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Expression data from control and Ascorbic Acid (AA) stimulated Mc-3T3-E1 osteoblasts

ABSTRACT: Despite advances in investigating functional aspects of osteoblast (OB) differentiation, especially studies on how bone proteins are deposited and mineralized, there has been little research on the intracellular trafficking of bone proteins during OB differentiation. Collagen synthesis and secretion is markedly upregulated upon Ascorbic Acid (AA) stimulation. Understanding the mechanism by which collagen is mobilized in specialized OB cells is important for both basic cell biology and diseases involving defects in bone secretion and deposition. RabGTPases are major regulators on protein trafficking throughout the cell. In this study, we identified the Rab GTPases that are upregulated during 5-day AA differentiation of OBs using microarray analysis, namely Rab1, Rab3d and Rab27b. We used microarrays to detail the global programme of gene expression underlying procollagen production and trafficking and identified up-regulated genes during this process.

ORGANISM(S): Mus musculus

PROVIDER: GSE37676 | GEO | 2012/05/02

SECONDARY ACCESSION(S): PRJNA162555

REPOSITORIES: GEO

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Project description:Osteolytic lesions with suppression of osteoblastic (OB) differentiation are hallmarks of multiple myeloma (MM). Most recently, Gln availability has been found to be essential to sustain bone mass formation in murine models. On the other hand, MM cells are Gln-addicted and consume huge amounts of the amino acid, leading to a partial Gln depletion in the bone marrow (BM) plasma. We hypothesize that MM-dependent Gln depletion contributes to the defective OB differentiation characteristic of MM. Conversely, GLS (both KGA and GAC isoforms) and SLC38A2, the gene for the concentrative Gln transporter SNAT2, were induced. Consistent with this conclusion, the activity of SNAT2 was absent in undifferentiated hMSCs but well detectable after 14 days of OB differentiation, when total Gln uptake was also increased. Under the same conditions, OB differentiation markers (RUNX2, COL1A1, ALPL expression and ALPL activity or staining) were significantly induced but their expression was blunted by incubation in low-Gln (0.4 mM) medium or in the presence of the SNAT2 inhibitor MeAIB. The incubation in Gln-free D-MEM suppressed the induction of GLS and SLC38A2 along with OB differentiation, which was restored by the supplementation of Non-Essential Amino Acids (NEAA). Among NEAA, only asparagine (Asn) was able to rescue OB differentiation in the absence of Gln. The determination of intracellular amino acids with MS indicated that OB differentiation was associated with the increase of cell Asn, without significant changes of Gln, glutamate (Glu) or aspartate (Asp). Asparagine Synthetase (ASNS), the Gln-dependent enzyme that accounts for Asn synthesis, was also found induced during OB differentiation of hMSCs. Gene Expression Profiles of primary BM hMSCs and OBs from bone biopsies of both healthy donors and MM patients indicated that GLS, ASNS, and SLC38A2 are more expressed in OBs, while the expression of GLUL, the gene for GS, is higher in undifferentiated hMSCs from healthy donors. Overall, these results indicate that (1) OB differentiation of hMSCs is Gln-dependent; (2) the partial Gln depletion, imposed by Gln-addicted MM cells in the BM microenvironment, contributes to the impairment of osteoblastic differentiation of hMSCs; (3) hindrance of differentiation may depend on the limited availability of intracellular Asn derived from Gln-dependent ASNS. These results support the evidence that Gln addiction of MM cells affects bone microenvironment leading to the inhibition of OB differentiation and, consequently, to the development of MM bone disease.

Project description:Col1a1 is located on mouse distal chromosome 11 (Celera 94607393-94622990bp; 15kb) and encodes type I procollagen that associates stoichiometrically with col1a2 in forming secreted type I mature collagen that self-assembles into a supramolecular fibrillar structure consisting of a protein family. Type I collagens are predominantly enriched in bone, cartilage, skin, tendons, and eye (i.e. in major fibrous tissues) making up the main extracellular matrix (ECM) component for support and scaffolding purposes. Col1a1 is known to be expressed in the embryo (E9.5; Northern dot blot), perioptic mesenchyme (E11), cornea (E11), skeleton (E14.5), brain meninges (E14.5), and cartilage (E14.5) at relatively early stages. Most evidence of type I collagen function is coupled to the latter stages of skeletal development, thus bypassing the major events of axis formation, tissue differentiation, and mesenchlymal-epithial interactions, for example, that are more symbolic of morphogens and growth factors. Members of the integrin cell surface receptor family of molecules mediate cell adhesion to ECM proteins such as collagen (mainly to type IV basement membrane collagen that forms into a polygonal meshwork), fibronectin, and laminin implicated in wound healing and inflammatory responses (e.g. Crohn disease, ulcerative colitis) in connective tissues. There is some evidence for protein-protein interactions between leukocyte-associated integrins and the interstitial matrix in promoting the migration and/or activation of extravasated leukocytes (e.g., T cells and monocytes) within the perivascular compartment, an ECM-rich environment. This region is surmised to be an important location where certain human leukocytes undergo differentiation (e.g. monocytes) and activation (neutrophils, monocytes, lymphocytes) upon extravascular migration. The von Willebrand factor (vWF), type C motif is found in various plasma proteins like complement factors, the integrins, collagen types, and other extracellular proteins. Thus, the majority of vWF-containing proteins are extracellular and a common feature appears to be involvement in multi protein complexes participating in numerous biological events (e.g. cell adhesion, migration, homing, pattern formation, and signal transduction). Three organs (lung, heart and bone) of 5 AGA002 mutant mice (age 2x 10 days and 3x 11 days) were analysed by cDNA microarray technology. As reference five mice, 11 days old were used 50% of the chip hybridisations are dye swap experiments.

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Expression data from control and Ascorbic Acid (AA) stimulated Mc-3T3-E1 osteoblasts

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