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Protein expression in primary murine dermal fibroblasts grown on fibronectin vs. plastic

ABSTRACT: We were interested in identifying proteins that are regulated in response to the extracellular matrix molecule, fibronectin, in dermal fibroblasts. To determine proteins regulated by exposure to fibronectin, primary murine dermal fibroblasts were cultured on tissue culture plastic or on tissue culture plates coated with a thin layer of fibronectin (2 μg/cm2). Fibroblasts were seeded at 35,000 cells/10 cm2 and were harvested 48 hours later. Cell pellets from these fibroblasts were shipped to Kinexus Bioinformatics Corp. (Vancouver, B.C.) where they were lysed and analyzed using the Kinex TM Antibody Microarray (KAM 1.2), which contains 800 (500 pan-specific and 300 phosphorylation site-specific) antibodies. This enabled us to identify candidate proteins regulated by exposure to fibronectin.

ORGANISM(S): Mus musculus Homo sapiens

PROVIDER: GSE30135 | GEO | 2011/06/22

SECONDARY ACCESSION(S): PRJNA143831

REPOSITORIES: GEO

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Project description:Abstract Background: Bone marrow stromal cells (BMSCs) are being used for immune modulatory, anti-inflammatory and tissue engineering applications, but the properties responsible for these effects are not completely understood. Human BMSCs were characterized to identify factors that might be responsible for their clinical effects and biomarkers for assessing their quality. Methods: Early passage BMSCs prepared from marrow aspirates of 4 healthy subjects were compared to 3 human embryonic stem cell (hESC) samples, CD34+ cells from 3 healthy subjects and 3 fibroblast cell lines. The cells were analyzed with oligonucleotide expression microarrays with more than 35,000 probes. Results: BMSC gene expression signatures of BMSCs differed from those of hematopoietic stem cells (HSCs), hESCs and fibroblasts. Genes up-regulated in BMSCs were involved with cell movement, cell-to-cell signaling and interaction and proliferation. The BMSC up-regulated genes were most likely to belong to integrin signaling, integrin linked kinase (ILK) signaling, NFR2-mediated oxidative stress response, regulation of actin-based motility by Rho, actin cytoskeletal signaling, caveolar-mediated endocytosis, clathrin-mediated endocytosis and Wnt/beta catenin signaling pathways. Among the most highly up-regulated genes were structural extracellular (ECM) proteins: alpha1 and beta1 integrin chains, fibronectin, collagen type IIIalpha1, and collagen type Valpha1 and functional EMC proteins: connective tissue growth factor (CTGF) and transforming growth factor beta induced protein (TGFBI) and ADAM12. Conclusions: Global analysis of human BMSCs suggests that they are mobile, metabolically active, proliferative and interactive cells that make use of integrins and integrin signaling. They produce abundant ECM proteins; some of which may contribute to their clinical immune modulatory and anti-inflammatory effects. Seven samples from early passage BMSCs were prepared from marrow aspirates of healthy subjects and compared to 3 human embryonic stem cell (hESC) samples, CD34+ cells from 3 healthy subjects and 3 fibroblast cell lines. Total RNA from a pool of PBMCs from six healthy subjects was extracted and amplified into aRNA to serve as a reference.

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Protein expression in primary murine dermal fibroblasts grown on fibronectin vs. plastic

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