Systematic Assessment of Human Osteoblast Transcriptome in Resting and Induced Primary Cells
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE10311 | GEO | 2008/02/01
SECONDARY ACCESSION(S): PRJNA108537
REPOSITORIES: GEO
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Project description:Osteoblasts are key players in bone remodeling. The accessibility of human primary osteoblast-like cells (HOb) from bone explants render them a lucrative model for studying molecular physiology of bone turnover, discovery of novel anabolic therapeutics and mesenchymal cell biology in general. Relatively little is known about resting and dynamic expression profiles of HObs and no studies have been conducted to date to systematically assess the osteoblast transcriptome. The aim of this study was to characterize HObs and investigate signaling cascades and gene networks using genomewide expression profiling in resting and Bone Morphogenic Protein (BMP)-2 and Dexamethasone induced cells. Our data showed a vast number of genes and networks expressed predominantly in HObs as compared to closely related cells such as fibroblasts or chondrocytes. For instance, genes in the insulin-like growth factor (IGF) signaling pathway were enriched in HObs (p=0.003) and included the binding proteins (IGFBP1, 2, 5) and IGF-2 and its receptor. Another HOb specific expression pattern included leptin and its receptor (p<10-8). Furthermore, after stimulating HObs with Dexamethasone or BMP-2, the expression of several interesting genes and pathways were observed where data supported the role of peripheral leptin signaling in bone cell function. In conclusion, we provide the landscape of tissue-specific and dynamic gene expression in HObs, a resource, which will allow utilization of osteoblasts as a model to study specific gene networks and gene families related to human bone physiology and diseases. Experiment Overall Design: Human trabecular bone from the proximal femoral shaft was collected from two male patients, both undergoing total hip replacement. The bone chips were minced thoroughly and washed with PBS and cultured in three biological replicates in cell medium containing alpha-MEM supplemented with 2 mmol/l L-Glutamine, 100U/mL penicillin, 100mg/mL streptomycin, and 10% fetal bovine serum and grown at 37 degreeC with 5% CO2 until confluence. At 70-80% confluence, each replicate was trypsinized and sub-cultured in 6-well plates (100 000 cells/well) for 12 days. Prior to treatment, the cells were starved for 20h by adding complete cell medium containing 0.5% fetal bovine serum. The cells were then incubated for 2h and 24h with 10-7 M of dexamethasone and 10-4 mg/ml of rhBMP-2 with the same concentration of vehicle, respectively. Experiment Overall Design: At the two time points, the cell medium was removed and the cells were harvested stored in -70 degreeC until RNA extraction. Experiment Overall Design: RNA was isolated from the cell lysates using the RNeasy Mini Kit and Experiment Overall Design: expression studies were performed in triplicate (biological replicates) using the Affymetrix Human Genome U133 plus 2.0 Array (Affymetrix, Santa Clara, CA, USA). One microgram of RNA was reverse transcribed into cDNA and in vitro transcription was performed to generate biotin-labeled cRNA for subsequent hybridization. Hybridized target cRNA was then stained with streptavidin phycoerythrin, and arrays were scanned using a GeneArray Scanner at an excitation wavelength of 488nm.
2008-06-11 | E-GEOD-10311 | biostudies-arrayexpress
Project description:Titanium is widely used in bone prostheses due to its excellent biocompatibility and osseointegration capacity. To understand the effect of sandblasted acid-etched (SAE) Ti implants on the biological responses of human osteoblast (HOb), their proteomic profiles were analysed using nLC-MS/MS. The cells were cultured with the implant materials, and 2544 distinct proteins were detected in samples taken after 1, 3 and 7 days. Comparative analyses of proteomic data were performed using Perseus software. The expression of proteins related to EIF2, mTOR, insulin-secretion and IGF pathways showed marked differences in cells grown with SAE-Ti in comparison with cells cultured without Ti. Moreover, the proteomic profiles obtained with SAE-Ti were compared over time. The affected proteins were related to adhesion, immunity, oxidative stress, coagulation, angiogenesis, osteogenesis and extracellular matrix formation functions. The proliferation, mineralisation and osteogenic gene expression in HObs cultured with SAE-Ti were characterised in vitro. The results showed that the osteoblasts exposed to this material increase their mineralisation rate and expression of COLI, RUNX2, SP7, CTNNB1, CAD13, IGF2, MAPK2 and mTOR. Overall, the observed proteomic profiles can explain the SAE-Ti osteogenic properties, widening our knowledge of key signalling pathways taking part in the early stages of the osseointegration process in this type of implantations.
2023-03-11 | PXD035986 | Pride
Project description:The osteogenic differentiation process consists in a well-coordinated multi-step sequence of events, in which each stage is characterized by the expression of distinct protein markers, as well as by individual morphological features. In order to precisely identify the step at which cells are permissive to MET-driven transformation, we used expression profiling. We compared the transcriptional profiles of MET-HOB and MSC cells where osteoblast differentiation was induced. Using microarrays, we evaluated the expression profiles of MET-HOB and MSC cells where osteoblast differentiation was induced, by culturing cells in the presence of ascorbic acid, α-glycerophosphate and dexamethasone for 7, 14 and 21 days
2013-02-28 | E-GEOD-28205 | biostudies-arrayexpress
Project description:We utilized RNA sequencing to provide the gene expression profile of mesenchymal stem cells (MSCs) derived osteoblasts in different differentiation stages as well as the gene alteration profile of H9 MSCs-derived osteoblasts following different gene regulation treatments, including SDC1 overexpression, knockout of CEBPD, knockout of IL1R1, and knockdown of CORIN. The commitment of stem cells to an osteoblastic lineage is a complex and tightly regulated process, involving coordination between extrinsic signals and intrinsic transcriptional machinery. While many rodent osteoblast studies abound, human osteoblastic signaling networks are not as well-researched due to limitations in cell sources and existing models. Here, we generated human pluripotent stem cell (hPSC)-derived osteoblasts and used this modeling platform to identify functional osteoblastic surface receptors and their downstream transcriptional networks involved in human osteogenesis. We systematically dissected osteoblastic gene expression patterns and identified critical clusters associated with osteogenesis. The osteoblast surface receptor signature study revealed enriched CORIN expression in osteoblasts and enriched SDC1 expression in MSCs. In vitro calcified staining and 3D biomimetic GelMA/microCT (μCT) studies demonstrated that depletion of CORIN as well as ectopic expression of SDC1 significantly impaired osteogenesis. Transcriptome analyses revealed that dysregulation of CORIN or SDC1 alters biological processes and pathways mainly involved in bone formation associated signaling including TGFβ regulating extracellular matrix and Wnt signaling. Genome-wide ChIP enrichment analysis further indicated that CEBPD is a downstream transcription factor involved in CORIN and SDC1-modulated osteogenesis. CEBPD ChIP-seq and RNA-seq validated its role in controlling extracellular matrix organization, bone mineralization, and TGFβ, BMP, and Wnt signaling. Depletion of CEBPD led to impairment of osteoblastic differentiation. Differential expression analysis of single-cell transcriptomes revealed enriched expression of CEBPD and its transcriptional targets during the different stages of osteoblast differentiation. In summary, our findings elucidated the vital signaling in osteoblast lineage commitment.
2023-04-28 | GSE202080 | GEO
Project description:A microRNA array was performed using human primary osteoblasts (hOB) obtained from trabecular bone of postmenopausal women after knee replacement due to osteoarthritis in order to determine the miRNAs expressed in these osteoblastic cells.
2015-12-21 | GSE74211 | GEO
Project description:We carried out CEBPD chromatin immunoprecipitation followed by next generation sequencing (ChIP-seq) and revealed that the top motif enriched in CEBPD sites are identical to the CEBPD family motif and osteoblast differentiation/bone formation motifs like Fos1, Fra1, JUNB, RUNX and STAT3. The commitment of stem cells to an osteoblastic lineage is a complex and tightly regulated process, involving coordination between extrinsic signals and intrinsic transcriptional machinery. While many rodent osteoblast studies abound, human osteoblastic signaling networks are not as well-researched due to limitations in cell sources and existing models. Here, we generated human pluripotent stem cell (hPSC)-derived osteoblasts and used this modeling platform to identify functional osteoblastic surface receptors and their downstream transcriptional networks involved in human osteogenesis. We systematically dissected osteoblastic gene expression patterns and identified critical clusters associated with osteogenesis. The osteoblast surface receptor signature study revealed enriched CORIN expression in osteoblasts and enriched SDC1 expression in MSCs. In vitro calcified staining and 3D biomimetic GelMA/microCT (μCT) studies demonstrated that depletion of CORIN as well as ectopic expression of SDC1 significantly impaired osteogenesis. Transcriptome analyses revealed that dysregulation of CORIN or SDC1 alters biological processes and pathways mainly involved in bone formation associated signaling including TGFβ regulating extracellular matrix and Wnt signaling. Genome-wide ChIP enrichment analysis further indicated that CEBPD is a downstream transcription factor involved in CORIN and SDC1-modulated osteogenesis. CEBPD ChIP-seq and RNA-seq validated its role in controlling extracellular matrix organization, bone mineralization, and TGFβ, BMP, and Wnt signaling. Depletion of CEBPD led to impairment of osteoblastic differentiation. Differential expression analysis of single-cell transcriptomes revealed enriched expression of CEBPD and its transcriptional targets during the different stages of osteoblast differentiation. In summary, our findings elucidated the vital signaling in osteoblast lineage commitment.
2023-04-28 | GSE201836 | GEO
Project description:<p>Mechanical force is critical for the development and remodeling of bone. Here we report that mechanical force regulates the production of the metabolite asymmetric dimethylarginine (ADMA) via regulating the hydrolytic enzyme dimethylarginine dimethylaminohydrolase 1 (Ddah1) expression in osteoblasts. The presence of -394 4N del/ins polymorphism of Ddah1 and higher serum ADMA concentration are negatively associated with bone mineral density. Global or osteoblast-specific deletion of Ddah1 leads to increased ADMA level but reduced bone formation. Further molecular study unveils that mechanical stimulation enhances TAZ/SMAD4-induced Ddah1 transcription. Deletion of Ddah1 in osteoblast-lineage cells fails to respond to mechanical stimulus-associated bone formation. Taken together, the study reveals mechanical force is capable of down-regulating ADMA to enhance bone formation.</p>
2021-11-23 | MTBLS3725 | MetaboLights
Project description:Osteoblasts are the only somatic cell type with bone-forming ability. While the cellular and molecular mechanisms underlying their differentiation and function have been identified, their dynamic control in vivo is unclear. By intravital multiphoton microscopy of live bone tissues of osteoblast fluorescent reporter mice, we visualized intact mature osteoblasts in vivo and found that they actively secrete and capture extracellular microvesicles. Microvesicles from mature osteoblasts contain the microRNA miR-143-3p, which inhibits osteoblast differentiation. Osteoblast-specific deletion of miR-143 increased bone formation and miR-143-deficient microvesicles induced recovery from bone defect. In sum, we report a novel mode of intercellular communication in vivo via microRNA-containing extracellular vesicles, which controls bone homeostasis.
2022-02-04 | GSE144512 | GEO
Project description:The αNAC (alpha chain of the Nascent polypeptide-Associated Complex) transcriptional coregulator is developmentally expressed in osteoblasts and regulates osteoblast differentiation in vitro and in vivo. αNAC can activate or repress gene transcription, a function that is dynamically regulated by post-translational modification. Phosphorylation of residue Ser132 stimulates the sumoylation of αNAC on Lys127 to repress gene expression. Using in vitro kinase assays, we show that Ser132 phosphorylation is mediated by the DNA-dependent protein kinase catalytic subunit (DNA-PKcs). Pharmacological inhibition of DNA-PKcs kinase activity or gene silencing of Prkdc (encoding DNA-PKcs) in murine osteoblastic MC3T3-E1 cells and human adipose-derived mesenchymal stromal cells markedly enhanced osteogenesis and the expression of osteoblast differentiation marker genes. ChIP-seq identified Ezh2 as a target of the αNAC/DNA-PKcs signaling pathway. Mechanistically, inhibition of DNA-PKcs repressed Ezh2 expression, induced cell cycle block, and increased osteogenesis by significantly enhancing the bone morphogenetic protein 2 (BMP-2) response in osteoblasts and other mesenchymal cells. Importantly, in vivo inhibition of the kinase enhanced bone biomechanical properties, and bones from osteoblast-specific conditional Prkdc-knockout mice exhibited increased stiffness. In conclusion, DNA-PKcs is a negative regulator of osteoblast differentiation, and therefore DNA-PKcs inhibitors may have therapeutic potential for bone regeneration and metabolic bone diseases.
2019-12-10 | GSE141686 | GEO
Project description:Osteoarthritis (OA) is the most common joint disease and this is a major cause of joint pain and disability in the aging population. Its etiology is multifactorial (i.e., age, obesity, joint injury, genetic predisposition), and the pathophysiologic process affects the entirety of the joint (Martel-Pelletier J et al. Osteoarthritis. Nature reviews Disease primers. 2016;2:16072). Although it is not yet clear if it precedes or occurs subsequently to cartilage damage, subchondral bone sclerosis is an important feature in OA pathophysiology (Goldring SR et al. Changes in the osteochondral unit during osteoarthritis: structure, function and cartilage-bone crosstalk. Nat Rev Rheumatol. 2016;12:632-44). It is characterized by local bone resorption and the accumulation of weakly mineralized osteoid substance (Bailey AJ et al. Phenotypic expression of osteoblast collagen in osteoarthritic bone: production of type I homotrimer. Int J Biochem Cell Biol. 2002;34:176-82). Subchondral bone sclerosis is suspected to be linked to cartilage degradation, not only by modifying the mechanical stresses transmitted to the cartilage, but also by releasing biochemical factors with an activity on cartilage metabolism (Sanchez C et al. Osteoblasts from the sclerotic subchondral bone downregulate aggrecan but upregulate metalloproteinases expression by chondrocytes. This effect is mimicked by interleukin-6, -1beta and oncostatin M pre-treated non-sclerotic osteoblasts. Osteoarthritis Cartilage. 2005;13:979-87; Sanchez C et al. Subchondral bone osteoblasts induce phenotypic changes in human osteoarthritic chondrocytes. Osteoarthritis Cartilage. 2005;13:988-97; Westacott CI et al J. Alteration of cartilage metabolism by cells from osteoarthritic bone. Arthritis Rheum. 1997;40:1282-91. We have previously demonstrated that osteoblasts isolated from subchondral OA bone exhibited an altered phenotype. More precisely, we showed that osteoblasts coming from the thickening (called sclerotic, SC) of subchondral bone located just below a cartilage lesion produced higher levels of alkaline phosphatase, interleukin (IL)-6, IL-8, prostaglandinE2, vascular endothelial growth factor (VEGF), matrix metalloproteinase (MMP)-9 and transforming growth factor(TGF)-β1 and type I collagen than osteoblasts coming from the non-thickening neighboring area (called non-sclerotic area, NSC) (Sanchez C et al. Phenotypic characterization of osteoblasts from the sclerotic zones of osteoarthritic subchondral bone. Arthritis Rheum. 2008;58:442-55; Sanchez C et al. Regulation of subchondral bone osteoblast metabolism by cyclic compression. Arthritis Rheum. 2012;64:1193-203.) To compare secretome of cells living in different in vivo conditions is useful, not only to better understand the pathological mechanisms underlying changes in OA subchondral bone, but also to identify soluble biomarkers potentially reflecting these changes. Using our well-characterised human subchondral osteoblast culture model, we compared the secretome of osteoblasts coming from sclerotic and non sclerotic OA subchondral bone. This approach allowed to identify changes in secretome that contribute to explain some subchondral bone abnormalities in OA and to propose osteomodulin and fibulin-3 as potential biomarkers of OA subchondral bone remodelling.
2018-03-20 | PXD008494 | Pride