Dataset Information

Interplay between estrogen receptor and AKT in estradiol-induced alternative splicing.

ABSTRACT:

Background

Alternative splicing is critical for generating complex proteomes in response to extracellular signals. Nuclear receptors including estrogen receptor alpha (ER?) and their ligands promote alternative splicing. The endogenous targets of ER?:estradiol (E2)-mediated alternative splicing and the influence of extracellular kinases that phosphorylate ER? on E2-induced splicing are unknown.

Methods

MCF-7 and its anti-estrogen derivatives were used for the majority of the assays. CD44 mini gene was used to measure the effect of E2 and AKT on alternative splicing. ExonHit array analysis was performed to identify E2 and AKT-regulated endogenous alternatively spliced apoptosis-related genes. Quantitative reverse transcription polymerase chain reaction was performed to verify alternative splicing. ER? binding to alternatively spliced genes was verified by chromatin immunoprecipitation assay. Bromodeoxyuridine incorporation-ELISA and Annexin V labeling assays were done to measure cell proliferation and apoptosis, respectively.

Results

We identified the targets of E2-induced alternative splicing and deconstructed some of the mechanisms surrounding E2-induced splicing by combining splice array with ER? cistrome and gene expression array. E2-induced alternatively spliced genes fall into at least two subgroups: coupled to E2-regulated transcription and ER? binding to the gene without an effect on rate of transcription. Further, AKT, which phosphorylates both ER? and splicing factors, influenced ER?:E2 dependent splicing in a gene-specific manner. Genes that are alternatively spliced include FAS/CD95, FGFR2, and AXIN-1. E2 increased the expression of FGFR2 C1 isoform but reduced C3 isoform at mRNA level. E2-induced alternative splicing of FAS and FGFR2 in MCF-7 cells correlated with resistance to FAS activation-induced apoptosis and response to keratinocyte growth factor (KGF), respectively. Resistance of MCF-7 breast cancer cells to the anti-estrogen tamoxifen was associated with ER?-dependent overexpression of FGFR2, whereas resistance to fulvestrant was associated with ER?-dependent isoform switching, which correlated with altered response to KGF.

Conclusion

E2 may partly alter cellular proteome through alternative splicing uncoupled to its effects on transcription initiation and aberration in E2-induced alternative splicing events may influence response to anti-estrogens.

SUBMITTER: Bhat-Nakshatri P

PROVIDER: S-EPMC3687557 | biostudies-literature | 2013 Jun

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Interplay between estrogen receptor and AKT in estradiol-induced alternative splicing.

Bhat-Nakshatri Poornima P Song Eun-Kyung EK Collins Nikail R NR Uversky Vladimir N VN Dunker A Keith AK O'Malley Bert W BW Geistlinger Tim R TR Carroll Jason S JS Brown Myles M Nakshatri Harikrishna H

BMC medical genomics 20130611

<h4>Background</h4>Alternative splicing is critical for generating complex proteomes in response to extracellular signals. Nuclear receptors including estrogen receptor alpha (ERα) and their ligands promote alternative splicing. The endogenous targets of ERα:estradiol (E2)-mediated alternative splicing and the influence of extracellular kinases that phosphorylate ERα on E2-induced splicing are unknown.<h4>Methods</h4>MCF-7 and its anti-estrogen derivatives were used for the majority of the assay ...[more]

PMID: 23758675

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Project description:Estrogens play an important role in breast cancer (BC) development and progression; when the two isoforms of the estrogen receptor (ER? and ER?) are co-expressed each of them mediate specific effects of these hormones in BC cells. ER? has been suggested to exert an antagonist role toward the oncogenic activities of ER?, and for this reason it is considered an oncosuppressor. As clinical evidence regarding a prognostic role for this receptor subtype in hormone-responsive BC is still limited and conflicting, more knowledge is required on the biological functions of ER? in cancer cells. We have previously described the ER? and ER? interactomes from BC cells, identifying specific and distinct patterns of protein interactions for the two receptors. In particular, we identified factors involved in mRNA splicing and maturation as important components of both ER? and ER? pathways. Guided by these findings, here we performed RNA sequencing to investigate in depth the differences in the early transcriptional events and RNA splicing patterns induced by estradiol in cells expressing ER? alone or ER? and ER?.Exon skipping was the most abundant splicing event in the post-transcriptional regulation by estradiol. We identified several splicing events induced by ER? alone and by ER?+ER?, demonstrating for the first time that ER? significantly affects estrogen-induced splicing in BC cells, as revealed by modification of a subset of ER?-dependent splicing by ER?, as well as by the presence of splicing isoforms only in ER?+cells. In particular, we observed that ER?+BC cell lines exhibited around 2-fold more splicing events than the ER?- cells. Interestingly, we identified putative direct targets of ER?-mediated alternative splicing by correlating the genomic locations of ER? and ER? binding sites with estradiol-induced differential splicing in the corresponding genes.Taken together, these results demonstrate that ER? significantly affects estrogen-induced early transcription and mRNA splicing in hormone-responsive BC cells, providing novel information on the biological role of ER? in these tumors.

Project description:The pregnancy-augmented uterine vasodilation is linked to increased AT2R (angiotensin type-2 receptor) that mediates the vasodilatory effects of angiotensin II. However, the mechanisms controlling AT2R expression during pregnancy remain unclear. Estrogens are known to play a role in vascular adaptations during pregnancy. We hypothesized that estrogen stimulates uterine artery AT2R expression via ER (estrogen receptor)-?-dependent transcription in a pregnancy-specific endothelium-dependent manner. Plasma estradiol levels increased and peaked in late pregnancy and returned to prepregnant levels post-partum, correlating with uterine artery AT2R and ER? upregulation. Estradiol stimulated AT2R mRNA expression in endothelium-intact but not endothelium-denuded late pregnant and nonpregnant rat uterine artery ex vivo. Consistently, estradiol stimulated AT2R mRNA expression in late pregnant but not nonpregnant primary human uterine artery endothelial cells in vitro, which was abolished by ER antagonist ICI 182,780. Higher ER? protein bound to ER-responsive elements in AT2R promoter in the nonpregnant arteries whereas higher ER? bound in the pregnant state. ER? protein levels were similar but higher ER? protein levels were expressed in pregnant versus nonpregnant human uterine artery endothelial cells. Estradiol stimulation recruited ER? to the AT2R promoter in the nonpregnant state and ER? to the AT2R promoter in pregnancy; however, only ER? recruitment mediated transactivation of the AT2R reporter gene in pregnant human uterine artery endothelial cells. Estradiol-induced AT2R expression was abolished by the specific ER? (not ER?) antagonist 4-[2-Phenyl-5,7-bis(trifluoromethyl)pyrazolo[1,5-a]pyrimidin-3-yl]phenol (PHTPP) and mimicked by the specific ER? (not ER?) agonist 2,3-bis(4-Hydroxyphenyl)-propionitrile (DPN) in pregnant human uterine artery endothelial cells in vitro. This study demonstrates a novel role of pregnancy-augmented ER? in AT2R upregulation in the uterine artery and provides new insights into the mechanisms underlying uterine vascular adaptation to pregnancy.

Dataset Information

Interplay between estrogen receptor and AKT in estradiol-induced alternative splicing.

Background

Methods

Results

Conclusion

Publications

Interplay between estrogen receptor and AKT in estradiol-induced alternative splicing.

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