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. 2010 May;24(5):969-80.
doi: 10.1210/me.2009-0148. Epub 2010 Mar 8.

Regulation of ERRalpha gene expression by estrogen receptor agonists and antagonists in SKBR3 breast cancer cells: differential molecular mechanisms mediated by g protein-coupled receptor GPR30/GPER-1

Yin Li  1 , Lutz Birnbaumer, Christina T Teng
Affiliations

Regulation of ERRalpha gene expression by estrogen receptor agonists and antagonists in SKBR3 breast cancer cells: differential molecular mechanisms mediated by g protein-coupled receptor GPR30/GPER-1

Yin Li et al. Mol Endocrinol. 2010 May.

Abstract

In selected tissues and cell lines, 17beta-estradiol (E2) regulates the expression of estrogen-related receptor alpha (ERRalpha), a member of the orphan nuclear receptor family. This effect is thought to be mediated by the estrogen receptor alpha (ERalpha). However in the ERalpha- and ERbeta-negative SKBR3 breast cancer cell line, physiological levels of E2 also stimulate ERRalpha expression. Here, we explored the molecular mechanism that mediates estrogen action in ER-negative breast cancer cells. We observed that E2, the ERalpha agonist, as well as the ERalpha antagonists ICI 182,780 and tamoxifen (TAM), a selective ER modulator, stimulate the transcriptional activity of the ERRalpha gene and increase the production of ERRalpha protein in SKBR3 cells. Moreover, the ERRalpha downstream _target genes expression and cellular proliferation are also increased. We show further that the G protein-coupled receptor GPR30/GPER-1 (GPER-1) mediates these effects. The GPER-1 specific ligand G-1 mimics the actions of E2, ICI 182,780, and TAM on ERRalpha expression, and changing the levels of GPER-1 mRNA by overexpression or small interfering RNA knockdown affected the expression of ERRalpha accordingly. Utilizing inhibitors, we delineate a different downstream pathway for ER agonist and ER antagonist-triggered signaling through GPER-1. We also find differential histone acetylation and transcription factor recruitment at distinct nucleosomes of the ERRalpha promoter, depending on whether the cells are activated with E2 or with ER antagonists. These findings provide insight into the molecular mechanisms of GPER-1/ERRalpha-mediated signaling and may be relevant to what happens in breast cancer cells escaping inhibitory control by TAM.

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Figures

Figure 1
Figure 1
E2, G-1, ICI, and TAM induce the expression of ERRα in ER negative SKBR3 breasts cancer cells. A, Dependence of mRNA induction on compound concentration and time of incubation. The experiments were repeated three times, and results are presented as fold induction ± sd. B, mRNA induction correlates with protein accumulation. C, E2, ICI, and TAM induce the expression of ATP5β and Cytc. D, E2, ICI, and TAM induce SKBR3 cell proliferation. The experiments were repeated three times and results are presented as induction ± sd. *, P < 0.05.
Figure 2
Figure 2
GPER-1 mediates the effect of E2, G-1, ICI, and TAM on ERRα expression in SKBR3 cells. A1, GPER-1 protein is lowered by GPER-1 _targeted siRNA (siGPER#1). β-Actin was used as a loading control. A2, Knock down of GPER-1 mRNA by siGPER#1 oligonucleotides blocks the effect of E2, G-1, ICI, and TAM on ERRα mRNA expression. B1, GPER-1 protein is lowered by GPER-1 _targeted siRNA (siGPER#2 and siGPER#3). β-Actin was used as a loading control. B2, Knock down of GPER-1 mRNA by siGPER#2 and #3 oligonucleotides blocks the effect of E2 and TAM on ERRα mRNA expression. The experiments were repeated three times and results are presented as induction ± sd. *, P < 0.05.
Figure 3
Figure 3
Ectopic expression of GPER-1 potentiates the ERRα mRNA inducing effects of E2, G-1, ICI, and TAM. SKBR3 cells were transfected with control plasmid (pcDNA3) or the plamsid pcDNA3 directing the expression of the GPER-1 cDNA under the control of the cytomegalovirus promoter (Western blotting; inset). The treatment with E2, G-1, ICI, or TAM was initiated 24 h after the transfection. Except for E2, the experiments were repeated three times, and results are presented as induction ± sd. The experiment with E2 was repeated two times, and the results are reported as fold induction ± 1/2 the range. *, P < 0.05.
Figure 4
Figure 4
PTX treatment, which inhibits the activation of the Gi/Go class of G proteins, blocks the induction of ERRα mRNA expression by E2, G-1, ICI, and TAM. SKBR3 cells were first incubated with 100 ng/ml PTX (Biological Laboratories, Inc.) and then treated with the indicated compounds for 8 or 18 h, followed by mRNA analysis by real-time PCR. The experiments were repeated three times, and results are presented as induction ± sd. *, P < 0.05.
Figure 5
Figure 5
Effects of signaling pathway inhibitors on the induction of ERRα mRNA expression and MAPK phosphorylation. A, Effect on ERRα mRNA induction by E2, G-1, ICI, and TAM. The Erk1/2 MAPK inhibitor PD (10 μm), the src family tyrosine kinase inhibitor PP2 (10 μm) and the PI3K inhibitor WM (0.5 μm) were added to SKBR3 cells 1 h before initiation of treatments with the indicated compounds. The experiments were repeated three times, and results are presented as induction ± sd. *, P < 0.05. B, Effect on activation of MAPK (ERK1/2) phosphorylation by TAM. Cells were cultured and lysed under the conditions described in Materials and Methods. Protein level of phospho-p44/42 and total p44/42 were detected with specific antibodies.
Figure 6
Figure 6
Effects of E2, G-1, ICI, and TAM on histone acetylation and transcription factor recruitment at the ERRα gene promoter. Diagram, Representation of the nucleosome positions at the ERRα promoter. Regions spanning DNA of nucleosome A and E were analyzed for presence of acetylated histones and the indicated transcription factors. Cells were treated with vehicle (control) or 100 nm of E2 or G-1 for 3 h or 1000 nm ICI or TAM for 6 h, followed by isolation of nuclei and either analysis of histones by native ChIP (upper panels) or of transcription factor association by conventional ChIP (lower panels). The experiments were repeated three times, and the results are presented as change in amount of nucleosomal DNA recovered in the immunoprecipitates relative to control ± sd.
Figure 7
Figure 7
A model of the organization of the signaling pathways that were addressed in the present experiments. The results delineate the existence of two signaling pathways down stream of the GPER-1. One, activated by E2 and G-1, does not depend of activation of a member of the src family of tyrosine kinases; the other, activated by the ERα antagonist ICI and the inhibitory SERM TAM, depends on activation of a PP2-sensitive tyrosine kinase. Activation of the Erk1/2 MAPKs is required for the action of all four GPER-1 ligands.
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