LOX inhibition (BAPN treatment) attenuates satellite cell differentiation
Ontology highlight
ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE147007 | GEO | 2020/03/17
REPOSITORIES: GEO
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Project description:Vestigial-like (Vgll) proteins are transcriptional co-factors that have been reported to bind Tead family transcription factors to regulate various functions ranging from wing development in the fly to muscle fibre composition and immune function in mice. Here, we characterise Vgll3 in the skeletal muscle lineage. Vgll3 is expressed at low levels in unchallenging muscle but its expression increases in mechanically stimulated and regenerating muscle. VGLL3 is also >3-fold more expressed in the muscles of Duchenne muscular dystrophy patients than in healthy controls and VGLL3 expression is highest in PAX3-FOXO1-associated alveolar rhabdomyosarcoma when compared to other rhabdomyosarcomas, related tumours and adult muscle. Immunoprecipitation of over expressed VGLL3-flag followed by proteomics reveals that VGLL3 binds Tead1,3,4 transcription factors in myoblasts and myotubes. However, unlike Yap, there is no evidence for interaction with a regulatory system such as a kinase cascade. VGLL3 overexpression affects reduces the expression of members of the Hippo negative feedback loop and affects the expression of genes with important functions in muscle or signalling such as Myf5, Pitx2/3 as well as genes that encode Wnt members and IGF-binding proteins. It mainly represses gene expression and regulates similar genes as YAP1 S127A and TAZ S89A but not in a clear agonistic or antagonistic fashion. A loss of Vgll3 almost completely blocks the proliferation of human myoblasts and VGLL3 overexpression promotes myoblasts differentiation. In vivo, the loss of Vgll3 does not prevent normal skeletal muscle development presumably due to feedback signalling and/or redundancy. Collectively, this work identifies Vgll3 as a disease-related transcriptional co-factor that competes with the Hippo effectors Yap and Taz to control myoblast proliferation and differentiation.
2019-06-17 | PXD012040 | Pride
Project description:Hippo effectors Yap and Taz, Vgll1-4 and Tead 1-4 transcription factors have been linked to satellite cell/ myoblast proliferation and differentiation, muscle function and disease. The molecular function of Vgll3 in skeletal muscle stem cells was investigated using analysis of gene expression by microarrays to determine Vgll3 mediated gene expression changes and compare those to previously determined Taz and Yap mediated changes. Satellite cell-derived myoblasts were transduced with Vgll3 retrovirus for 24h or 48h, with empty retrovirus vector as control. Triplicate microarray analysis of empty vector controls and Vgll3 transgenic primary myoblasts were conducted.
2019-06-17 | E-MTAB-7614 | biostudies-arrayexpress
Project description:During myogenic differentiation the architecture and proteome of muscle stem cells undergo extensive remodelling. These processes are only partially understood and display impairments in age and disease. Using mass spectrometry to analyse protein dynamics during myogenic differentiation, we identified the actin nucleator Leiomodin 1 (LMOD1) increasing in abundance in early phases of myogenic differentiation. Knockdown of LMOD1 in primary myoblasts severely affected myogenic differentiation and fusion of myotubes, while overexpression had the opposite effect. Mechanistically, we show that LMOD1 interacts with Sirtuin1 (SIRT1) and that both follow similar changes in nuclear/cytoplasmic localization during differentiation. We demonstrate that LMOD1 influences SIRT1 localization and the expression of a subset of its target genes. Consistently, depletion or pharmacological inhibition of SIRT1 partially rescues the effects of LMOD1 knockdown. Our work identifies a novel regulator of myogenic differentiation that might be a potential target to improve muscle regeneration in aging and disease.
2024-08-31 | GSE254443 | GEO
Project description:Myogenic differentiation of iPSCs has been done by gene–overexpression or directed differentiation. However, viral integration, long-term culture and the presence of unwanted cells are the main obstacles. By using CRISPR/Cas9n, a novel double reporter hESC line was generated for PAX7/MYF5, allowing prospective readout. This strategy allowed pathway screen to define efficient myogenic induction in hESC/iPSCs. Next, surface marker screen allowed identification of CD10 and CD24 for purification of myogenic progenitors. CD10 expression was also confirmed on human satellite cells and muscle progenitors. In vivo studies using transgene/reporter-free hESC/iPSCs further validated myogenic potential of the cells by dystrophin restoration and satellite cell engraftment in NSG-mdx4cv mice. In addition, side-by-side in vitro and in vivo comparison proved superior specificity of CD10/CD24 compared to recently reported markers (ERBB3/NGFR). This study provides new biological insights for myogenic differentiation using a new cell resource and identifies CD10 as a potential myogenic marker.
2018-12-03 | GSE121154 | GEO
Project description:Objectives: Bone marrow (BM) stroma and microenvironmental factors in the BM milieu of myeloid neoplasms are increasingly being recognized as novel perspective therapeutic targets. Lysyl oxidases (LOX/LOXL) are crucial enzymes that cross-link collagen and contribute to the deposition of aberrant stiff extracellular matrix (ECM) in the BM of patients with myelodysplastic syndromes (MDS) and myeloproliferative neoplasms (MPN). Here, we studied whether inhibition of LOX/LOXL enzymes produced by BM-derived MSCs favors the differentiation of MDS/MPN hematopoietic stem cell and progenitor cells (HPSCs) and enhances the effects of 5-azacytidine (5-AZA).
Methods: Primary co-cultures of MSCs and HPSCs were established from n=31 MDS/MPN patients and treated with the pan-LOX/LOXL inhibitor PXS-5505, 5-AZA or the combination of both for 4 days. At the end of co-culture, HSPC differentiation was assessed by CFU assays and flow cytometry. In some of the experiments, MSCs monolayers were pre-treated or not with PXS-5505 for 6 days and decellularized using 20 mM ammonia. Subsequently, autologous HSPCs were cultured on ECM matrices in the presence of 5-AZA or PXS-5505 + 5-AZA combination with or without inhibitors of integrin signaling such as blebbistatin, Y-27632 and αvβ3-integrin blocking antibodies. Patient-derived xenografts (PDX) were established in NSG mice based on intrafemoral co-injection of HSPCs and autologous MSCs. PDX were treated with 3 cycles of 5-AZA (75 mg/m2), PXS-5505 (30 mg/kg on alternate days for the duration of 5-AZA treatment) or combination of both.
Results: MSCs of MDS and MPN patients displayed significant overexpression of LOX/LOXL transcripts as compared to healthy age-matched controls. Remarkably, LOX/LOXL inhibition in MSCs/HSPCs co-cultures increased erythroid differentiation of patient HSPCs that had suboptimal or no response to 5-AZA alone. From a total of n=31 advanced stage MDS/MPN patients, n=11 (35.5%) responded to the combination treatment, which was evidenced by a significant increase of CD235a+CD45- erythroid cell production compared to 5-AZA/PXS/untreated arms (p<0.05). Of note, combination treatment with 5-AZA + PXS-5505 facilitated differentiation of CD235+ erythroid progenitors from HSPCs sub-clones with low mutational VAFs. The presence of MSCs was necessary for the effects of the combination treatment and required direct cell contacts between MSCs and HSPCs. Moreover, PXS-5505-mediated alterations of the ECM composition alone were sufficient to induce erythroid
differentiation. Inhibition of integrin-mediated signaling using blebbistatin, Y-27632 and αvβ3-integrin antibodies completely abrogated the effects of PXS-5505 induced erythroid differentiation. In vivo, the combination of PXS-5505 + 5-AZA favored erythroid differentiation of HSPCs and confirmed the in-vitro results. The combination treatment of 5-AZA + PXS-5505 induced the greatest reduction of disease burden as assessed by decrease of spleen indexes, total engraftment rates, BM fibrosis and mutational VAF profiles.
Conclusions: The combination of 5-AZA + LOX/LOXL inhibitor PXS-5505 in MDS and MPN synergistically restores erythropoietic differentiation of primary HSPCs. The effect is dependent of contact interaction between HSPCs and niche components, such as MSCs and MSCs-derived ECM. Our data propose a strong rationale for a BM niche targeted combination treatment with 5-AZA + PXS-5505 in transfusion dependent MDS and MPN, which will be assessed in forthcoming clinical trials.
| EGAS00001006174 | EGA
Project description:To address the regulation of interferon-stimulated genes expression by VGLL3 in fibroblast-like synoviocytes (FLS) of rheumatoid arthritis (RA) patients, VGLL3 was overexpressed in RA-FLS via a lentiviral vector. After four days of transfection, VGLL3-overexpressed RA-FLS and vector-transfected RA-FLS were subjected to RNA sequencing.
2022-08-17 | GSE200030 | GEO
Project description:System-level analysis of the (phospho)proteome during muscle formation and its interplay with epigenetic factors is critical to understand muscular diseases. Using stable isotope labeling (SILAC) and nano liquid chromatography-tandem mass spectrometry (nLC-MS/MS), we analyzed the (phospho)proteome during myogenesis of LHCN-M2 human skeletal myoblast cell line. First, enriched phosphorylation motifs suggested that PKC, cyclin-dependent kinase and MAPK are regulatory kinases during myodifferentiation. Then, we uncovered that the drugs known to inhibit these kinases either promoted myogenesis (PD0325901 and GW8510) or stall differentiation (CHIR99021 and roscovitine). We identified differentiation-specific myogenic and chromatin-related proteins, including histone methyltransferases. We then analyzed histone post-translational modifications (PTMs), and observed regulation of two gene-silencing marks, H3K9me3 and H4K20me3, in a correlated manner with the observed phenotypes. Chromatin immunoprecipitation-quantitative PCR (ChIP-qPCR) confirmed that H3K9me3 is erased from the myogenic regulatory factors (MRFs) MyoD, MyoG and Myf5 in differentiating myotubes. Together, our work demonstrates that the integration of histone PTM, phosphoproteomics and full proteome analysis gives a comprehensive understanding of the close connection between signaling pathways and epigenetics during differentiation of myotube in vitro.
2019-03-14 | PXD007589 | Pride
Project description:Rhabdomyosarcoma (RMS) is a frequent non-epithelial tumor of soft tissue that originates from a myogenic differentiation defect. Expression of SNAIL transcription factor is elevated in the alveolar subtype of RMS, characterized by a low myogenic differentiation status and high aggressiveness. SNAIL affects RMS metastasis by reorganization of actin cytoskeleton, regulation of ezrin expression and chemotaxis to HGF and SDF-1. The differentiation of human RMS diminishes SNAIL level. SNAIL silencing completely abolishes the growth of human RMS xenotransplants. SNAIL inhibits myogenic differentiation of RMS by binding to the MYF5 promoter, suppressing its expression, displacing MYOD from canonical to alternative E-box sequences and regulating myomiRs expression. SNAIL silencing allows the re-expression of MYF5 and canonical MYOD binding, promoting RMS cell myogenic differentiation. These novel results open potential avenues for the development of innovative therapeutic strategies based on SNAIL silencing.
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Project description:Identifying tissue and condition-specific gene regulatory elements and the mechanisms by which they associate with their target genes in human cells remains a significant challenge, largely due to the vast amount of non-protein-coding sequence in the human genome. Despite increasing evidence of physical interactions between distant regulatory regions and gene promoters in a variety of mammalian cell types, many searches for regulatory regions still consider only genomic regions proximal to the gene promoter. We identify a set of putative cis-regulatory modules (CRMs) of human skeletal muscle differentiation by combining new ChIP-chip data measuring the binding of myogenic TFs MyoG, MyoD, and SRF over the timecourse of differentiation with previously generated histone modification data. A substantial proportion of these putative CRMs are located distant (> 20 kb) from muscle gene promoters, and these distantly located CRMs are more likely than proximal promoter regions to show differentiation-specific changes in myogenic TF binding. Examination of two distant CRMs, which were previously shown to activate transcription in differentiating muscle cells, revealed that they interact physically with their upstream or downstream gene promoters (PDLIM3 and ACTA1) specifically upon myogenic differentiation. Our results highlight the importance of considering CRMs located distant from their target genes in investigations of mammalian gene regulation and further support the hypothesis that enhancer-promoter looping contacts are a general mechanism of regulation by distant CRMs. ChIP-chip experiments were performed using a custom Agilent 1x244K oligonucleotide array format (AMADID # 015037) to achieve 25-bp resolution tiling of 100 kb surrounding each of 104 selected human genes as well as positive and negative control regions. Biological and technical replicates of ChIP-chip experiments were performed using antibodies for MyoG and SRF in primary human skeletal muscle myoblasts crosslinked at 0 and 48 h relative to the removal of serum to induce differentiation. Additional technical replicate ChIP-chip experiments were performed for MyoD at 0 and 48 hours post-differentiation and p300 at 48 h post-differentiation. Total Input DNA for each experiment was labeled with Cy3 and used to create log-ratios with the Cy5 labeled IP data. Negative control no antibody mock-IP ChIP experiments were performed at 0 and 48 h post-differentiation.
2012-02-01 | E-GEOD-29865 | biostudies-arrayexpress
Project description:This model is from the article:
Reduction of off-flavor generation in soybean homogenates: a mathematical model.
Mellor N , Bligh F , Chandler I , Hodgman C
J. Food Sci.2010 Sep; 75(7): R131-8; PMID: 2153556,
Abstract:
The generation of off-flavors in soybean homogenates such as n-hexanal via the lipoxygenase (LOX) pathway can be a problem in the processed food industry. Previous studies have examined the effect of using soybean varieties missing one or more of the 3 LOX isozymes on n-hexanal generation. A dynamic mathematical model of the soybean LOX pathway using ordinary differential equations was constructed using parameters estimated from existing data with the aim of predicting how n-hexanal generation could be reduced. Time-course simulations of LOX-null beans were run and compared with experimental results. Model L(2), L(3), and L(12) beans were within the range relative to the wild type found experimentally, with L(13) and L(23) beans close to the experimental range. Model L(1) beans produced much more n-hexanal relative to the wild type than those in experiments. Sensitivity analysis indicates that reducing the estimated K(m) parameter for LOX isozyme 3 (L-3) would improve the fit between model predictions and experimental results found in the literature. The model also predicts that increasing L-3 or reducing L-2 levels within beans may reduce n-hexanal generation. PRACTICAL APPLICATION: This work describes the use of mathematics to attempt to quantify the enzyme-catalyzed conversions of compounds in soybean homogenates into undesirable flavors, primarily from the compound n-hexanal. The effect of different soybean genotypes and enzyme kinetic constants was also studied, leading to recommendations on which combinations might minimize off-flavor levels and what further work might be carried out to substantiate these conclusions.
2024-09-02 | BIOMD0000000415 | BioModels