Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Expression data from hypervitaminosis A rat diaphyseal bone

ABSTRACT: Vitamin A is the only known compound that produces spontaneous fractures in rats. In an effort to resolve the molecular mechanism behind this effect, we fed young rats high doses of vitamin A and performed a global transcriptional analysis of diaphyseal bone after one week, i.e. just before the first fractures appeared. Microarray gene expression analysis revealed that 68 transcripts were differentially expressed in hypervitaminotic cortical bone and 118 transcripts were found when the bone marrow was also included. 98% of the differentially expressed genes in the bone marrow sample were up-regulated. In contrast, hypervitaminotic cortical bone without marrow showed reduced expression of 37% of differentially expressed genes. Gene Ontology (GO) analysis revealed that only samples containing bone marrow were associated to a GO term, which principally represented extracellular matrix (ECM). This is consistent with the histological findings of increased endosteal bone formation. Four of the genes in this ECM cluster and four other genes, including Cyp26b1 which is known to be up-regulated by vitamin A, were selected and verified by real-time PCR. In addition, immunohistochemical staining of bone sections confirmed that the bone-specific molecule, osteoadherin (Omd) was up-regulated. Further analysis of the major gene expression changes revealed distinct differences between cortical bone and bone marrow, e.g. there appeared to be augmented Wnt signaling in the bone marrow but reduced Wnt signaling in cortical bone. Moreover, induced expression of hypoxia-associated genes was only found in samples containing bone marrow. Together, these results corroborate our previous observations of compartment-specific effects of vitamin A, with reduced periosteal but increased endosteal bone formation, and suggest important roles for Wnt signaling and hypoxia in the processes leading to spontaneous fractures. Rat diaphyseal bones from hypervitaminosis A and controls (8week old) were extracted for RNA and hybridized on Affymetrix microarrays. Humeri were isolated, and all connective tissue, including periosteum, was completely removed as were both epiphyses, including growth plates. Samples of cortical bone including marrow were snap-frozen in liquid nitrogen and quickly crushed into a fine powder using a mortar and pestle followed by total RNA extraction with TRI Reagent® (Sigma-Aldrich). For cortical bone without marrow, diaphyseal bones were cut into pieces, vortexed in ice-cold PBS three times for 10 s to remove marrow cells followed by snap-freezing in liquid nitrogen, crushing into a fine powder and RNA extraction. Isolated RNA was quantified using spectrophotometry by measuring the absorbance at 260 nm, and the 260/280 nm ratio was calculated. RNA was kept only when this ratio was 1.9-2.0 to ensure the absence of protein contamination and limited RNA degradation. The integrity of sample RNAs was confirmed by capillary electrophoresis separating 18 S and 28 S ribosomal RNA on an Agilent Technologies 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA). Notably, the metaphyses and growth plates were not included in theses samples. Importantly the amount of RNA extracted from cortical bone including marrow was approximately four times the amount of RNA extracted from the pure cortical bone and was therefore considered herein to mainly represent bone marrow cells.

ORGANISM(S): Rattus norvegicus

SUBMITTER: Anders Isaksson

PROVIDER: E-GEOD-29859 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:We used gene array analysis of cortical bone to identify Phex-dependent gene transcripts regulating Fgf23 production and mineralization in Hyp mice. We discovered that activation of Fgf receptor- and Wnt-pathways contribute to increased Ffg23 gene transcription in Hyp bone. We found evidence in Hyp bone for increased expression of Fgf1, Fgf7, and Egr2 in the Fgf-signaling pathway and decrements in Sost and Cpz and increments in Sfrp1 and 4 in the Wnt-signaling pathway. Moreover, activation of Fgf and Wnt-signaling stimulated, whereas Tgf β inhibited Fgf23 promoter activity in osteoblasts. We also observed reductions in Bmp1, a metalloproteinase that metabolizes the Fgf23 regulatory extracellular matrix protein Dmp1. These findings suggest that elevation of Fgf23 expression in osteocytes is regulated by interactions between cell surface expression of Phex, extracellular matrix proteins and paracrine effects of Fgf and Wnt. Alterations were also found in enzymes regulating the posttranslational processing and stability of Fgf23, including decrements in the glycosyltransferase Galnt3 and the proprotein convertase Pcsk5. In addition, we found that the Pcsk5 and the glycosyltransferase Galnt3 were decreased in Hyp bone, suggesting that reduced post-translational processing of FGF23 may also contribute to increased Fgf23 levels in Hyp mice. With regards to mineralization, we identified additional candidates to explain the intrinsic mineralization defect in Hyp osteoblasts, including increases in the mineralization inhibitors Mgp and Thbs4, as well as increases in local pH altering factors, carbonic anhydrase 12 (Car12) and 3 (Car3) and the sodium-dependent citrate transporter (Slc13a5). These studies demonstrate the complexity of gene expression alterations in bone that accompanies inactivating Phex mutations and identify novel pathways that may coordinate Fgf23 expression and mineralization of extracellular matrix in Hyp bone. We isolated total RNAs from long bones of both WT and Hyp mice at 12 days of age. Since the RNA yields from the long bones are very low, we combined 2 bone samples with same genotype (WT or Hyp) for one RNA extraction. We will compare the difference of the gene expressions between Hyp and WT. We will use 4 samples in each animal condition.

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Expression data from hypervitaminosis A rat diaphyseal bone

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