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Estrogen Regulates MAPK-Related Genes through Genomic and Nongenomic Interactions between IGF-I Receptor Tyrosine Kinase and Estrogen Receptor-Alpha Signaling Pathways in Human Uterine Leiomyoma Cells.

ABSTRACT: Estrogen and growth factors play a major role in uterine leiomyoma (UtLM) growth possibly through interactions of receptor tyrosine kinases (RTKs) and estrogen receptor-alpha (ER?) signaling. We determined the genomic and nongenomic effects of 17?-estradiol (E(2)) on IGF-IR/MAPKp44/42 signaling and gene expression in human UtLM cells with intact or silenced IGF-IR. Analysis by RT(2) Profiler PCR-array showed genes involved in IGF-IR/MAPK signaling were upregulated in UtLM cells by E(2) including cyclin D kinases, MAPKs, and MAPK kinases; RTK signaling mediator, GRB2; transcriptional factors ELK1 and E2F1; CCNB2 involved in cell cycle progression, proliferation, and survival; and COL1A1 associated with collagen synthesis. Silencing (si)IGF-IR attenuated the above effects and resulted in upregulation of different genes, such as transcriptional factor ETS2; the tyrosine kinase receptor, EGFR; and DLK1 involved in fibrosis. E(2) rapidly activated IGF-IR/MAPKp44/42 signaling nongenomically and induced phosphorylation of ER? at ser118 in cells with a functional IGF-IR versus those without. E(2) also upregulated IGF-I gene and protein expression through a prolonged genomic event. These results suggest a pivotal role of IGF-IR and possibly other RTKs in mediating genomic and nongenomic hormone receptor interactions and signaling in fibroids and provide novel genes and targets for future intervention and prevention strategies.

SUBMITTER: Yu L

PROVIDER: S-EPMC3474284 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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Estrogen Regulates MAPK-Related Genes through Genomic and Nongenomic Interactions between IGF-I Receptor Tyrosine Kinase and Estrogen Receptor-Alpha Signaling Pathways in Human Uterine Leiomyoma Cells.

Yu Linda L Moore Alicia B AB Castro Lysandra L Gao Xiaohua X Huynh Hoang-Long C HL Klippel Michelle M Flagler Norris D ND Lu Yi Y Kissling Grace E GE Dixon Darlene D

Journal of signal transduction 20121009

Estrogen and growth factors play a major role in uterine leiomyoma (UtLM) growth possibly through interactions of receptor tyrosine kinases (RTKs) and estrogen receptor-alpha (ERα) signaling. We determined the genomic and nongenomic effects of 17β-estradiol (E(2)) on IGF-IR/MAPKp44/42 signaling and gene expression in human UtLM cells with intact or silenced IGF-IR. Analysis by RT(2) Profiler PCR-array showed genes involved in IGF-IR/MAPK signaling were upregulated in UtLM cells by E(2) including ...[more]

PMID: 23094148

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Project description:ERα36 is a naturally occurring, membrane-associated, isoform of estrogen receptor α. The expression of ERα36 is due to alternative splicing and different promoter usage. ERα36 is a dominant-negative effector of ERα66-mediated transactivational activities and has the potential to trigger membrane-initiated mitogenic, nongenomic, estrogen signaling; however, the subcellular localization of ERα36 remains controversial. To determine the cellular localization of ERα36 in estrogen-responsive human uterine smooth muscle (ht-UtSMC) and leiomyoma (fibroid; ht-UtLM) cells, we conducted systematic confocal microscopy and subcellular fractionation analysis using ERα36 antibodies. With Image J colocalizaton analysis plugin, confocal images were analyzed to obtain a Pearson's Correlation Coefficient (PCC) to quantify signal colocalization of ERα36 with mitochondrial, endoplasmic reticulum, and cytoskeletal components in both cell lines. When cells were double-stained with an ERα36 antibody and a mitochondrial-specific dye, MitoTracker, the PCC for the two channel signals were both greater than 0.75, indicating strong correlation between ERα36 and mitochondrial signals in the two cell lines. A blocking peptide competition assay confirmed that the mitochondria-associated ERα36 signal detected by confocal analysis was specific for ERα36. In contrast, confocal images double-stained with an ERα36 antibody and endoplasmic reticulum or cytoskeletal markers, had PCCs that were all less than 0.4, indicating no or very weak signal correlation. Fractionation studies showed that ERα36 existed predominantly in membrane fractions, with minimal or undetected amounts in the cytosol, nuclear, chromatin, and cytoskeletal fractions. With isolated mitochondrial preparations, we confirmed that a known mitochondrial protein, prohibitin, was present in mitochondria, and by co-immunoprecipitation analysis that ERα36 was associated with prohibitin in ht-UtLM cells. The distinctive colocalization pattern of ERα36 with mitochondria in ht-UtSMC and ht-UtLM cells, and the association of ERα36 with a mitochondrial-specific protein suggest that ERα36 is localized primarily in mitochondria and may play a pivotal role in non-genomic signaling and mitochondrial functions.

Project description:Uterine leiomyomas or fibroids are the most common tumors of the female reproductive tract. Estrogen (E2), a steroid-derived hormone, and its receptors (ERs), particularly ER-α, are important drivers for the development and growth of leiomyomas. We previously demonstrated that simvastatin, a drug used for hyperlipidemia, also possesses anti-leiomyoma properties. The aim of this work is to investigate the impact of simvastatin on ER-α signaling in leiomyoma cells, including its expression, downstream signaling, transcriptional activity, post-translational modification, trafficking and degradation. Primary and immortalized human uterine leiomyoma (HuLM) cells were used for in vitro experiments. Immunodeficient mice xenografted with human leiomyoma tissue explants were used for in vivo studies. Leiomyoma samples were obtained from patients enrolled in an ongoing double-blinded, phase II, randomized controlled trial. Here, we found that simvastatin significantly reduced E2-induced proliferation and PCNA expression. In addition, simvastatin reduced total ER-α expression in leiomyoma cells and altered its subcellular localization by inhibiting its trafficking to the plasma membrane and nucleus. Simvastatin also inhibited E2 downstream signaling, including ERK and AKT pathways, E2/ER transcriptional activity and E2-responsive genes. To explain simvastatin effects on ER-α level and trafficking, we examined its effects on ER-α post-translational processing. We noticed that simvastatin reduced ER-α palmitoylation; a required modification for its stability, trafficking to plasma membrane, and signaling. We also observed an increase in ubiquitin-mediated ER-α degradation. Importantly, we found that the effects of simvastatin on ER-α expression were recapitulated in the xenograft leiomyoma mouse model and human tissues. Thus, our data suggest that simvastatin modulates several E2/ER signaling targets with potential implications in leiomyoma therapy and beyond.

Dataset Information

Estrogen Regulates MAPK-Related Genes through Genomic and Nongenomic Interactions between IGF-I Receptor Tyrosine Kinase and Estrogen Receptor-Alpha Signaling Pathways in Human Uterine Leiomyoma Cells.

Publications

Estrogen Regulates MAPK-Related Genes through Genomic and Nongenomic Interactions between IGF-I Receptor Tyrosine Kinase and Estrogen Receptor-Alpha Signaling Pathways in Human Uterine Leiomyoma Cells.

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