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. 2017 Aug 1;158(8):2427-2435.
doi: 10.1210/en.2017-00349.

Role of ERα in Mediating Female Uterine Transcriptional Responses to IGF1

Sylvia C Hewitt  1 , Wipawee Winuthayanon  1   2 , Sydney L Lierz  1 , Katherine J Hamilton  1 , Lauren J Donoghue  1 , J Tyler Ramsey  1 , Sara A Grimm  3 , Yukitomo Arao  1 , Kenneth S Korach  1
Affiliations

Role of ERα in Mediating Female Uterine Transcriptional Responses to IGF1

Sylvia C Hewitt et al. Endocrinology. .

Abstract

Estrogen (E2) signaling through its nuclear receptor, E2 receptor α (ERα) increases insulinlike growth factor 1 (IGF1) in the rodent uterus, which then initiates further signals via the IGF1 receptor. Directly administering IGF1 results in similar biological and transcriptional uterine responses. Our studies using global ERα-null mice demonstrated a loss of uterine biological responses of the uterus to E2 or IGF1 treatment, while maintaining transcriptional responses to IGF1. To address this discrepancy in the need for uterine ERα in mediating the IGF1 transcriptional vs growth responses, we assessed the IGF1 transcriptional responses in PgrCre+Esr1f/f (called ERαUtcKO) mice, which selectively lack ERα in progesterone receptor (PGR) expressing cells, including all uterine cells, while maintaining ERα expression in other tissues and cells that do not express Pgr. Additionally, we profiled IGF1-induced ERα binding sites in uterine chromatin using chromatin immunoprecipitation sequencing. Herein, we explore the transcriptional and molecular signaling that underlies our findings to refine our understanding of uterine IGF1 signaling and identify ERα-mediated and ERα-independent uterine transcriptional responses. Defining these mechanisms in vivo in whole tissue and animal contexts provides details of nuclear receptor mediated mechanisms that impact biological systems and have potential applicability to reproductive processes of humans, livestock and wildlife.

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Figures

Figure 1.
Figure 1.
ERα antagonist does not inhibit IGF1-induced uterine growth or gene regulation. (a) Ki67 proliferative marker immunohistochemistry of uterine samples from WT mice treated for 24 hours with saline V, E2, or IGF1 either alone (–ICI) or in combination with the ERα antagonist ICI 182,780 (+ICI). Scale bars = 60 µM. (b) RT-PCR shows that transcripts that are induced (Cdkn1a, Nr4a1) or repressed (Txnip, Sox4,) by E2 (2 hours) are also regulated by IGF1 (2 hours). ICI inhibits the E2-mediated upregulation, and partially inhibits the E2-mediated downregulation, but does not affect the IGF1-mediated regulation. Black bars: no ICI; gray bars: with ICI. Two-way analysis of variance with posttest *P < 0.05, ***P < 0.001, ****P < 0.0001 vs V; +P < 0.05, ++P < 0.01 no ICI vs with ICI; #### P < 0.0001 E2 vs IGF1. N = 4 or 5 for all samples.
Figure 2.
Figure 2.
Microarray analysis of E2- and IGF1-regulated transcripts. (a) Principal component analysis of all sample means from WT (diamond), ERαUtcKO (triangle), and Ex3αERKO (square) uterine RNA from V, E2, or IGF1 treatments for 2 hours or 24 hours. Red = E2 (24 hours), blue = E2 (2 hours), green = IGF1 (24 hours), purple = IGF1 (2 hours), orange = V. Numbers of each sample are detailed in Supplemental Table 1. (b) Hierarchical cluster of DE (intensity >100, twofold, false discovery rate <0.05) probes. Green box, probes DE by IGF1; yellow box, probes DE only by E2.
Figure 3.
Figure 3.
ERα-independent transcriptional responses. (a) Left: Venn diagram showing selection of 654 ERα-independent probes from overlap of transcripts DE by IGF1 in WT and ERαUtcKO, but not Ex3αERKO. Right: Hierarchical cluster of the 654 transcripts. (b) RT-PCR showing IGF1 (2 hours) regulation of Fos, Ppargc1a, and Adamts5 is ERα independent. Black = V, gray = E2, white = IGF1. Two-way analysis of variance with posttest. *P < 0.05, **P <0.01, ***P <0.001, ****P <0.0001 vs V; +P < 0.05, ++P < 0.01, +++P < 0.001, ++++P <0.0001 vs WT; #P < 0.05, ##P < 0.01, ###P < 0.001, ####P < 0.0001 E2 vs IGF1. N = 3 to 7 for all samples. (c) ICI does not inhibit IGF1 (2 hours) regulation of Fos, Ppargc1a, and Adamts5 consistent with an ERα-independent mechanism. Black = no ICI, gray = with ICI. Two-way analysis of variance with posttest *P < 0.05, **P < 0.01, ***P < 0.001 vs V; +P < 0.05, ++++P < 0.0001 no ICI vs with ICI; ##P < 0.01 E2 vs IGF1. N = 4 to 5 for all samples.
Figure 4.
Figure 4.
Selecting ERα-dependent transcriptional responses. (a) Left: Venn diagram showing selection of 230 ERα-dependent probes from overlap of transcripts DE by E2 and IGF1 in WT, but not by IGF1 in ERαUtcKO. Right: Hierarchical cluster of the 230 transcripts. (b) Venn diagram and table comparing E2- and IGF1-induced ERα binding sites and their enriched DNA motifs. (c) Venn diagram comparing the 1826 TSS closest to overlapping E2 and IGF1 ERα ChIP-peaks and the 132 coding RNA transcripts from the 230 transcripts selected in (a). The symbols of the 25 overlapping genes are listed. RT-PCR tested shaded genes.
Figure 5.
Figure 5.
Verification of ERα-dependent transcriptional responses. (a) RT-PCR showing E2 (2 hours) and IGF1 (2 hours) induction of Dhcr24, Wnt4, and Ngfr and repression of Map2k6 is ERα dependent. Two-way analysis of variance with posttest *P < 0.05, **P < 0.01, ***P < 0.001 vs V; ++P < 0.01, +++P < 0.001, ++++P <0.0001 vs WT; ###P < 0.001, ####P < 0.0001 E2 vs IGF1. N = 3 to 7 for all samples. Black = V, gray = E2, white = IGF1. (b) ICI inhibits E2 (2 hours) and IGF1 (2 hours) induction of Dhcr24, Wnt4 and Ngfr, consistent with a two-way analysis of variance with posttest *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 vs V; +P < 0.05, +++P < 0.001 vs –ICI; #P < 0.05 E2 vs IGF1. N = 4 to 5 for all samples. Black = no ICI, gray = with ICI. (c) ChIP-PCR confirms binding of ERα to sites near the Dhcr24, Ngfr, and Wnt4 transcripts. All three tested sites have basal ERα binding (V) that is increased by E2 or IGF1 activation. *P < 0.05, ****P < 0.0001 vs V; #### P < 0.0001 E2 vs IGF1. Black = V, gray = E2, white = IGF1. A pool of three uterine samples was tested per treatment; assay done in triplicate.
Figure 6.
Figure 6.
Model: Role of ERα in adult uterine growth and IGF1 crosstalk. Left: ERα-independent response to IGF1. E2, through action of ERα in uterine stromal cells, induces IGF1, which then binds to IGF1 receptors on epithelial cells. The IGF1 receptor signaling leads to regulation of gene products. Direct treatment with IGF1 induces similar transcriptional responses, and does not require uterine ERα. Right: ERα-dependent/transcriptional cross talk in uterine cells. Both E2 and IGF1 can induce genes through the ERα. IGF1/IGF1R signals through ERα. E2 and IGF1 induce similar responses but require the ERα gene products are mostly upregulated.
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References

    1. Cooke PS, Buchanan DL, Young P, Setiawan T, Brody J, Korach KS, Taylor J, Lubahn DB, Cunha GR. Stromal estrogen receptors mediate mitogenic effects of estradiol on uterine epithelium. Proc Natl Acad Sci USA. 1997;94(12):6535–6540. - PMC - PubMed
    1. Murphy LJ, Ghahary A. Uterine insulin-like growth factor-1: regulation of expression and its role in estrogen-induced uterine proliferation. Endocr Rev. 1990;11(3):443–453. - PubMed
    1. Winuthayanon W, Hewitt SC, Orvis GD, Behringer RR, Korach KS. Uterine epithelial estrogen receptor α is dispensable for proliferation but essential for complete biological and biochemical responses. Proc Natl Acad Sci USA. 2010;107(45):19272–19277. - PMC - PubMed
    1. Zhu L, Pollard JW. Estradiol-17beta regulates mouse uterine epithelial cell proliferation through insulin-like growth factor 1 signaling. Proc Natl Acad Sci USA. 2007;104(40):15847–15851. - PMC - PubMed
    1. Richards RG, DiAugustine RP, Petrusz P, Clark GC, Sebastian J. Estradiol stimulates tyrosine phosphorylation of the insulin-like growth factor-1 receptor and insulin receptor substrate-1 in the uterus. Proc Natl Acad Sci USA. 1996;93(21):12002–12007. - PMC - PubMed

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