Modulation of LRH-1 expression in transcriptionally regulates GREB1.

<p>(a) Changes in LRH-1 mRNA and (b) protein levels in MCF-7 cells transfected with siRNA for LRH-1 (−LRH-1) or control; with pcDNA only or LRH-1-pcDNA (+LRH-1) constructs 24 h post transfection. (c) The expression levels of GREB1 in response to LRH-1 knockdown (siRNA) and over-expression (+LRH-1). Data were presented as % fold change compared to controls of the normalized expression levels, as mean ± SD, n = 3 separate experiments.</p

LRH-1 induces cell proliferation in 17β-estradiol and ICI 182,780 treated cells.

<p>Cell proliferation was measured in pcDNA alone transfected, estrogen-deprived MCF-7 cells (control) or LRH-1 over-expressing (+LRH-1) MCF-7 cells treated with vehicle, 10 nM 17β-estradiol (E2) or 10 nM 17β-estradiol and 1 nM ICI 182,780, an ERα antagonist for 5 days. Data is presented as mean+SEM, n = 3 separate experiments, triplicate treatments per experiment, ***P&lt;0.001 compared to control transfected cells; a,b P&lt;0.001 compared to vehicle control.</p

LRH-1 regulation of GREB1 expression in ER negative breast cancer cells.

<p>MDA-MB-231 cells were transfected with empty vector (C) or expression vectors for LRH-1 alone (L), ERα alone (E) or both LRH-1 and ERα (L+E). Cells were treated with vehicle or 10 nM 17β-estradiol (E2) for 16 h. Quantitation of (a) LRH-1, (b) ERα and (c) GREB1 mRNA expression. Data is presented as mean+SE, n = 3 separate experiments, ***P&lt;0.001 compared to vehicle control.</p

LRH-1 acts synergistically with ERα to activate ERE containing promoters.

<p>Transcriptional activation of (a) 2×ERE and (b) GREB-ERE2 luciferase reporters by ERα and LRH-1 with vehicle (veh) or 10 nM 17β-estradiol (E2). Estrogen-deprived MCF-7 cells were over expressed with LRH-1 or ERα alone, or in combination with the appropriate reporter construct. Cells were treated with 17β-estradiol for 16 h prior to luciferase assays. Data is presented as mean+SE, n = 3 separate experiments, treatments in triplicate per experiment. *P&lt;0.05, *P&lt;0.01, ***P&lt;0.001 compared to vehicle control unless indicated by reference line.</p

LRH-1 binds to specific ERE sequences of the GREB1 and pS2 promoters.

<p>(a) EMSA showing binding of LRH-1 to the EREs present in the GREB1 promoter. Radiolabeled ERE1-GREB1, ERE2-GREB1 and ERE3-GREB1 probes were incubated with <i>in vitro</i> translated LRH-1 protein. <i>In vitro</i> translation of the empty vector was used as a negative control. Anti-LRH-1 antibody was added in addition to the probe and the LRH-1 protein to indicate specificity of protein binding. (b) EMSA showing binding of LRH-1 to the EREs present in the GREB1 and pS2 promoters. Radiolabeled LRHRE probe (containing the LRH-1 response element derived from the aromatase promoter), whole cell nuclear extracts infected with a LRH-1 viral construct were incubated with various oligonucleotides (as listed in the figure) including unlabeled LRHRE, mutated LRHRE, ERE1-GREB1, ERE2-GREB1, ERE3-GREB1 and ERE-pS2 which were added in 200 fold excess. Anti-LRH-1 antibody and IgG were also added in addition to the probe and the nuclear extract to indicate specificity of protein binding.</p

LRH-1 binds to three ERE sites within the GREB1 promoter.

<p>(a) Location of regulatory EREs on the distal and proximal GREB1 promoter, highlighting (bold) sequence similarity of the LRH-1 nuclear receptor half site within the ERE palindrome. (b) Chromatin immunoprecipitation (ChIP) showing occupancy of LRH-1 on the three EREs where ERα binds in the presence or absence of estradiol. Immunoprecipitation was performed with anti-LRH-1 and ERα antibodies on chromatin isolated from MCF-7 cells treated with vehicle or 10 nM 17β-estradiol for 45 mins. (c) The precipitated chromatin was analyzed by quantitative real-time PCR to demonstrate relative occupancy using the delta delta C_t method. Data is normalised to 10% of input. Data is represented from 3 or more separate treatments and separate ChIP experiments. (d) Sequential ChIP demonstrating co-localisation of ERα and LRH-1 on ERE1 of the <i>GREB1</i> promoter. Figures are representative of 3 or more separate ChIP experiments.</p

Synergistic effects of LRH-1 and 17β-estradiol treatment on GREB1 expression.

<p>Quantitation of (a) LRH-1, (b) GREB1 and (c) ERα mRNA expression in estrogen-deprived MCF-7 cells (control) or LRH-1 over-expressing (+LRH-1) MCF-7 cells treated with vehicle (veh) or 10 nM 17β-estradiol (E2) for 16 h. Data is presented as mean+SE, n = 3 separate experiments, triplicate treatments per experiment, **P&lt;0.01, ***P&lt;0.001 compared to vehicle control.</p