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GPM6B regulates osteoblast differentiation and induction of mineralization by controlling cytoskeleton and matrix vesicle release.

ABSTRACT: Analysis of GPM6B silenced osteogenic hMSC at gene expression level. Analysis is showing significant changes in genes involved in cytoskeleton organization and biogenesis. Immunocytochemistry confirms changed distribution of actin filaments and change in shape and in size of focal adhesions upon GPM6B silencing. Moreover, we demonstrated that production and release of ALP-positive matrix vesicles (MVs) was reduced. In conclusion, we identified GPM6B as a novel regulator of osteoblast differentiation and bone formation and thereby demonstrating the significance of cytoskeleton organization for MV production and eventual mineralization.

ORGANISM(S): Homo sapiens

PROVIDER: GSE26004 | GEO | 2011/06/01

SECONDARY ACCESSION(S): PRJNA135513

REPOSITORIES: GEO

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GPM6B regulates osteoblast differentiation and induction of mineralization by controlling cytoskeleton and matrix vesicle release.

Project description:Analysis of GPM6B silenced osteogenic hMSC at gene expression level. Analysis is showing significant changes in genes involved in cytoskeleton organization and biogenesis. Immunocytochemistry confirms changed distribution of actin filaments and change in shape and in size of focal adhesions upon GPM6B silencing. Moreover, we demonstrated that production and release of ALP-positive matrix vesicles (MVs) was reduced. In conclusion, we identified GPM6B as a novel regulator of osteoblast differentiation and bone formation and thereby demonstrating the significance of cytoskeleton organization for MV production and eventual mineralization. Total RNA obtained from osteogenic hMSC, 7days after shRNAi lenti viral transductions with 3 different GPM6B silencing constructs (sh1, sh2, sh3) compared to 3 different controls (shC:non-silencing shRNA, tGFP: tGFP control vector and Mock: mock transduced cells)

2011-06-01 | E-GEOD-26004 | biostudies-arrayexpress

1α,25-Dihydroxyvitamin D3 Regulates Packaging of microRNAs within Extracellular Matrix Vesicles and Their Release in the Matrix

Project description:Growth plate chondrocytes are regulated by numerous factors and hormones as they mature during endochondral bone formation. Chondrocytes in the growth plate’s growth zone (GC cells) produce and export matrix vesicles (MVs) under the regulation of 1α,25-dihydroxyvitamin D3 [1α,25(OH)2D3]. 1α,25(OH)2D3 secreted by the cells acts on the MV membrane, releasing its contents. This study examined the regulatory role 1α,25(OH)2D3 has over the production and packaging of microRNA into MVs by GC cells and the ability to release microRNA from MVs once produced. We treated GC cells with 1α,25(OH)2D3 and then sequenced the microRNA in the cells and MVs. We also treated MVs with 1α,25(OH)2D3 and determined if the microRNA was released. To assess whether MVs can act directly with chondrocytes and if this is regulated by 1α,25(OH)2D3, we stained MVs with a membrane dye and treated GC cells with them. 1α,25(OH)2D3 regulated the production and packaging of microRNA into matrix vesicles. MVs did not release microRNA when treated with 1α,25(OH)2D3, indicating a heterogeneous MV population or a protective factor. Stained MVs were endocytosed by GC cells and this was increased with 1α,25(OH)2D3 treatment. This study adds new regulatory roles for 1α,25(OH)2D3 with respect to packaging and transport of MV microRNAs.

2021-12-06 | GSE190020 | GEO

Influence of Erythropoietin on Microvesicles Derived from Mesenchymal Ste cells(MSC) protecting renal function of Chronic Kidney Disease

Project description:miRNA profiles of the MSC-MVs and EPO-MVs were analyzed with a quantitative PCR (qPCR)-based array of the whole mice genome. Further analysis revealed differences in the miRNAs of 212 EPO-MVs (fold change ≥ 1.5 compared to the MSC-MVs), which constituted approximately 22.64% of all of the evaluated mouse miRNAs. Of all of the differences, 70.28% of the changes in the EPO-MV group involved upregulation To study the differential miRNA expression in MV and EPO-MV which might contributed to the better treatment in chronic kidney disease, we performed miRNA expression profiling of the culture supernatant of MSC with or without EPO incubation using the miRCURY LNA Array (v.18.0) (Exiqon, Vedbaek, Denmark).

2015-05-09 | E-GEOD-68665 | biostudies-arrayexpress

Influence of Erythropoietin on Microvesicles Derived from Mesenchymal Ste cells(MSC) protecting renal function of Chronic Kidney Disease

2015-05-09 | GSE68665 | GEO

TaqMan microRNA array for microvesicles released from adipose derived stem cells

Project description:Adipose derived stem cells (ASCs) were preconditioned with endothelial differentiation medium (EDM) for four days and then incubated in endothelial basal medium (EBM) supplemented with 1% microvesicle (MV)-free FBS for two days. The conditioned medium was harvested for MV isolation. The small RNA extracted from the MVs was used for microRNA array. qPCR gene expression profiling; Two equal-amount small RNA samples were from two independent experiments.

2014-08-08 | E-GEOD-59811 | biostudies-arrayexpress

Analysis of the Escherichia coli membrane vesicle proteome identifies markers of purity and culture conditions

Project description:Bacteria release nano-sized membrane vesicles (MVs) into the extracellular milieu. Bacterial MVs contain molecular cargo originating from the parent bacterium and have important roles in bacterial survival and pathogenesis. Using 8-plex iTRAQ approaches, we profiled the MV proteome of two Escherichia coli strains - uropathogenic E. coli (UPEC) 536 and probiotic Nissle 1917. For these strains, we compared the difference in the MV proteome between a crude input MV prepared by ultracentrifugation alone with that from MVs that were further purified by either density gradient centrifugation (DGC) or size exclusion chromatography (SEC); and also compared MVs from bacterial cultures that were grown in iron-restricted (R) and ironsupplemented (RF) conditions. We found that overall, outer membrane components were highly enriched, and bacterial inner membrane components were significantly depleted in both UPEC and Nissle MVs, in keeping with an outer membrane origin. In addition, we found enrichment of ribosome-related Gene Ontology terms in UPEC MV, and proteins involved in glycolytic process and ligase activity in Nissle MV. We have identified that three proteins (RbsB of UPEC in R; YoeA of UPEC in RF; BamA of Nissle in R) were consistently enriched in the DGC- and SEC-purified MV samples in comparison to their crude input MV, whereas conversely the 60 kDa chaperonin GroEL was enriched in the crude input MVs for both UPEC and Nissle in R condition. Such proteins may have a potential utility as technical markers for assessing the purity from contaminating non-vesicular protein after MV purification. Several proteins were changed in their abundance depending on the iron availability in the media.

2019-07-15 | PXD011345 | Pride

Proteomic analysis of Streptomyces coelicolor vesicles

Project description:Streptomyces coelicolor is a model organism for the study of Streptomyces, a genus of Gram-positive bacteria that undergoes a complex life cycle and produces an enormous repertoire of bioactive metabolites and extracellular enzymes. This study investigated the production and characterization of membrane vesicles (MVs) in liquid cultures of S. coelicolor M145 from a structural and biochemical point of view using microscopic, physical and -omics analyzes. Two main populations of MVs, F3 and F4 MVs, with different size and cargo were isolated and purified. S. coelicolor MV cargo is complex and contains many “messages” such as proteins and metabolites. A total of 166 proteins involved in cell metabolism and differentiation, molecular processing and transport was identified in MVs. In particular, a subset of these proteins was protected from the degradation after proteinase K treatment, indicating their localization inside the vesicles. Vesicle proteome includes many stress response proteins which also play an important role in S. coelicolor morpho- physiological differentiation. Moreover, MVs packaged with an array of metabolites such as antibiotics, vitamins, amino acids and components of carbon metabolism. The analysis of S. coelicolor MV cargo will provide informations to elucidate their biogenesis and functions

2021-11-02 | PXD024085 | Pride

Proteomic analysis of MRSA252 secretome and membrane vesicles

Project description:The secretome of the Staphylococcus aureus includes specific pathway-exported proteins, as well as cytoplasmic proteins with unknown mechanisms of secretion. As one component of the S. aureus secretome, extracellular membrane vesicles (MVs) are also comprised of secreted and cytoplasmic proteins. However, the contribution of MVs to the secretome of S. aureus has not been investigated. To address this, we performed a proteomic analysis of MVs purified from S. aureus strain MRSA252 along with a similar analysis of the culture supernatans before (S+MV) and after (S-MV) depletion of MVs.

2023-01-26 | PXD035662 | Pride

Adherent and suspension baby hamster kidney cells have a different cytoskeleton and surface receptor repertoire

Project description:Animal cell culture, with single cells growing in suspension, ideally in a chemically defined environment, is the workhorse of biopharmaceutical production. The synthetic environment lacks exogenous growth factors and usually requires a time-consuming adaptation process to select cell clones that proliferate in suspension to high cell numbers. The molecular mechanisms that facilitate the adaptation and that take place inside the cell are largely unknown. Especially for cell lines that are used for virus antigen production such as baby hamster kidney (BHK) cells, the restriction of virus growth through the evolution of undesired cell characteristics properties is highly unwanted. The comparison between adherently growing BHK cells and suspension cells with different susceptibility to foot-and-mouth disease virus revealed differences in the expression of cellular receptors such as integrins and heparan sulfates, and in the organization of the actin cytoskeleton. Transcriptome analyses and growth kinetics demonstrated the diversity of BHK cell clones and confirmed the importance of well-characterized parental cell clones and mindful screening to make sure that essential cellular features do not get lost during adaptation.

2021-05-15 | E-MTAB-10417 | biostudies-arrayexpress

Integration of functional imaging, cytometry and unbiased proteomics reveals new features of endothelial-to-mesenchymal transition in ischemic mitral valve regurgitation in human patients

Project description:Background: Following myocardial infarction, mitral regurgitation (MR) is a common complication. Previous animal studies demonstrated the association of endothelial-to-mesenchymal transition (EndMT) with mitral valve (MV) remodeling. Nevertheless, little is known about how MV tissue responds to ischemic heart changes in humans. Methods: MVs were obtained by the Cardiothoracic Surgical Trials Network from 17 patients with ischemic mitral regurgitation (IMR). Echo-doppler imaging assessed MV function at time of resection. Cryosections of MVs were analyzed using a multi-faceted histology and immunofluorescence examination of cell populations. MVs were further analyzed using unbiased label-free proteomics. Echo-Doppler imaging, histo-cytometry measures and proteomic analysis were then integrated. Results: MVs from patients with greater MR exhibited proteomic changes associated with proteolysis-, inflammatory- and oxidative stress-related processes compared to MVs with less MR. Cryosections of MVs from patients with IMR displayed activated valvular interstitial cells (aVICs) and double positive CD31+ αSMA+ cells, a hallmark of EndMT. Univariable and multivariable association with echocardiography measures revealed a positive correlation of MR severity with both cellular and geometric changes (e.g., aVICs, EndMT, leaflet thickness, leaflet tenting). Finally, proteomic changes associated with EndMT showed gene-ontology enrichment in vesicle-, inflammatory- and oxidative stress-related processes. This discovery approach indicated new candidate proteins associated with EndMT regulation in IMR. Conclusion: We describe an atypical cellular composition and distinctive proteome of human MVs from patients with IMR, which highlighted new candidate proteins implicated in EndMT-related processes, associated with maladaptive MV fibrotic remodeling.

2021-08-13 | PXD025096 | Pride

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