Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

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. 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)

ORGANISM(S): Homo sapiens

SUBMITTER: Jeroen van de Peppel

PROVIDER: E-GEOD-26004 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

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

Drabek Ksenija K van de Peppel Jeroen J Eijken Marco M van Leeuwen Johannes P T M JP

Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research 20110901 9

Neuronal membrane glycoprotein gene (GPM6B) encodes a membrane glycoprotein that belongs to the proteolipid protein family. We identified GPM6B as a gene that is strongly upregulated during osteoblast differentiation. To investigate the role of GPM6B in the process of bone formation, we silenced GPM6B expression during osteogenic differentiation of human mesenchymal stem cells (hMSCs). GPM6B silencing in hMSCs resulted in reduced alkaline phosphate (ALP) activity along with reduced mineralizatio ...[more]

PMID: 21638316

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Project description:Background: Ectopic vascular calcifications represent a major clinical problem associated with cardiovascular disease and mortality. However, the mechanisms underlying pathological vascular calcifications are largely unknown hampering the development of therapies to tackle this life threatening medical condition. Results: In order to gain insight into the genes and mechanisms driving this pathological calcification process we analyzed the transcriptional profile of calcifying vascular smooth muscle cells (C-VSMCs). These profiles were compared to differentiating osteoblasts, cells that constitute their physiological calcification counterparts in the body. Overall the transcriptional program of C-VSMC and osteoblasts did not overlap. Several genes, some of them relevant for bone formation, were distinctly modulated by C-VSMCs which did not necessarily lose their smooth muscle cell markers while calcifying. Bioinformatics gene clustering and correlation analysis disclosed limited bone-related mechanisms being shared by two cell types. Extracellular matrix (ECM) and biomineralization genes represented common denominators between pathological vascular and physiological bone calcifications. These genes constitute the strongest link between these cells and represent potential drivers for their shared end-point phenotype. Conclusions: the analyses support the hypothesis that VSMC trans-differentiate into C-VSMCs keeping their own identity while using mechanisms that osteoblasts use to mineralize. The data provide novel insights into groups of genes and biological processes shared in MSC and VSMC osteogenic differentiation. The distinct gene regulation between C-VSMC and osteoblasts might hold clues to find cell-specific pathway modulations, opening the possibility to tackle undesired vascular calcifications without disturbing physiologic bone formation and vice versa. Total RNA obtained from hMSC and hVSMC cultured in osteogenic differentiation medium supplemented with 1.8 mM Ca2+ for 0, 2, 8, 12 or 25 days respectively. For each timepoint 3 replicates were used, with exception for day 0 where 4 replicates were collected.

Project description:Rosiglitazone-targeted transcriptional networks as gene expression signature of physiological and pathological mineralization – a top-down approach to identify novel targets for bone and vascular health Clinical studies have recently revealed detrimental skeletal and vascular effects of the insulin sensitizer rosiglitazone. At the cellular level, we have shown earlier that rosiglitazone accelerates osteoblast differentiation from human mesenchymal stem cells (hMSC) at the expense of increased oxidative stress and apoptosis. In calcifying human vascular cells, rosiglitazone stimulates pathological mineralization, an effect that was diminished by the antioxidant resveratrol. In order to elucidate the transcriptional networks underlying the rosiglitazone-enhanced mineralization phenotype, we performed genome-wide transcriptional profiling of osteogenic hMSCs treated with rosiglitazone for short-term periods of 1 up to 48h during the first two days of culture time, a phase that we show is sufficient to mediate the stimulatory effect of rosiglitazone on mineralization. Microarray-based mRNA expression analysis revealed 190 probes that were differently expressed in at least one condition compared to vehicle-treated control. This rosiglitazone gene signature contained confirmed primary PPAR targets and was also endogenously regulated during osteogenic differentiation from hMSCs and osteoblast-like differentiation of vascular smooth muscle cells (VSMCs) into calcifying vascular cells (CVCs). Further comparative analysis revealed rosiglitazone targets that were commonly enriched in osteoblasts and CVCs or specifically enriched in either osteoblasts or CVCs. Finally, we compared the expression patterns of CVC-specific genes with patient expression data from carotid plaque versus intact adjacent tissue, and identified five rosiglitazone targets that were differentially regulated in CVCs and carotid plaque but not osteoblasts when compared to their respective non-mineralizing counterparts. These targets, i.e PDK4, SDC4, SPRY4, TCF4, and DACT1 may specifically control extracellular matrix mineralization in vascular cells and hence provide interesting candidates for further investigations to improve vascular health.

Dataset Information

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

Publications

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

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