OC syrup (100 and 200 mg/kg BP plus 1 mL OC/daily/gavage/90 days). On the 30th, 60th, and 90th days, radiography of the lesion was done. Bone mineral density (BMD) was measured by dual-energy X-ray absorptiometry during the mentioned days. At the end of the study, blood calcium (Ca), phosphorus (P), and alkaline phosphatase (ALP), along with the activity of glutathione peroxidase (GPx), superoxide dismutase (SOD), and catalase (CAT) enzymes, and nitric oxide levels, were measured. Calcitonin and parathyroid hormone levels were measured with enzyme-linked immunosorbent assay (ELISA) kits. In order to evaluate the stimulation of osteoprogenitors, the expression of osteoprotegerin (OPG), receptor activator of nuclear factor kappa-B (RANK), receptor activator of nuclear factor kappa-B ligand (RANKL), and bone morphogenetic protein-2 (BMP-2) genes and proteins in bone tissue was measured. Results The evaluations of this study showed that BP could improve BMD parameters through stimulation of the OPG/RANK/BMP-2 pathway. BP also improved serum levels of biochemical factors (Ca, P, and ALP) and hormones related to osteogenesis. Conclusion BP can be used for bone fractures and disorders related to osteoporosis.

BMP suppresses WNT to integrate patterning of orthogonal body axes in adult planarians.

Adult regeneration restores patterning of orthogonal body axes after damage in a post-embryonic context. Planarians regenerate using distinct body-wide signals primarily regulating each axis dimension: anteroposterior Wnts, dorsoventral BMP, and mediolateral Wnt5 and Slit determinants. How regeneration can coordinate perpendicular tissue axes without symmetry-breaking embryonic events is not fully understood. Here, we report that the planarian dorsoventral regulator bmp4 suppresses the posterior determinant wnt1 to provide patterning input to the anteroposterior axis. Double-FISH identified distinct anteroposterior domains within dorsal midline muscle that express either bmp4 or wnt1. Homeostatic inhibition bmp4 and smad1 expanded the wnt1 expression anteriorly, while elevation of BMP signaling through nog1;nog2 RNAi reduced the wnt1 expression domain and elevated bmp4 expression. Homeostatic BMP signal perturbation broadly affected anteroposterior identity as measured by expression of posterior Wnt pathway factors, and caused mislocalization of AP-regionalized pharynx progenitors, without strongly affecting expression domains of anterior regulators. Additionally, wnt1 inhibition elevated bmp4 expression in the tip of the tail. Therefore, dorsal BMP signals and posterior wnt1 mutually antagonize for patterning the tail. Furthermore, homeostatic bmp4 RNAi caused medial expansion of the lateral determinant wnt5 and reduced expression of the medial regulator slit. By contrast, nog1;nog2 RNAi restricted wnt5 expression. Double RNAi of bmp4 and wnt5 resulted in lateral ectopic eye phenotypes, suggesting bmp4 acts upstream of wnt5 to pattern the mediolateral axis. These results indicate bmp4 controls dorsoventral information and also, through suppression of Wnt signals, influences anteroposterior and mediolateral identity. Based on related functions across vertebrates and Cnidarians, Wnt and BMP cross-regulation could form an ancient mechanism for coordinating orthogonal axis patterning.

A periosteum-derived cell line to study the role of BMP/TGFβ signaling in periosteal cell behavior and function.

The periosteum is a thin tissue surrounding each skeletal element that contains stem and progenitor cells involved in bone development, postnatal appositional bone growth, load-induced bone formation, and fracture repair. BMP and TGFβ signaling are important for periosteal activity and periosteal cell behavior, but thorough examination of the influence of these pathways on specific cell populations resident in the periosteum is lacking due to limitations associated with primary periosteal cell isolations and in vitro experiments. Here we describe the generation of a novel periosteum-derived clonal cell (PDC) line from postnatal day 14 mice and use it to examine periosteal cell behavior in vitro. PDCs exhibit key characteristics of periosteal cells observed during skeletal development, maintenance, and bone repair. Specifically, PDCs express established periosteal markers, can be expanded in culture, demonstrate the ability to differentiate into chondrocytes, osteoblasts, and adipocytes, and exhibit an osteogenic response to physical stimulation. PDCs also engage in BMP and/or TGFβ signaling when treated with the activating ligands BMP2 and TGFβ-1, and in response to mechanical stimulation via fluid shear. We believe that this PDC line will be useful for large-scale, long-term experiments that were not feasible when using primary periosteal cells. Anticipated future uses include advancing our understanding of the signaling interactions that occur during appositional bone growth and fracture repair and developing drug screening platforms to discover novel growth and fracture healing factors.

SMAD4 maintains the fluid shear stress set point to protect against arterial-venous malformations.

Vascular networks form, remodel, and mature under the influence of both fluid shear stress (FSS) and soluble factors. Physiological FSS promotes and maintains vascular stability via synergy with bone morphogenic proteins 9 and 10 (BMP9 and BMP10). Conversely, mutation of the BMP receptors activin-like kinase 1 (ALK1), endoglin (ENG), or the downstream effector, SMAD family member 4 (SMAD4) leads to hereditary hemorrhagic telangiectasia (HHT), characterized by fragile and leaky arterial-venous malformations (AVMs). How endothelial cells (ECs) integrate FSS and BMP signals in vascular development and homeostasis and how mutations give rise to vascular malformations is not well understood. Here, we aimed to elucidate the mechanism of synergy between FSS and SMAD signaling in vascular stability and how disruption of this synergy leads to AVMs. We found that loss of Smad4 increased the sensitivity of ECs to flow by lowering the FSS set point, with resulting AVMs exhibiting features of excessive flow-mediated morphological responses. Mechanistically, loss of SMAD4 disinhibits flow-mediated KLF4-TIE2-PI3K/Akt signaling, leading to cell cycle progression-mediated loss of arterial identity due to KLF4-mediated repression of cyclin dependent Kinase (CDK) inhibitors CDKN2A and CDKN2B. Thus, AVMs caused by Smad4 deletion are characterized by chronic high flow remodeling with excessive EC proliferation and loss of arterial identity as triggering events.

Presynaptic Ube3a E3 ligase promotes synapse elimination through down-regulation of BMP signaling.

Inactivation of the ubiquitin ligase Ube3a causes the developmental disorder Angelman syndrome, whereas increased Ube3a dosage is associated with autism spectrum disorders. Despite the enriched localization of Ube3a in the axon terminals including presynapses, little is known about the presynaptic function of Ube3a and mechanisms underlying its presynaptic localization. We show that developmental synapse elimination requires presynaptic Ube3a activity in Drosophila neurons. We further identified the domain of Ube3a that is required for its interaction with the kinesin motor. Angelman syndrome-associated missense mutations in the interaction domain attenuate presynaptic targeting of Ube3a and prevent synapse elimination. Conversely, increased Ube3a activity in presynapses leads to precocious synapse elimination and impairs synaptic transmission. Our findings reveal the physiological role of Ube3a and suggest potential pathogenic mechanisms associated with Ube3a dysregulation.

DMRT1 regulates human germline commitment

Germline commitment following primordial germ cell (PGC) specification during early human development establishes an epigenetic programme and competence for gametogenesis. Here we follow the progression of nascent PGC-like cells derived from human embryonic stem cells in vitro. We show that switching from BMP signalling for PGC specification to Activin A and retinoic acid resulted in DMRT1 and CDH5 expression, the indicators of migratory PGCs in vivo. Moreover, the induction of DMRT1 and SOX17 in PGC-like cells promoted epigenetic resetting with striking global enrichment of 5-hydroxymethylcytosine and locus-specific loss of 5-methylcytosine at DMRT1 binding sites and the expression of DAZL representing DNA methylation-sensitive genes, a hallmark of the germline commitment programme. We provide insight into the unique role of DMRT1 in germline development for advances in human germ cell biology and in vitro gametogenesis.

Time space and single-cell resolved tissue lineage trajectories and laterality of body plan at gastrulation

Understanding of the molecular drivers of lineage diversification and tissue patterning during primary germ layer development requires in-depth knowledge of the dynamic molecular trajectories of cell lineages across a series of developmental stages of gastrulation. Through computational modeling, we constructed at single-cell resolution, a spatio-temporal transcriptome of cell populations in the germ-layers of gastrula-stage mouse embryos. This molecular atlas enables the inference of molecular network activity underpinning the specification and differentiation of the germ-layer tissue lineages. Heterogeneity analysis of cellular composition at defined positions in the epiblast revealed progressive diversification of cell types. The single-cell transcriptome revealed an enhanced BMP signaling activity in the right-side mesoderm of late-gastrulation embryo. Perturbation of asymmetric BMP signaling activity at late gastrulation led to randomization of left-right molecular asymmetry in the lateral mesoderm of early-somite-stage embryo. These findings indicate the asymmetric BMP activity during gastrulation may be critical for the symmetry breaking process.

ADAM11 a novel regulator of Wnt and BMP4 signaling in neural crest and cancer.

Introduction: Cranial neural crest (CNC) cells are induced at the border of the neural plate by a combination of FGF, Wnt, and BMP4 signaling. CNC then migrate ventrally and invade ventral structures where they contribute to craniofacial development. Methods: We used loss and gain of function experiments to determine phenotypes associated with the perturbation of Adam11 expression in Xenopus Laevis. Mass spectrometry to identify partners of Adam11 and changes in protein expression in CNC lacking Adam11. We used mouse B16 melanoma to test the function of Adam11 in cancer cells, and published database analysis to study the expression of ADAM11 in human tumors. Results: Here we show that a non-proteolytic ADAM, Adam11, originally identified as a putative tumor suppressor binds to proteins of the Wnt and BMP4 signaling pathway. Mechanistic studies concerning these non-proteolytic ADAM lack almost entirely. We show that Adam11 positively regulates BMP4 signaling while negatively regulating β-catenin activity. In vivo, we show that Adam11 influences the timing of neural tube closure and the proliferation and migration of CNC. Using both human tumor data and mouse B16 melanoma cells, we further show that ADAM11 levels similarly correlate with Wnt or BMP4 activation levels. Discussion: We propose that ADAM11 preserves naïve cells by maintaining low Sox3 and Snail/Slug levels through stimulation of BMP4 and repression of Wnt signaling, while loss of ADAM11 results in increased Wnt signaling, increased proliferation and early epithelium to mesenchyme transition.

Acquisition of neural fate by combination of BMP blockade and chromatin modification

Neural induction is a process where naive cells are converted into committed cells with neural characteristics, and it occurs at the earliest step during embryogenesis. Although the signaling molecules and chromatin remodeling for neural induction have been identified, the mutual relationships between these molecules are yet to be fully understood. By taking advantage of the neural differentiation system of mouse embryonic stem (ES) cells, we discovered that the BMP signal regulates the expression of several polycomb repressor complex (PRC) component genes. We particularly focused on Polyhomeotic Homolog 1 (Phc1) and established Phc1-knockout (Phc1-KO) ES cells. We found that Phc1-KO failed to acquire the neural fate, and the cells remained in pluripotent or primitive non-neural states. Chromatin accessibility analysis suggests that Phc1 is essential for chromatin packing. Aberrant upregulation of the BMP signal was confirmed in the Phc1 homozygotic mutant embryos. Taken together, Phc1 is required for neural differentiation through epigenetic modification.

P10.05.A INDUCED SENESCENCE AND CALCIFICATION TO ANAPLASTIC MENINGIOMA

Abstract BACKGROUND Although the majority of meningiomas are slow-growing and benign, atypical and anaplastic meningiomas behave aggressively with a penchant for recurrence. The 5-year risk of progression for incompletely resected meningiomas is over 80 percents. Specifically, the 5-year survival rates are 78-91% and 41-65% for atypical and anaplastic meningiomas, respectively. On the other hand, it has been reported that benign meningiomas with psammoma bodies or those with bone hyperplasia at the attachment site and subtype of high calcification have a low growth rate. Here we show that BMP signals essential for bone formation, especially GREM2 upstream inhibitor of BMP signals, are involved in meningioma growth, senescence and calcification. MATERIAL AND METHODS 1) A long-term primary cultured meningioma and commercially available anaplastic meningioma (IOMM-lee and HKB-MM) are used. For immunohistochemistry and expression analysis, 87 surgical samples operated in our hospital were used.2) We observed changes in phenotype, proliferation rate and cell cycle by inhibiting BMP signal. Overexpression of GREM2, an upstream inhibitor of BMP signals, was performed in anaplastic meningiomas and analyzed comprehensively. Downloaded RNA-seq data from GREIN, a public database, and performed a comprehensive analysis of the extent of GREM2 expression and gene expression related to cellular senescence. RESULTS 1) Inhibition of BMP signaling reduced proliferation. Moreover, long term inhibition of BMP signals induced calcification. 2) Expression of GREM2 related to proliferation in surgical samples. GREM2 mainly localized surrounding of psammoma bodies. 3)Overexpression of GREM2 increased sub-G1 population and inhibits cell proliferation. 4)Expression of GREM2 shifts tryptophan catabolism from Kynurenine/NAD/NADH to serotonin production. 5) Comprehensive analysis using RNA-seq data from GREIN showed that high expression of GREM2 decreases oxidative phosphorylation. 6)Senescence assay showed that inhibition of BMP signaling induced senescence. CONCLUSION Inhibition of BMP signaling reduced proliferation and induced calcification. The underlying mechanism is shifting tryptophan catabolism from Kynurenine/NADH/NAD to serotonin production.

Dermal white adipose tissue development and metabolism: The role of transcription factor Foxn1.

Intradermal adipocytes form dermal white adipose tissue (dWAT), a unique fat depot localized in the lower layer of the dermis. However, recognition of molecular factors regulating dWAT development, homeostasis, and bioactivity is limited. Using Foxn1-/- and Foxn1+/+ mice, we demonstrated that epidermally expressed Foxn1 regulates dWAT development and defines the adipogenic capacity of dermal fibroblasts. In intact and post-wounded skin, Foxn1 contributes to the initial stimulation of dWAT adipogenesis and participates in the modulation of lipid metabolism processes. Furthermore, Foxn1 activity strengthens adipogenic processes through Bmp2 and Igf2 signaling and regulates lipid metabolism in differentiated dermal fibroblasts. The results reveal the contribution of Foxn1 to dWAT metabolism, thus identifying possible targets for modulation and regulation of dWAT in physiological and pathological processes in the skin.

Wnt and BMP signalling direct anterior-posterior differentiation in aggregates of mouse embryonic stem cells.

Stem-cell-based embryo models have allowed greater insight into peri-implantation mammalian developmental events that are otherwise difficult to manipulate due to the inaccessibility of the early embryo. The rapid development of this field has resulted in the precise roles of frequently used supplements such as N2, B27 and Chiron in driving stem cell lineage commitment not being clearly defined. Here, we investigate the effects of these supplements on embryoid bodies to better understand their roles in stem cell differentiation. We show that Wnt signalling has a general posteriorising effect on stem cell aggregates and directs differentiation towards the mesoderm, as confirmed through the upregulation of posterior and mesodermal markers. N2 and B27 can mitigate these effects and upregulate the expression of anterior markers. To control the Wnt gradient and the subsequent anterior versus posterior fate, we make use of a BMP4 signalling centre and show that aggregates in these conditions express cephalic markers. These findings indicate that there is an intricate balance between various culture supplements and their ability to guide differentiation in stem cell embryo models.

Panicum miliaceum L. and Bone Healing Properties in Rat Model of Femur Fracture by Activating Phosphate Stimulating Macrophages via BMP2 and RANK Signaling Pathway

Objectives The aim of this study was to investigate the effect of Panicum miliaceum involved in the repair of femur bone fracture in rats. Materials and Methods In this study, 40 Wistar rats in five groups were studied as healthy, rats with lesion induced by surgery in femur (FX), FX rats treated with P. miliaceum extract (FX +100 and 200 P. miliaceum) and FX rats treated with Osteocare syrup (FX + OC). After 40 days, the serum levels of blood calcium (Ca), phosphorus (P), and alkaline phosphatase (ALP) along with activity of glutathione peroxidase (GPx), superoxide dismutase (SOD), and catalase (CAT) enzymes, and nitric oxide (NO) level were measured. The serum level of cytokines (IL-6, C-reactive protein [CRP], IL-10, IL-1β, and tumor necrosis factor-α [TNF-α]) was also measured by ELISA. The expression of OPG, RANK, RANKL, and BMP-2 genes and proteins were evaluated by real-time PCR and Western blotting techniques, respectively. Results The results showed that P. miliaceum was able to decrease the activity of ALP enzyme in addition to increasing serum Ca and P levels and GPx, SOD, and CAT activity. P. miliaceum reduces serum IL-6, IL-1β, and TNF-α, and NO levels. Also, the extract of this plant could increase the expression of OPG/RANK/BMP-2 pathway genes and proteins. Conclusion P. miliaceum can be used as a prodrug in traditional medicine to repair bone fractures and osteoporosis.