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

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 the 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 for 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.

Previously, Mig-6 d/d mice are 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 hours. The 772 genes were identified as differentially expressed when comparing the nonhormone-stimulated Mig-6 f/f to Mig-6 d/d uterus ( Supplementary Table 2 ). This difference could be accounted for by the impact of Mig-6 in the uterus. To determine which pathways are regulated by Mig-6 ablation, we performed pathway analysis using Ingenuity Systems Software. The altered pathways included G1/S checkpoint regulation, G2/M DNA damage checkpoint regulation, cell cycle regulation, ILK signaling, p53 signaling, HIF1α signaling, and Wnt/β-catenin signaling ( Supplementary Table 3 ). Among these, we observed an increase in genes necessary for cell cycle progression in the Mig-6 d/d uterus compared to Mig-6 f/f . To validate our microarray analysis, we determined the transcript levels on uteri of Mig-6 f/f and Mig-6 d/d mice treated with vehicle for 6 hours. The mRNA expression level of Ccnd1, Ccnd2, E2f3, and Rbl1, cell cycle G1/S checkpoint regulation genes, Ccnb2, Cdk1, Chek1, Cks1b, Cks2, and Top2a, cell cycle G2/M DNA damage checkpoint regulation genes, and Ccna2 and Ppp2r2b, cell-cycle-related genes were significantly increased and Rprm was significantly decreased in the Mig-6 d/d uteri as compared with Mig-6 f/f ( Fig. 1 ). Immunohistochemical staining of KI67 as a proliferation marker showed that proliferation was significantly increased in the epithelium of Mig-6 d/d mice compared with Mig-6 f/f mice ( Fig. 2A and B ). These results suggest that Mig-6 has a negative regulator for cell cycle progression and epithelial cell proliferation. Previously, we have shown that Mig-6 -ablated mice uteri increased estrogen signaling in the presence of P4, which typically antagonizes ESR activity [ 19 , 20 ]. The overall goal of this investigation was to identify genes that are regulated by the P4- Mig-6 axis. A total of 324 genes were common in both vehicle-treated vs. P4-treated control Mig-6 f/f mice and P4-treated Mig-6 d/d vs. Mig-6 f/f mice ( Fig. 3A ). Of the 324 genes, 107 genes were more highly expressed in the P4 treated control uteri and 217 genes decreased. Of the 107 genes, 39 genes were markedly decreased in the P4-treated Mig-6 d/d mice compare to P4-treated Mig-6 f/f mice ( Fig. 3B ). A complete list is presented in Supplementary Table 4 . To validate our microarray analysis, we performed qPCR analysis on the uteri of Mig-6 f/f and Mig-6 d/d mice treated with vehicle or P4 for 6 hours. The transcript levels of FK506 binding protein 5 ( Fkbp5 ), interleukin 13 receptor, alpha 2 ( Il13ra2 ), Cbp/p300-interacting transactivator, with Glu/Asp-rich carboxy-terminal domain, 2 ( Cited2 ), and genes regulated by estrogen in breast cancer protein ( Greb1 ) were significantly increased in the uteri of Mig-6 f/f mice treated with P4 compared to vehicle. However, these inductions were significantly decreased in the Mig-6 d/d mice treated with P4 compared to Mig-6 f/f mice ( Fig. 3C ). Cited2 is a member of the Cited transcription coactivator family that binds the p300/corticosterone-binding protein coactivator and a P4- target gene. To determine the role of Cited2 in the uterus, we generated the mice with Cited2 conditionally ablated in progesterone receptor-positive cells ( PGR cre/+ Cited2 f/f ; Cited2 d/d ) [ 24 , 25 ]. P4 signaling is crucial for decidualization of the endometrial stromal cells for successful pregnancy [ 29 ]. To determine whether decidualization of the uterus is affected by Cited2 ablation, we examined the ability of Cited2 d/d mice to undergo a well-characterized induced decidualization after artificial hormonal induction. Ovariectomized female Cited2 f/f and Cited2 d/d mice were provided with E2+P4, and the uterus mechanically stimulated to mimic the presence of an implanting embryo and induce decidualization. First, we checked the transcript levels of Cited2 in uteri from Cited2 f/f and Cited2 d/d mice after artificially induced decidualization by real-time PCR Analysis. Cited2 mRNA expression was significantly lower in Cited2 d/d mice compared with Cited2 f/f mice in the uterine horn with/without stimuli ( Fig. 4A ). Cited2 f/f mice showed a decidual uterine horn that responded well to stimuli for artificial induction. However, Cited2 d/d mice displayed remarkable reduction in the decidual response ( Fig. 4B ). Weight ratio of stimulated to control horn was significantly decreased in Cited2 d/d mice compared to Cited2 f/f control mice ( Fig. 4C ). Histological analysis confirmed that well-developed decidual cells were detected in the decidual uterine horn of Cited2 f/f control mice, but differentiated decidual stromal cells were decreased in the decidual uterine horn of Cited2 d/d mice ( Fig. 4D ). In addition, the expression of the known decidualization marker, bone morphogenetic protein 2 ( Bmp2 ), was reduced in the decidual uterine horn of the Cited2 d/d mice compared with the decidual uterine horn of Cited2 f/f control mice ( Fig. 4E ). Collectively, the uteri of Cited2 d/d mice were impaired to undergo a hormonally induced decidual reaction. These results suggest that Cited2 plays an important role for decidualization as a P4 and Mig-6 target gene.

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 Mig-6 ablation. The choice of conducting the microarray analysis on uteri isolated for 6 hours after a single dose of P4 was for the purpose of identifying direct targets of P4 activity. Previously, we showed that Mig-6 is a P4 signaling mediator that suppresses E2 signaling in the uterus [ 19 , 20 , 31 ]. However, the majority of genes are altered in ovariectomized Mig-6 d/d mice treated with vehicle (772 genes). Our microarray analysis showed an increase in genes necessary for cell cycle progression, such as cell cycle G1/S checkpoint regulation genes, cell cycle G2/M DNA damage checkpoint regulation genes, and cell-cycle-related genes in the Mig-6 d/d uterus compared to Mig-6 f/f . Furthermore, proliferation of luminal epithelium was significantly increased in the uterus of Mig-6 d/d mice treated with vehicle. These results showed that Mig-6 has an intrinsic hormone-independent tumor suppressor role in cell cycle progression and epithelial cell proliferation. This microarray analysis identified 324 genes in which the ablation of Mig-6 impacted the P4 regulation of genes in ovariectomized mice given a pharmacological dose of P4. We compared and analyzed direct P4-regulated genes and P4 regulated genes in Mig-6 response to identify the P4-responsive transcriptome that require Mig-6 . The genes whose regulation by P4 is altered by Mig-6 ablation reflect metabolic proteins, cell cycle, structural proteins, transcription factors, and transport protein. Investigation of the expression pattern of these genes during natural pregnancy combined with functional analysis of the role of these genes in the establishment and maintenance of pregnancy will determine the significance of the Mig-6 regulation of these genes during pregnancy. The 39 of 324 genes showed Mig-6 dependent P4-regulation in the uterus. As validation of our microarray analysis, the transcriptional regulation of several P4 responsive genes such as Fkbp5 Il13ra2 Cited2 , and Greb1 [ 31 , 32 ] are significantly attenuated in the uterus of the Mig-6 d/d mice compare to Mig-6 f/f . Therfore, the identification of the precise mechanism of P4-PGR regulation in the endometrium is crucial for understanding the causes of impairments in proper uterine functioning. Cited2 is a member of the Cited (CBP/p300-Interacting Transactivators with glutamic acid (E)/aspartic acid (D)-rich C-terminal domain) transcription coactivator family. CITED2 can bind to DNA binding proteins, such as LHX2, TFAP2, PPARα, PPARγ, and Smads, recruit the histone acetyltransferases CBP/p300, and act as a transcriptional regulator [ 33 , 34 , 35 , 36 ]. Cited2 is induced by the P4-PGR signaling and expressed in the uterus during early pregnancy [ 31 ]. Cited2 is identified as a Mig-6 target gene in our microarray analysis. However, the function of Cited2 in reproductive biology is elusive. To explore the function of Cited2 in the uterus, we generated uterine specific Cited2 ablation mice since targeted deletion of Cited2 results in embryonic lethality [ 35 ]. We show that Cited2 d/d mice show a decrease in the uterine response to P4 during the decidual reaction. Our results suggest that Cited2 plays an important role for P4-dependent decidualization. In summary, the Mig-6 -dependently regulated target genes have been identified by microarray analysis. Epithelial cell proliferation of the ovarian steroid hormone independently increased in Mig-6 d/d mice. The uteri of Mig-6 d/d mice showed an increase of gene expression including cell cycle G1/S checkpoint regulation genes, cell cycle G2/M DNA damage checkpoint regulation genes, and cell-cycle-related genes. The alteration of P4-responsive genes in Mig-6 d/d mice suggest a dual Mig-6 function as a positive regulator as well as negative regulator for P4 functioning. The results of our investigation provide significant insights into our understanding of the importance of Mig-6 in pregnancy and female reproduction.

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 in 100 µl) was administered into ovariectomized mice via s.c. injection. At 6 hours following the P4 or vehicle injection, mice were euthanized for tissue collection. Cited2 f/f mice [ 25 ] were bred with PGR-Cre mice [ 24 ] to generate PGR cre/+ Cited2 f/f ( Cited2 d/d ) mice. Uterine tissues were immediately frozen at the time of dissection and stored at −80°C for RNA extraction or fixed with 4% (v/v) paraformaldehyde for immunohistochemistry. Total RNA was extracted from the uterine tissues using the RNeasy total RNA isolation kit (Qiagen, Valencia, CA). RNA was pooled from the uteri of 3 mice per genotype and treatment. All RNA samples were analyzed with a Bioanalyzer 2100 (Agilent Technologies, Wilmington, DE) before microarray hybridization. Microarray data analysis was performed as previously described [ 26 ]. To adjust arrays to a common baseline using invariant set normalization, DNA-Chip Analyzer dChip was used and the perfect match ( PM ) model described by Li and Wong [ 27 ] was used to estimate expression. We selected differentially expressed genes within each treatment in the Mig-6 f/f and Mig-6 d/d mice using a two-sample comparison according to the following criteria: lower boundary of 90% confidence interval of fold change greater than 1.2 and an absolute value of difference between group means greater than 80. Differentially expressed genes were classified according to canonical pathway analyzed by Ingenuity System Software (Ingenuity Systems Inc., Redwood City, CA). Total RNA was extracted from the uterine tissues using the RNeasy total RNA isolation kit (Qiagen, Valencia, CA). The expression levels of mRNA were measured by real-time PCR TaqMan analysis using an Applied Biosystems StepOnePlusTM system (Applied Biosystems, Foster City, CA) and real-time PCR SYBR Green detection system (Bio-Rad, Hercules, CA) according to the manufacturer's instructions. mRNA quantities were normalized against the housekeeping gene, 18S RNA. Primer sequences used in these studies are shown in Supplementary Table 1 . Immunohistochemistry analysis was performed as previously described [ 20 ]. Uterine sections from paraffin-embedded tissue were preincubated with 10% normal serum in phosphate-buffered saline (PBS) and incubated with anti-KI67 (ab15580; Abcam, Cambridge, MA) antibody in 10% normal serum in PBS. On the following day, sections were washed in PBS and incubated with a secondary antibody (Vector Laboratories, Burlingame, CA) for 1 h at room temperature. Immunoreactivity was detected using the Vectastain Elite DAB kit (Vector Laboratories). The hormonally induced decidual response has been previously described [ 28 ]. The ovariectomized Cited2 f/f and Cited2 d/d female mice were subjected to the following hormonal regimen: 100 ng of E2 per day for three days; two days rest; then three daily injections of 1 mg of P4 plus 6.7 ng of E2. At 6 hours following the third P4 and E2 injection, the left uterine horn was mechanically stimulated by scratching the full length of the anti-mesometrial side with a burred needle. The other horn was left unstimulated as a control. Daily injections of P4 (1 mg/mouse) plus E2 (6.7 ng/mouse) were continued for 5 days to maximize the decidual response. The mice were sacrificed on day 5. The uteri were then excised, weighed and fixed in 4% paraformaldehyde for histological analysis. For data with two groups, Student’s t test was used. For data containing more than two groups, one way ANOVA was used, followed by Tukey’s post hoc multiple range. All data are p resented as means ± SEM. p < 0.05 was considered statistically significant. All statistical analyses were performed using the Instat package from GraphPad (San Diego, CA, USA).