Growth regulation by estrogen in breast cancer 1 (GREB1) is a novel progesterone-responsive gene required for human endometrial stromal decidualization

Abstract

Study question: Is Growth Regulation by Estrogen in Breast Cancer 1 (GREB1) required for progesterone-driven endometrial stromal cell decidualization?

Summary answer: GREB1 is a novel progesterone-responsive gene required for progesterone-driven human endometrial stromal cell (HESC) decidualization.

What is known already: Successful establishment of pregnancy requires HESCs to transform from fibroblastic to epithelioid cells in a process called decidualization. This process depends on the hormone progesterone, but the molecular mechanisms by which it occurs have not been determined.

Study design, size, duration: Primary and transformed HESCs in which GREB1 expression was knocked down were decidualized in culture for up to 6 days. Wild-type and progesterone receptor (PR) knockout mice were treated with progesterone, and their uteri were assessed for levels of GREB1 expression.

Participants/materials, setting, methods: Analysis of previous data included data mining of expression profile data sets and in silico transcription factor-binding analysis. Endometrial biopsies obtained from healthy women of reproductive age during the proliferative phase (Days 8-12) of their menstrual cycle were used for isolating HESCs. Experiments were carried out with early passage (no more than four passages) HESCs isolated from at least three subjects. Transcript levels of decidualization markers prolactin (PRL) and insulin-like growth factor-binding protein-1 (IGFBP-1) were detected by quantitative RT-PCR as readouts for HESC decidualization. Cells were also imaged by phase-contrast microscopy. To assess the requirement for GREB1, PR and SRC-2, cells were transfected with specifically targeted small interfering RNAs. Results are shown as mean and SE from three replicates of one representative patient-derived primary endometrial cell line. Experiments were also conducted with transformed HESCs.

Main results and the role of chance: Progesterone treatment of mice and transformed HESCs led to an ~5-fold (5.6 ± 0.81, P < 0.05, and 5.2 ± 0.26, P < 0.01, respectively) increase in GREB1 transcript levels. This increase was significantly reduced in the uteri of PR knock-out mice (P < 0.01), in HESCs treated with the PR antagonist RU486 (P < 0.01), or in HESCs in which PR expression was knocked down (P < 0.05). When GREB1 expression was knocked down, progesterone-driven decidualization markers in both immortalized and primary HESCs was significantly reduced (P < 0.05 and P < 0.01). Finally, GREB1 knock down signficantly reduced expression of the PR target genes WNT4 and FOXOA1 (P < 0.05 and P < 0.01, respectively).

Large scale data: This study used the Nuclear Receptor Signaling Atlas.

Limitations, reasons for caution: Although in vitro cell culture studies indicate that GREB1 is required for endoemtrial decidualization, the in vivo role of GREB1 in endometrial function and dysfunction should be assessed by using knock-out mouse models.

Wider implications of the findings: Identification and functional analysis of GREB1 as a key molecular mediator of decidualization may lead to improved diagnosis and clinical management of women with peri-implantation loss due to inadequate endometrial decidualization.

Study funding and competing interest(s): This research was funded in part by: a National Institutes of Health (NIH)/ National Institute of Child Health and Human Development (NICHD) grant (R00 HD080742) and Washington University School of Medicine start-up funds to R.K., an NIH/NICHD grant (RO1 HD-07857) to B.W.O.M., and a NIH/NICHD grant (R01 HD-042311) to J.P.L. The authors declare no conflicts of interests.

Keywords: decidualization; differentiation; endometrium; gene transcription; progesterone.

Figure 1

Greb1 is rapidly induced by progesterone in the mouse and human endometrium. (A) Graph depicting the Greb1 probes found to be induced by progesterone in expression profiling data sets in the Nuclear Receptor Signaling Atlas Transcriptomine database. (B) Greb1 transcript levels in uteri from ovariectomized mice treated with vehicle or progesterone (P4) (1 mg) for 6, 12 or 24 h. Results represent the mean ± SE; n = 3 mice/group. (C) Relative levels of GREB1 and FOXO1A (positive control) transcripts from transformed HESCs treated with vehicle (control) or MPA (1 μM) for indicated time points. Results represent the mean ± SE from three biological replicates; **P < 0.01.

Figure 2

Greb1 is induced by progesterone and PR in the mouse uterus and HESCs. (A) Relative levels of Greb1 and Areg (positive control) transcripts in uteri from ovariectomized wild-type (WT) and PR knockout (PRKO) mice treated for 6 h with P4 (1 mg). Results represent the mean ± SE; n = 3 mice/group. (B) Distribution of binding sites for PR (orange arrows) on Greb1 locus from murine uterine tissues treated for 6 h with P4 (1 mg) or vehicle (oil). (C) Relative transcript levels of GREB1 and FOXO1A from transformed HESCs (three biological replicates) treated with vehicle (control), MPA or MPA plus RU486 (1 μM) for 4 h. Note: The RU486 was added 1 h before MPA. (D) Relative transcript levels of GREB1 and PR from transformed HESCs (three biological replicates) transfected with control siRNA or PGR siRNA treated with vehicle or MPA (1 μM) for 4 h. Results represent the mean ± SE; *P < 0.05 and **P < 0.01. HESC, human endometrial stromal cell; PR, progesterone receptor.

Figure 3

PR and its coactivator SRC-2 regulate GREB1 expression in decidualizing HESCs. (A) Levels of GREB1 and PR transcripts in primary HESCs (three biological replicates) transfected with control or PR siRNAs and induced to decidualize for the indicated lengths of time. (B) Levels of GREB1 and SRC-2 transcripts in primary HESCs transfected with control or SRC-2 siRNAs and induced to decidualize for the indicated lengths of time. Representative data from three replicates from one patient sample are shown as mean ± SE; *P < 0.05 and **P < 0.01.

Figure 4

GREB1 is required for decidualization of immortalized human endometrial stromal cells. (A) Levels of GREB1 and PRL transcripts from immortalized human endometrial stromal cells induced to decidualize for the indicated lengths of time. (B) Levels of GREB1 and PRL transcripts in immortalized human endometrial stromal cells transfected with control or GREB1 siRNAs treated and induced to decidualize for the indicated lengths of time. Results stated as the mean ± SE from three biological replicates from a representative experiment (experiment repeated three times); *P < 0.05, **P < 0.01, and ***P < 0.001.

Figure 5

GREB1 is essential for primary human endometrial stromal cell decidualization. HESCs were transfected with negative control siRNA or GREB1 siRNA as indicated. (A) Left, morphology of HESCs transfected with control or GREB1 siRNA after zero or six days of culture in decidualization conditions. Right, levels of GREB1 transcripts confirm the effective knockdown of GREB1 in HESCs. (B) Levels of IGFBP-1 and PRL transcripts in HESCs transfected with control or GREB1 siRNAs and induced to decidualize for the indicated lengths of time. from three replicates from one patient sample are shown as mean ± SE; **P < 0.01.

Figure 6

GREB1 is required for the induction of key decidual genes in decidualizing HESCs. HESC cells were transfected with negative control siRNA or GREB1 siRNA as indicated. Transcript levels of FOXO1A, WNT4 (P4 targets) and PR (estrogen target) in HESCs transfected with control or GREB1 siRNAs and induced to decidualize for the indicated lengths of time. Representative data from three replicates from one patient sample are shown as mean ± SE; *P < 0.05 and **P < 0.01.