Mig-6 regulates endometrial genes involved in cell cycle and progesterone signaling

doi:10.1016/j.bbrc.2015.04.146

Biochemical and Biophysical Research Communications

Volume 462, Issue 4, 10 July 2015, Pages 409-414

https://doi.org/10.1016/j.bbrc.2015.04.146 Get rights and content

Highlights

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We identify Mig-6- and P4-regulated uterine genes by microarray analysis.
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Mig-6 suppresses cell cycle progression and epithelial cell proliferation in uterus.
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We identify the Mig-6 dependent induced genes by P4.
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Cited2 plays an important role for decidualization as a P4 and Mig-6 target gene.

Abstract

Mitogen inducible gene 6 (Mig-6) is an important mediator of progesterone (P4) signaling to inhibit estrogen (E2) signaling in the uterus. Ablation of Mig-6 in the murine uterus leads to the development of endometrial hyperplasia and E2-induced endometrial cancer. To identify the molecular pathways regulated by Mig-6, we performed microarray analysis on the uterus of ovariectomized Mig-6^f/f and PGR^cre/+Mig-6^f/f (Mig-6^d/d) mice treated with vehicle or P4 for 6 h. The results revealed that 772 transcripts were significantly regulated in the Mig-6^d/d uterus treated with vehicle as compared with Mig-6^f/f mice. The pathway analysis showed that Mig-6 suppressed the expression of gene-related cell cycle regulation in the absence of ovarian steroid hormone. The epithelium of Mig-6^d/d mice showed a significant increase in the number of proliferative cells compared to Mig-6^f/f mice. This microarray analysis also revealed that 324 genes are regulated by P4 as well as Mig-6. Cited2, the developmentally important transcription factor, was identified as being regulated by the P4-Mig-6 axis. To determine the role of Cited2 in the uterus, we used the mice with Cited2 that were conditionally ablated in progesterone receptor-positive cells (PGR^cre/+Cited2^f/f; Cited2^d/d). Ablation of Cited2 in the uterus resulted in a significant reduction in the ability of the uterus to undergo a hormonally induced decidual reaction. Identification and analysis of these responsive genes will help define the role of P4 as well as Mig-6 in regulating uterine biology.

Introduction

The uterus is an important hormone-responsive reproductive organ in mammals. The ovarian steroid hormones progesterone (P4) and estrogen (E2) are essential mediators of reproductive events associated with the establishment and maintenance of pregnancy [1], [2]. E2 stimulates the proliferation of both uterine luminal and glandular epithelium [3]. In contrast, P4 inhibits E2-mediated proliferation of the luminal and glandular epithelium [4], [5]. P4 is a critical regulator of reproductive events associated with embryo implantation, decidualization of the endometrial stromal cells and maintenance of pregnancy [2], [6]. The physiological effects of P4 are mediated through its cognate receptor, the progesterone receptor (PGR) [7]. The fertility defects exhibited by the progesterone receptor knockout (PRKO) mice unequivocally demonstrated the critical importance of P4 and its receptor in the establishment and maintenance of pregnancy [2], [8].

Progestin is a synthetic progesterone. Progestin has been used in the conservative endocrine treatment of early endometrial cancer patients in order to preserve their fertility, as well as in palliative treatment of advanced-stage patients [9], [10]. Interruption of P4 signaling, occurring from the loss of the PGR itself or through the loss of its interacting partners or downstream effectors, leads to a physiological state of P4 resistance [11]. P4 resistance is seen in a wide variety of diseases. P4 resistance is a hallmark of endometriosis [12], [13]. P4 resistance is also seen in the endometrium of women with polycystic ovary syndrome (PCOS) [14], [15]. Expression of the progesterone receptor (PGR) was known to be positively correlated with a good prognosis and response to progestin treatment [16]. However, more than 30% of patients with progestin treatment did not respond to progestin due to de novo or acquired progestin resistance [9], [17], [18]. The mechanism of progestin resistance is still unknown. Understanding the precise mechanism of P4 regulation in the endometrium is of critical importance in developing therapeutic approaches to alleviate this women's health crisis.

Mig-6 has been shown to be critical for uterine functioning because conditional ablation of Mig-6 (PGR^cre/+Mig-6^f/f; Mig-6^d/d) in the mouse uterus results in infertility due to a defect of embryo implantation [19], [20]. Additionally, Mig-6^d/d mice leads to the development of animals with epithelial hyperplasia, adenoma and adenocarcinomas in organs, such as the uterus, lung, gallbladder and bile duct [19], [21], [22], [23]. Endometrial tumorigenesis is accelerated by double ablation of Mig-6 and Pten compared to single ablation of Mig-6 or Pten [20]. However, the precise mechanism of Mig-6 in endometrial cancer remains poorly understood.

Here, we identified Mig-6 regulated uterine genes using Mig-6^d/d mice in combination with high density DNA microarray analysis. This analysis indicates that Mig-6 plays an important role in uterine functioning by modulating the regulation of cell cycle related genes and the ability of P4 to regulate specific genes. The results of our investigation provide significant insights into our understanding of the importance of steroid hormone regulation in female reproduction and endometrial cancer.

Section snippets

Animals and tissue collection

Mig-6 “floxed” (Mig-6^f/f) and PGR^cre/+Mig-6^f/f (Mig-6^d/d) mice [22], [24] were maintained in the designated animal care facility according to the Michigan State University institutional guidelines. All animal procedures were approved by the Institutional Animal Care and Use Committee of Michigan State University. Eighteen Mig-6^f/f and 18 Mig-6^d/d mice were ovariectomized at 6 weeks of age. After 2 weeks of rest, vehicle (sesame oil) or P4 (in sesame oil; Sigma–Aldrich, St. Louis, MO; 1 mg/mouse

Mig-6 suppresses cell cycle progression

Previously, Mig-6^d/d mice were infertile due to an inability of the uterus to undergo embryo implantation [19], [20]. To identify the molecular pathways regulated by Mig-6 in a steroid-hormone independent and dependent manner, we performed high-density DNA microarray analysis on the uteri of ovariectomized Mig-6^f/f and Mig-6^d/d mice treated with vehicle or progesterone (P4) for 6 h. The 772 genes were identified as differentially expressed when comparing the nonhormone-stimulated Mig-6^f/f to

Discussion

The ablation of Mig-6 increased proliferation of endometrial epithelial cells [19], [30]. However, the molecular mechanism of Mig-6 has not been studied in uterine biology. In this study, we have identified Mig-6- and P4-regulated uterine genes using the Mig-6^d/d mouse and high-density DNA microarray analysis. The design of our microarray analysis was to determine the role of Mig-6 in the uterus. This analysis identified P4-dependent as well as -independent genes whose expression was altered by

Acknowledgments

We would like to thank John P. Lydon, Ph.D. and Francesco J. DeMayo, Ph.D. (Baylor College of Medicine, Houston, TX) for contributing the PGR^cre/+ mice and Amanda Sterling for manuscript preparation. This work was supported by a NIH Grant R01HD057873 and an American Cancer Society Research Scholar Grant RSG-12-084-01-TBG (to J.W.J.), and an Australian Research Council grant DP0346729 and a National Health and Medical Research Senior Research Fellowship ID1042002 (to S.LD).

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View full text

Mig-6 regulates endometrial genes involved in cell cycle and progesterone signaling

Highlights

Abstract

Introduction

Section snippets

Animals and tissue collection

Mig-6 suppresses cell cycle progression

Discussion

Acknowledgments

J. Steroid Biochem. Mol. Biol.

Steroids

J. Biol. Chem.

FEBS Lett.

Progestin regulation of cellular proliferation

Endocr. Rev.

Mice lacking progesterone receptor exhibit pleiotropic reproductive abnormalities

Genes. Dev.

The influence of 17 beta-estradiol on patterns of cell division in the uterus

Endocrinology

The inhibition by progesterone of uterine epithelial proliferation in the mouse

J. Endocrinol.

Epithelial progesterone receptor exhibits pleiotropic roles in uterine development and function

FASEB J.

Molecular cues to implantation