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