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Status |
Public on Jul 07, 2008 |
Title |
Liver expression of Bmp6, Smad7, Id1 and Atoh8 is regulated by iron stores |
Organism |
Mus musculus |
Experiment type |
Expression profiling by array
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Summary |
Background & Aims: Although hepcidin expression was shown to be induced by the BMP signaling pathway, it is not yet known how iron regulates hepcidin and which of the BMP molecules is the endogenous regulator of iron homeostasis in vivo. We therefore assessed liver transcription profiles of mice fed an iron-deficient or an iron-enriched diet and looked for genes that were regulated similarly to hepcidin in that context. Methods: Genome-wide liver expression profiles of mice of the B6 and D2 genetic backgrounds subjected to iron-deficient, -balanced, or -enriched diets were obtained using Agilent Whole Genome microarrays. Real-time quantitative-PCR and western-blots were used to confirm microarray results and compare gene expression variations induced by secondary iron deficiency or iron overload with those consecutive to Smad4 or Hamp1-deficiency. Results: Among 1419 transcripts significantly modulated by the dietary iron content, four were regulated similarly to the hepcidin genes Hamp1 and Hamp2. They are coding for Bmp6, the regulator of Bmp/Smad signal transduction Smad7, the negative regulator of basic helix-loop-helix (bHLH) proteins Id1, and a protein with a bHLH domain, Atoh8. The iron overload developed by Smad4 and Hamp1-deficient mice also increased Bmp6 transcription. Body iron stores influence Smad1/5/8 phosphorylation and, as shown by analysis of mice with liver-specific disruption of Smad4, the binding partner for the receptor-activated Smads is necessary for activation of Smad7, Id1, and Atoh8 transcription by iron. Conclusions: Liver expression of Bmp6, Smad7, Id1, and Atoh8 is regulated by body iron stores and may participate in hepcidin regulation through the Bmp/Smad pathway. Keywords: response to dietary iron content
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Overall design |
Total RNA was extracted and purified using the RNeasy Lipid Tissue kit (Qiagen, Courtaboeuf, France). RNA quality was checked on RNA 6000 Nano chips using a Bioanalyzer 2100 (Agilent Technologies, Palo Alto, CA, USA). RNA samples used for chip experiments all had RNA Integrity Numbers (RIN) 16 greater than 9. Agilent’s Low RNA Input Linear Amplification Kit PLUS (One-color) was used to generate fluorescent cRNA. The amplified cyanine 3-labeled cRNA samples were then purified using Qiagen’s RNeasy mini spin colums and hybridized to Agilent Whole Mouse Genome Microarrays, 4x44K. Microarray slides were washed and scanned with an Agilent Scanner, according to the standard protocol of the manufacturer. Information from probe features was extracted from microarray scan images using the Agilent Feature Extraction software v.9.5.1. All the analyses were performed using Bioconductor, an open source software for the analysis of genomic data rooted in the statistical computing environment R. Normalization between arrays was performed using the quantile method. Genes for which the background-corrected signal intensities were not greater than 2.6 standard deviations above the average background in at least 3 B6 and 3 D2 mice were assumed not to be expressed in the liver and were excluded from further analysis. The R/MAANOVA package implemented in Bioconductor was used to perform a two-way analysis of variance in which the log2-transformed expression level was considered to be a function of diet, strain, and the effects of the interaction between these two factors. Fs tests, based on the James-Stein shrinkage estimates of the error variance, were computed on a gene-by-gene basis, and p values obtained by permutation analysis. The proportion of false positives among all the genes initially identified as being differentially expressed (FDR) was assessed using the procedure described by Storey. When influence of diet on expression levels was significant, t-tests were performed to investigate specific effects of iron deficiency and iron enrichment.
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Contributor(s) |
Kautz L, Meynard D, Darnaud V, Bouvet R, Monnier A, Mosser J, Coppin H, Roth M |
Citation(s) |
18539898 |
Submission date |
Feb 07, 2008 |
Last update date |
May 10, 2018 |
Contact name |
Marie-Paule Roth |
E-mail(s) |
roth@cict.fr
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Phone |
33 5 62744508
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Fax |
33 5 62744558
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Organization name |
Inserm
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Department |
U563
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Lab |
Génétique
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Street address |
Chu Purpan
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City |
Toulouse |
ZIP/Postal code |
31024 |
Country |
France |
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Platforms (1) |
GPL4134 |
Agilent-014868 Whole Mouse Genome Microarray 4x44K G4122F (Feature Number version) |
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Samples (30)
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Relations |
BioProject |
PRJNA108071 |
Supplementary file |
Size |
Download |
File type/resource |
GSE10421_RAW.tar |
203.2 Mb |
(http)(custom) |
TAR (of TXT) |
Processed data included within Sample table |
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