Unknown,Transcriptomics,Genomics,Proteomics

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Expression profiles of human primary osteoblasts (HOB) and MET transformed osteoblast clones

ABSTRACT: We studied MET-transformed human primary osteoblasts (MET-HOBs), which we previously turned into osteosarcoma cells by LV driven over-expression of MET oncogene. We obtained distinct MET transformed HOB clones derived from independent events of transgene integration. To characterise the phenotype of the MET-HOB clones we used oligonucleotide microarrays. Expression profiles of MET-HOBs and parental HOBs were compared. To characterise the phenotype of the MET-HOB clones we used oligonucleotide microarrays

ORGANISM(S): Homo sapiens

SUBMITTER: Raffaele Calogero

PROVIDER: E-GEOD-28256 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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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.

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Expression profiles of human primary osteoblasts (HOB) and MET transformed osteoblast clones

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