Notion d'expéditeur : Cass., com., 28 octobre 2008, pourvoi n°07-20.786, SARL Transports Page contre SA Charal

<p>Most Highly Regulated Genes in the Nuclear ERE-dependent Dataset.</p

De la délégation en cascade

<p>Selected Pathways of Interest with Known Function in Bone in the “Nuclear ERE-independent” Dataset.</p

Dissection of Estrogen Receptor Alpha Signaling Pathways in Osteoblasts Using RNA-Sequencing

<div><p>The effects of 17-β-estradiol in osteoblasts are primarily mediated by the nuclear transcription factors, estrogen receptor (ER)α and ERβ. ERs function through three general modes of action: DNA-binding dependent through estrogen response elements (EREs; designated nuclear ERE signaling); nuclear signaling via protein-protein interactions to other transcription factors (nuclear non-ERE signaling); and extra-nuclear signaling (membrane-bound functions of ERs). Identification of the specific transcriptional signatures regulated by each of these modes of action should contribute to an enhanced understanding of estrogen signaling in osteoblasts. To achieve this goal, we utilized specific mutations of ERα that eliminate the ability of the receptor to signal through a specific mode of action. The non-classical ERα knock-in (NERKI) mutation is incapable of signaling through direct DNA binding to EREs and the nuclear only ERα (NOER) mutation eliminates all membrane-localized signaling. Comparison of the gene expression patterns elicited by these mutations with the wild-type ERα (WT) pattern provides mode-specific data concerning transcriptional regulation by ERα. We expressed these constructs in the ER-negative osteoblastic cell line hFOB (−/+ estrogen) and performed global RNA-sequencing. Using a series of pair-wise comparisons, we generated three lists of genes that were regulated either by the nuclear ERE-dependent, nuclear ERE-independent, or extra-nuclear actions of ERα. Pathway and gene ontology analyses revealed that genes regulated through the nuclear ERE and nuclear non-ERE pathways were largely involved in transcriptional regulation, whereas genes regulated through extra-nuclear mechanisms are involved in cytoplasmic signaling transduction pathways. We also intersected our data with genes linked to bone density and fractures from a recent genome-wide association study and found 25 of 72 genes (35%) regulated by estrogen. These data provide a comprehensive list of genes and pathways targeted by these specific modes of ERα action and suggest that “mode-specific” ligands could be developed to modulate specific ERα functionality in bone.</p></div

Flow-chart detailing our data analysis strategy (described in detail in the text).

<p>Flow-chart detailing our data analysis strategy (described in detail in the text).</p

Comparison of the RNAseq datasets to gene with known involvement in bone biology from genome-wide association studies (GWAS).

<p>The nuclear ERE-dependent, nuclear ERE-independent, and extra-nuclear datasets were intersected with the 72 genes identified by GWAS studies. The overlapped genes for each mode of ERα action are listed and color-coded for clarity. The directionality of the estrogen-dependent regulation is denoted with an arrow following the gene symbol.</p

Estrogen receptor expression in hFOB cells.

<p>A) hFOB cells were infected with Ad-ERα, Ad-NERKI and Ad-NOER and cultured for 24 h. Protein extracts were prepared and a western blot was performed using the anti-FLAG and anti-β-actin antibodies. B) Densitometry was performed and the data are expressed as ER expression relative to the β-actin control.</p

Effects of Age and Estrogen on Skeletal Gene Expression in Humans as Assessed by RNA Sequencing

<div><p>Precise delineation of the specific genes and pathways altered with aging and estrogen (E) therapy may lead to new skeletal biomarkers and the development of novel bone therapeutics. Previous human bone studies, however, have been limited by only examining pre-specified genes and pathways. High-throughput RNA sequencing (RNAseq), on the other hand, offers an unbiased approach to examine the entire transcriptome. Here we present an RNAseq analysis of human bone samples, obtained from iliac crest needle biopsies, to yield the first <i>in vivo</i> interrogation of all genes and pathways that may be altered in bone with aging and E therapy in humans. 58 healthy women were studied, including 19 young women (mean age ± SD, 30.3 ± 5.4 years), 19 old women (73.1 ± 6.6 years), and 20 old women treated with 3 weeks of E therapy (70.5 ± 5.2 years). Using generally accepted criteria (false discovery rate [<i>q</i>] < 0.10), aging altered a total of 678 genes and 12 pathways, including a subset known to regulate bone metabolism (e.g., Notch). Interestingly, the <i>LEF1</i> transcription factor, which is a classical downstream target of the Wnt/β-catenin signaling pathway, was significantly downregulated in the bones from the old versus young women; consistent with this, <i>LEF1</i> binding sites were significantly enriched in the promoter regions of the differentially expressed genes in the old versus young women, suggesting that aging was associated with alterations in Wnt signaling in bone. Further, of the 21 unique genes altered in bone by E therapy, the expression of <i>INHBB</i> (encoding for the inhibin, beta B polypeptide), which decreased with aging (by 0.6-fold), was restored to young adult levels in response to E therapy. In conclusion, our data demonstrate that aging alters a substantial portion of the skeletal transcriptome, whereas E therapy appears to have significant, albeit less wide-ranging effects. These data provide a valuable resource for the potential identification of novel biomarkers associated with age-related bone loss and also highlight potential pathways that could be targeted to treat osteoporosis.</p><p>Trial Registration</p><p>ClinicalTrials.gov <a href="https://clinicaltrials.gov/ct2/show/NCT02349113" target="_blank">NCT02349113</a></p></div

Flow diagram for enrollment and allocation of the subjects in the E arm of the study.

<p>Flow diagram for enrollment and allocation of the subjects in the E arm of the study.</p

Effects of age and estrogen (E) on serum sclerostin levels.

<p>Serum sclerostin levels in the young versus old subjects (A) and in the old versus E-treated subjects (B) by the Biomedica and Meso Scale Discovery (MSD) assays. Data are mean ± SEM; note the difference in scales for the two sclerostin assays. *<i>p</i> < 0.05; ^†<i>p</i> < 0.01.</p

Effects of age and estrogen (E) on gene expression of <i>INHBA</i> and <i>INHBB</i>.

<p>Bone <i>INHBB</i> and <i>INHBA</i> gene expression levels by RNAseq in the old relative to young women (A) and in the E-treated relative to untreated old women (B). Data are shown as median fold changes (95% CIs). ^†<i>p</i> < 0.01; ^‡<i>p</i> < 0.001.</p