Dissection of estrogen receptor alpha signaling pathways in osteoblasts using RNA-sequencing.

ABSTRACT: The effects of 17-?-estradiol in osteoblasts are primarily mediated by the nuclear transcription factors, estrogen receptor (ER)? and ER?. 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 ER? that eliminate the ability of the receptor to signal through a specific mode of action. The non-classical ER? knock-in (NERKI) mutation is incapable of signaling through direct DNA binding to EREs and the nuclear only ER? (NOER) mutation eliminates all membrane-localized signaling. Comparison of the gene expression patterns elicited by these mutations with the wild-type ER? (WT) pattern provides mode-specific data concerning transcriptional regulation by ER?. 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 ER?. 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 ER? action and suggest that "mode-specific" ligands could be developed to modulate specific ER? functionality in bone.

SUBMITTER: Roforth MM 

PROVIDER: S-EPMC4002480 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature