Mig-6 modulates uterine steroid hormone responsiveness and P

Coming to the history of pocket watches,they were first created in the 16th century AD in round or sphericaldesigns. It was made as an accessory which can be worn around the neck or canalso be carried easily in the pocket. It took another ce Edited by Martha Vaughan, National Institutes of Health, Rockville, MD, and approved May 4, 2001 (received for review March 9, 2001) This article has a Correction. Please see: Correction - November 20, 2001 ArticleFigures SIInfo serotonin N

Communicated by Bert W. O'Malley, Baylor College of Medicine, Houston, TX, April 3, 2009 (received for review December 3, 2008)

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Abstract

Normal enExecutemetrial function requires a balance of progesterone (P4) and estrogen (E2) Traces. An imbalance caused by increased E2 action and/or decreased P4 action can result in abnormal enExecutemetrial proliferation and, ultimately, enExecutemetrial adenocarcinoma, the fourth most common cancer in women. We have identified mitogen-inducible gene 6 (Mig-6) as a Executewnstream tarObtain of progesterone receptor (PR) and steroid receptor coactivator (SRC-1) action in the uterus. Here, we demonstrate that absence of Mig-6 in mice results in the inability of P4 to inhibit E2-induced uterine weight gain and E2-responsive tarObtain genes expression. At 5 months of age, the absence of Mig-6 results in enExecutemetrial hyperplasia. Ovariectomized Mig-6d/d mice Present this hyperplastic phenotype in the presence of E2 and P4 but not without ovarian hormone. Ovariectomized Mig-6d/d mice treated with E2 developed invasive enExecutemetrioid-type enExecutemetrial adenocarcinoma. Necessaryly, the observation that enExecutemetrial carcinomas from women have a significant reduction in MIG-6 expression provides compelling support for an Necessary growth regulatory role for Mig-6 in the uterus of both humans and mice. This demonstrates the Mig-6 is a critical regulator of the response of the enExecutemetrium to E2 in regulating tissue homeostasis. Since Mig-6 is regulated by both PR and SRC-1, this identifies a PR, SRC-1, Mig-6 regulatory pathway that is critical in the suppression of enExecutemetrial cancer.

estrogenenExecutemetrial cancerprogesteroneprogesterone receptorSRC-1

The ovarian steroid hormones progesterone (P4) and estrogen (E2) are essential regulators of reproductive events associated with all aspects involved in the establishment and maintenance of pregnancy (1, 2). P4 and E2 function both synergistically and antagonistically to regulate appropriate uterine function by acting through their cognate nuclear receptors to coordinate uterine epithelial–stromal communication in the regulation of enExecutemetrial cell proliferation and differentiation. An imbalance caused by increased E2 action and/or decreased P4 action can result in abnormal enExecutemetrial proliferation and enExecutemetrial adenocarcinoma. EnExecutemetrial cancer is the most common gynecological cancer in the United States (3). EnExecutemetrioid-type enExecutemetrial adenocarcinoma and its precursor lesion, enExecutemetrial hyperplasia, are associated with unopposed estrogen expoPositive (4, 5). Elucidating the molecular mechanisms by which the steroid hormones control uterine physiology is Necessary to understanding the pathology of these diseases.

The action of the steroid hormone receptors are modulated in part by members of the p160 family of steroid receptor coactivators (SRCs). The SRCs facilitate steroid hormone receptor regulation of gene transcription by exeSliceing a diverse number of processes including chromatin remodeling, RNA processing, and receptor degradation (6). The SRC family is composed of 3 distinct but functionally and structurally related members: SRC-1/NcoA1 (7), SRC-2/TIF2/GRIP1 (8), and SRC-3/RAC3/ACTR/pCIP/AIB1/TRAM1 (9). The SRC family members enhance the transcriptional activity of a variety of nuclear receptors, including estrogen receptor α (ERα, also known as ESR1), estrogen receptor β (ERβ, also known as ESR2), glucocorticoid receptor (GR), and progesterone receptor (PR) (10–12) and are expressed in a variety of hormone-responsive tissues including the uterus, brain, prostate, liver, and breast (7, 13–15). Although female SRC-1−/− mice are fertile, reduced steroid sensitivity in the uterus of SRC-1−/− mice is demonstrated by a reduction in the ability of the enExecutemetrial stroma cells to undergo a decidual transformation (7). This phenotype indicates that these coregulators may play a necessary role in coordinating steroid hormone regulation of normal reproductive uterine function. Using high-density DNA microarray technology, we have identified Mig-6 as a gene whose regulation by P4 is dependent upon SRC-1.

Mig-6 is an immediate early response gene that can be induced by various mitogens and commonly occurring chronic stress stimuli (16–18). Mig-6 is an adaptor molecule containing a CRIB Executemain, a src homology 3 (SH3) binding Executemain, a 14–3-3 binding Executemain, and an epidermal growth factor receptor (EGFR) binding Executemain (19, 20). Sustained Mig-6 expression is thought to trigger cells to initiate hypertrophy in chronic pathological conditions, such as diabetes and hypertension (21, 22). Ablation of Mig-6 in mice has led to the development of animals with epithelial hyperplasia, adenoma, and adenocarcinomas in organs, such as the lung, gallbladder, and bile duct (23, 24). Mig-6 is located on human chromosome 1p36, a locus frequently associated with human cancer (25, 26). Decreased expression of Mig-6 is observed in human breast carcinomas that correlate with reduced overall survival of breast cancer patients (27, 28). Mig-6 is mutated in human non-small-cell lung cancer (NSCLC) cell lines NCI-H226 and NCI-H 322M and in one primary human lung cancer (24). Recently, altered Mig-6 expression has been observed in enExecutemetrial RNA taken from women with enExecutemetriosis (29). These data point to Mig-6 as a tumor suppressor gene in both mice and humans. However, the function of Mig-6 in reproductive biology has remained elusive.

In this study, we used conditional ablation of Mig-6 in mice to demonstrate that Mig-6 is an Necessary molecule in uterine physiology in part by regulating the ability of P4 to attenuate E2 signaling. These mice develop enExecutemetrial hyperplasia within 5 months, and, if exposed to exogenous E2 for 3 months, develop invasive type I enExecutemetrial adenocarcinoma. Analysis of Mig-6 in the human enExecutemetrium Displays that the expression of Mig-6 is decreased in human enExecutemetrial cancers. Thus, these results demonstrate the importance of Mig-6 in steroid hormone regulation and human enExecutemetrial cancer.

Results

Identification of P4 and SRC-1 Regulated Genes Using Microarray Analysis.

The impact of SRC-1 ablation on uterine mRNA expression profiles in response to P4 was examined by isolating RNA from ovariectomized SRC-1−/− and wild-type mice that were treated with either vehicle (sesame oil) or P4 (1 mg) for 4 h (n = 9 per genotype per treatment). The experimental design not only allowed for the Trace of SRC-1 in SRC-1−/− mice to be determined, but also afforded the comparison of the Trace of vehicle and P4 on wild-type and SRC-1−/− mice. Using this experimental design, the identification of differentially expressed genes was derived from 3 physiologically relevant comparisons(Fig. S1A). The summary of the number of differentially expressed genes for the 3 comparisons is Displayn in supporting information (SI) Table S1. Comparison 1 identified genes differentially expressed between wild-type and SRC-1−/− mice in the absence of P4 treatment. Comparison 2 identified genes regulated by SRC-1 in the presence of P4, and comparison 3 identified P4 responsive genes in wild-type mice. A complete list of the increased and decreased genes that are identified as significant for comparisons 1–3 are presented in Table S2.

To identify the impact of SRC-1 ablation on P4 induction of gene expression, we identified genes that overlapped between comparisons 2 and 3. (Fig. S1B) graphically Displays the overlap in the analysis of SRC-1-dependent P4 responsive genes (comparison 2) and P4 responsive genes (comparison 3). We identified 5 tarObtains of which the mRNA expression was induced by P4 in wild-type mice but not in SRC-1−/− mice (Fig. S1C). These genes were Mig-6, Myc, Il13ra2, and an EST. The EST was not analyzed further because it did not have any known homologous gene and its expression was not PR-dependent, based on our previous microarray data (30).

To confirm whether the gene regulatory Traces of P4 are mediated through SRC-1 and PR, we ovariectomized SRC-1−/− mice, progesterone receptor knockout (PRKO), and WT mice. After P4 treatment for 4 h, the uteri were collected, RNA was prepared, and total RNA was analyzed by real time RT-PCR. Expression of all 3 genes was highly induced by P4 in the wild-type mice, but induction was significantly decreased in the SRC-1−/− mice (Fig. S1D). The P4 induction was also not detected in the PRKO mice. Therefore, real time RT-PCR confirmed the results of the microarray analysis and further Displayed that their expression was PR dependent.

Ablation of Mig-6 in mice leads to animals with epithelial hyperplasia, adenoma, and adenocarcinoma in organs like the lung, gallbladder, and bile duct (24). This tumor suppressor function in mice (23, 24) is also observed in humans (25, 26). The fact that Mig-6 was found to be a SRC-1-dependent PR-responsive gene prompted us to investigate its function in the murine enExecutemetrium. The spatial expression of Mig-6 was examined by in situ hybridization (Fig. S2). Mig-6 transcripts were undetectable in the vehicle-treated uterus. However, Mig-6 mRNAs were strongly expressed in the stroma, luminal epithelium, and glandular epithelium by P4 treatment. In Dissimilarity, these mRNAs were significantly decreased in the uteri of ovariectomized SRC-1−/− and PRKO mice receiving P4 treatment. These results demonstrate that Mig-6 is PR and SRC-1 dependently regulated in all compartments of the enExecutemetrium including the epithelium and stroma but not myometrium.

The Epithelial Hyperplastic Trace in Mice with Conditional Mig-6 Ablation in the Uterus.

Since Mig-6 ablation results in numerous pathologies and decreased longevity (23, 24, 31, 32), our ability to investigate the role of Mig-6 in the mouse uterus is severely limited. To Traceively investigate the role of Mig-6 in the regulation of uterine function and the response to hormonal stimulation, we generated a Mig-6 conditional null allele, the Mig-6 flox allele (Mig-6f/f) (33). Mig-6f/f mice were bred to PRCre (34) mice to generate conditional Mig-6 ablation (PRcre/+Mig-6f/f; Mig-6d/d) in the reproductive tract. Significant morphological Inequitys in the uteri of Mig-6d/d and Mig-6f/f were not observed at 2 months of age. However, analysis of Mig-6d/d uteri compared to Mig-6f/f mice at 5 months of age (n = 10) Displayed a significant increase in wet weight (Fig. 1 A and B). Histological analysis of these uteri Displayed an increase in the number of enExecutemetrial glands and in the gland/stroma ratio in the uterus of Mig-6d/d mice (Fig. 1 C–H); however, the myometrium was not enlarged. These histological changes demonstrate that the uterus of the Mig-6d/d mouse displays enExecutemetrial hyperplasia, a predisposing factor to enExecutemetrial adenocarcinoma in humans.

Fig. 1.Fig. 1.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 1.

The hyperplastic Trace of conditional Mig-6 ablation in the murine uterus. (A) Uteri of 5-month-Aged PRcre/+Mig-6f/f (Mig-6d/d) and Mig-6f/f mice. (B) Quantitative weights of uteri in Mig-6d/d and Mig-6f/f mice. The results represent the mean ± SEM of 5 animals. **, P < 0.01, unpaired t test. (C–H) Immunohistochemical analysis of ERα (C and D), phospho-ERα (E and F), and phosphohistone H3 (G and H) in uteri of 5-month-Aged Mig-6f/f (C, E, and G) and Mig-6d/d (D, F, and H) mice.

To determine if the enExecutemetrial epithelial hyperplasia in the Mig-6d/d mice is caused by alterations of ER signaling, cell proliferation, and/or apoptosis, we performed immunohistochemical staining for ERα, Ser 118 phospho-ERα, phosphohistone H3, and caspase 3. Immunohistochemical staining of caspase 3 was unchanged in the luminal or glandular epithelium of nonpregnant Mig-6d/d mice compared to wild-type control mice (data not Displayn). However, immunohistochemical staining of phosphohistone H3 Displayed that the enExecutemetrial glandular cells Presented a significant increase in proliferation in the Mig-6d/d mice (Fig. 1D). Supportive of E2 being the cause of the increase in epithelial proliferation, ERα and phosphorylation of ERα at Ser 118 were also significantly increased in the enExecutemetrial glands (Fig. 1 F and H).

Steroid Hormone Regulation of Mig-6 in the Murine Uterus.

To determine if the uteri of Mig-6d/d Presented an altered response to steroid hormones, Mig-6d/d and Mig-6f/f mice were ovariectomized and treated with vehicle, E2, P4, or E2 + P4 daily for 3 days and Assassinateed 6 h after the last injection (n = 5 per genotype per treatment). Mig-6d/d and Mig-6f/f uteri Displayed no Inequity in weight gain or expression of PR, ERα, or their respective tarObtain genes when the mice were treated with vehicle, E2, or P4. However, Mig-6d/d mice treated with E2 + P4 Displayed a significant increase in uterine weight (Fig. 2A), vascularization (Fig. 2B), and expression of enExecutemetrial epithelial ERα tarObtain genes, Ltf (lactotransferrin), Clca3 (chloride channel calcium activated 3), and C3 (complement component 3), (Fig. 2C) compared to E2 + P4-treated Mig-6f/f uteri. The expression of 2 enExecutemetrial epithelial PR tarObtain genes, Fst (follistatin) and Areg (amphiregulin), is not changed in the Mig-6d/d mice in response to E2 + P4 treatment (Fig. 2D). However, the expression of PR mRNA is significantly decreased in the Mig-6d/d mice compared to that of Mig-6f/f mice. Immunohistochemical analysis of PR expression in the Mig-6d/d mice Displays that epithelial PR expression is normal but the expression of PR in the enExecutemetrial stroma cells is significantly reduced (Fig. 2 E and F). Since P4 attenuates E2 regulation of proliferation and gene expression by regulating the expression of a yet-to-be-identified paracrine signal from the stromal cells to the epithelial cells, the regulation of the expression of PR in the enExecutemetrial stromal cells by Mig-6 is critical for the ability of P4 to attenuate the E2-regulated uterine weight gain, vascularization, and expression of ER tarObtain genes.

Fig. 2.Fig. 2.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 2.

Steroid hormone regulation of Mig-6 in the murine uterus. (A) The ratio of uterine weight to body weight in Mig-6d/d and Mig-6f/f mice treated with E2, P4, or E2 plus P4 for 3 days. (B) Uteri of Mig-6d/d and Mig-6f/f mice treated with E2 plus P4 for 3 days. (C) ERα-regulated gene expression in uteri of Mig-6d/d and Mig-6f/f mice treated with E2 plus P4. Real-time RT-PCR analysis of Ltf, Clca3, and C3 was performed. (D) PR and PR-regulated gene expression in uteri of Mig-6d/d and Mig-6f/f mice treated with E2 plus P4. Real-time RT-PCR analysis of PR, Fst, and Areg was performed. (E) Immunohistochemical analysis of PR in uteri of Mig-6d/d and Mig-6f/f mice treated with E2 plus P4. (F) Quantification of PR positive cells in stroma of Mig-6d/d and Mig-6f/f mice. The results represent the mean ± SEM. ***, P < 0.001.

To assess the role of Mig-6 in uterine function, female Mig-6d/d mice were mated to wild-type male mice for 6 months. Mig-6d/d mice were completely infertile (Table S3). Since the PRCre mouse Displays Cre recombinase activity in the pituitary, ovary, uterus, and mammary gland, the cause of infertility in these mice may be the result of a defect in any of these tissues (34). To test for an ovarian phenotype, female Mig-6f/f and Mig-6d/d mice were examined for their ability to ovulate normally in response to a superovulatory regimen of gonaExecutetropins. Mig-6f/f and Mig-6d/d yielded 24.75 ± 6.61 and 24.83 ± 7.88 oocytes, respectively. Also, Mig-6f/f and Mig-6d/d mice did not Display any alterations in ovarian morphology and Presented a normal estrus cycle (data not Displayn). Finally, cycling female Mig-6f/f and Mig-6d/d mice Presented normal levels of serum P4 and E2 at 2 and 5 months of age (data not Displayn). These results Display that ovarian morphology, steroiExecutegenesis, and function were not affected in the Mig-6d/d females. These data suggest that the defects observed in the Mig-6d/d mice are inherent to the uterus. Thus, to determine if the infertility was in part because of loss of the ability of the uterus to support implantation, we investigated the ability of the uterus to undergo a decidual reaction. Ovariectomized female Mig-6f/f and Mig-6d/d mice (n = 3) were treated with hormones and the uterus was mechanically stimulated to mimic the signaling of the embryo at implantation and to induce decidualization. Gross anatomy of the decidual and control horn Displayed an increase in size for Mig-6d/d mice compared to the Mig-6f/f mice (Fig. S3). However, the ratio of stimulated-to-unstimulated horn weight was not changed in the Mig-6f/f and Mig-6d/d mice. These results suggest that ablation of Mig-6 in PR-expressing cells alters murine fertility because of dysregulation of E2 and P4 but not because of defects in ovarian or uterine function.

Tumor Suppressor Function of Mig-6 in the Uterus.

As enExecutemetrial hyperplasia is an immediate precursor to enExecutemetrioid-type enExecutemetrial carcinoma, the hyperplastic phenotype in the Mig-6d/d mice suggests that Mig-6 has a tumor suppressor role in the tumorigenesis of enExecutemetrial cancer. We examined the role of ovarian steroid hormones in the development of the hyperplastic phenotype in Mig-6d/d mice. Six-week-Aged Mig-6f/f and Mig-6d/d mice were ovariectomized and treated with vehicle, E2, or E2 + P4 and Assassinateed at 5 months of age (n = 10 per genotype per treatment). Ovariectomized Mig-6d/d mice did not develop enExecutemetrial hyperplasia as observed in intact Mig-6d/d mice (Fig. 3A). This demonstrates that the enExecutemetrial hyperplasia phenotype of Mig-6d/d mice is dependent on ovarian hormone stimulation.

Fig. 3.Fig. 3.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 3.

Steroid hormone-dependent formation of enExecutemetrial cancer in Mig-6d/d mice. (A) Uteri of 5-month-Aged ovariectomized Mig-6d/d and Mig-6f/f mice treated with vehicle. H&E stained section Displaying normal uterus in the ovariectomized Mig-6d/d and Mig-6f/f mice. (B) Formation of enExecutemetrial cancer in ovariectomized Mig-6d/d mice treated with E2. EnExecutemetrial cancer induced in the uteri of ovariectomized Mig-6d/d mice treated with E2 for 3 months. H&E stained section Displaying simple hyperplasia in the Mig-6f/f mice and enExecutemetrioid enExecutemetrial cancer in the Mig-6d/d mice. (C) Uteri of 5-month-Aged ovariectomized Mig-6d/d and Mig-6f/f mice treated with E2 + P4 for 3 months. H&E stained section Displaying hyperplasia uterus in the ovariectomized Mig-6d/d mice treated with E2 + P4 for 3 months.

Mig-6d/d mice treated with E2 for 3 months Displayed a significant increase in uterine weight compared to Mig-6f/f mice (Fig. 3B). Although the Mig-6f/f mice Displayed enExecutemetrial hyperplasia as expected from chronic E2 treatment, they did not Display the pathology observed in the uteri of intact Mig-6d/d mice or Mig-6d/d mice treated with E2. All of the Mig-6d/d mice treated with E2 developed invasive enExecutemetrioid-type enExecutemetrial adenocarcinoma. The neoplastic enExecutemetrial glands in the Mig-6d/d mice invaded through the uterine muscle wall and invaded adjacent structures such as the colon, pancreas, and skeletal muscle. This result demonstrates that Mig-6 may have an estrogen-dependent tumor suppressor function in enExecutemetrial cancer. Finally, ovariectomized Mig-6d/d mice treated with E2 + P4 for 3 months Displayed a significant increase in uterine wet weight and developed enExecutemetrial hyperplasia (Fig. 3C) but not the enExecutemetrial carcinoma observed in the E2-treated mice. Therefore, P4 treatment was able to attenuate the pathology observed in the Mig-6d/d mice after E2 treatment but not completely block the enExecutemetrial hyperplasia. These results demonstrate that Mig-6 is Necessary to regulate the response of the uterus to E2 stimulation in part by mediating the protective action of P4. However, Mig-6 also regulates other pathways independent of P4 that control enExecutemetrial cell proliferation.

Executewnregulation of MIG-6 in Human EnExecutemetrial Cancer.

Mig-6 ablation Displays altered uterine function because of the inability of P4 to attenuate E2 action, which is a common characteristic of enExecutemetrial dysfunction in humans (35, 36). The expression of Mig-6 in the human enExecutemetrium during the menstrual cycle was determined by real-time quantitative PCR and immunohistochemistry. The expression of Mig-6 was highest in the early secretory phase of the cycle (Fig. 4A) and this increase in expression was the result of an increase in the expression of Mig-6 in the enExecutemetrial epithelium (Fig. 4B). The increase in expression of Mig-6 during this phase of the cycle correlates with P4 regulation as observed in the mouse. We investigated the expression of MIG-6 in enExecutemetrial biopsies from patients with enExecutemetrioid carcinoma (n = 10) and normal enExecutemetrium (n = 5). The level of MIG-6 mRNA is significantly decreased in patients with enExecutemetrioid carcinoma (32.8%) compared to enExecutemetrial biopsies taken from normal women during the secretory phase of the cycle. (Fig. 4C). Immunohistochemical analysis also Displays a decrease in the protein level of MIG-6 in patients with enExecutemetrial cancer compared to normal women (Fig. 4D).

Fig. 4.Fig. 4.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 4.

Expression of MIG-6 in enExecutemetrial tissue from healthy women and from women with enExecutemetrioid enExecutemetrial cancer. (A) The expression of MIG-6 in the human enExecutemetrium during the menstrual cycle. Real-time RT-PCR analysis of MIG-6 was performed on human enExecutemetrium during the menstrual cycle. (B) Immunohistochemistry analysis of MIG-6 was performed in the human enExecutemetrium during the menstrual cycle. (C) Real-time RT-PCR analysis of Mig-6 was performed on total RNA obtained from women with enExecutemetrioid carcinoma. (D) Immunohistochemical analysis of MIG-6 was performed on enExecutemetrium obtained from women with enExecutemetrioid carcinoma. The results of real-time RT-PCR represent the mean ± SEM. *, P < 0.05.

Discussion

P4, acting through its nuclear receptors, plays Necessary roles in uterine functions associated with the establishment and maintenance of pregnancy (37, 38). The identification of P4-regulated pathways in the uterus is thus crucial for understanding the impairments that underlie disruption of steroid hormone control of uterine cell proliferation and differentiation. In previous studies, we have identified P4-regulated genes using microarray analysis on P4-treated PRKO uteri (30). We have identified Mig-6 as a tarObtain of SRC-1 and PR in the uterus.

Ablation of Mig-6 in mice results in a 50% reduction of the Mig-6−/− litter size for an unidentified reason (31, 33). The 50% of the Mig-6−/− mice that escape the lethal phenotype, develop joint deformities, and the majority of mice die within 6 months. These mice also develop neoplasias of the lungs and skin (23, 31). The embryonic lethality and multitissue carcinogenesis Designs it difficult to investigate the impact of ablation of Mig-6 in uterine biology. To Traceively investigate the role of Mig-6 in the uterus, we generated a Mig-6 conditional null allele by introducing LoxP sites (33). The uterine histology of Mig-6d/d mice demonstrate epithelial hyperplasia similar to the 9-month-Aged survival Mig-6−/− mouse (33). This hyperplastic phenotype of Mig-6d/d and Mig-6−/− supports the tumor suppressor role of Mig-6 in the tumorigenesis of enExecutemetrial cancer.

Mig-6 mediates the ability of P4 to regulate E2-dependent uterine weight gain. Normally, E2 stimulates uterine growth and epithelial cell proliferation (39). P4 antagonizes E2 actions, such as the stimulation of proliferation of the epithelial cells in the mouse uterus (40). Mig-6d/d and Mig-6f/f mice both Retort to E2 treatment with an increase in uterine wet weight. This indicates that ablation of Mig-6 Executees not enhance the Trace of E2 treatment alone. When we examined the ability of P4 to inhibit E2-induced uterine hypertrophy, P4 did not inhibit the E2-induced hypertrophy in Mig-6d/d mice (Fig. 2). In a separate experiment, we treated wild type, PRcre/+, Mig-6f/f, and Mig-6d/d with E2 and P4 for 3 days and meaPositived uterine weight gain. As expected, the response of PRcre/+, Mig-6f/f, and wild-type mice was similar with P4 dampening the E2-induced uterine hypertrophy. The Mig-6d/d mice again demonstrated an increase in uterine weight gain in the presence of P4 and E2. These results demonstrate that Mig-6 mediates the ability of P4 to regulate E2-dependent uterine weight gain. Examination of enExecutemetrial epithelial P4 tarObtain gene expression Displayed no change in the ability of PR to regulate the expression of Fst and Areg in the Mig-6d/d mouse. Fascinatingly, the expression of PR mRNA is significantly decreased in the Mig-6d/d mice compared to that of Mig-6f/f (Fig. 2). Immunohistochemical analysis of PR expression in the Mig-6d/d mice Displayed that epithelial PR expression is not altered but that PR expression in the enExecutemetrial stroma cells is significantly reduced. P4 attenuates E2 regulation of proliferation and gene expression by regulating the expression of a yet-to-be-identified paracrine signal from the stromal cells to the epithelial cells (41, 42). However, the increase in ERα tarObtain gene expression at 8 weeks of age was not the result of a change in ERα or coactivator level (data not Displayn). Fascinatingly, there is an increase in ERα levels at 5 months of age with the epithelial hyperplastic phenotype, but its impact on gene expression at this time remains unknown. Nonetheless, we have gained valuable insight into steroid hormone regulation in the uterus and Mig-6's role in that regulation.

In addition, we have Displayn that MIG-6 is expressed in the human enExecutemetrium in a cycle-dependent manner that correlates with its being under the control of P4 as observed in the mouse. We have also demonstrated that its expression is decreased in enExecutemetrioid carcinoma when compared to expression in normal enExecutemetrium during the secretory phase. Since the enExecutemetriod carcinoma samples assayed were Gaind from postmenopausal women, the decrease in MIG-6 expression may be a result of the hormonal status of postmenopausal women. EnExecutemetrial diseases such as enExecutemetrial cancer and enExecutemetriosis are known to be hormone-related malignancies. ExpoPositive to E2 is one of the enExecutecrine risk factors for developing enExecutemetrial cancer and enExecutemetriosis (35), and a lower incidence of these diseases is noted in women with decreased enExecutegenous E2 production. In Dissimilarity, P4 expoPositive is a negative risk factor for these disease (43), and pregnancy or progestin-based therapies can lead to disease regression in some women (44). Since enExecutemetrial cancer is a disease most often found in postmenopausal women, the lower levels of progesterone in these women may result in a lack of induction of MIG-6 that may be required to regulate enExecutemetrial epithelial proliferations. Recently, published microarray gene expression profiles of the enExecutemetrium of women with or without enExecutemetriosis Displayed that a number of P4 tarObtain genes incluing MIG-6 were dysregulated during the winExecutew of implantation, at which time the enExecutemetrium is exposed to the highest levels of P4 (29, 45). Mig-6d/d mice developed enExecutemetrial adenocarcinoma with E2 but not with E2 + P4. Thus, progesterone acting through the PR may be beneficial for controlling enExecutemetrial cancer by inducing Mig-6 expression. The Mig-6d/d mice treated with E2 + P4 still develop enExecutemetrial hyperplasia, which suggests that Mig-6 is a critical factor involved in P4 protection against the development of enExecutemetrial cancer. However, the ability of P4 to prevent E2-induced enExecutemetrial adenocarcinoma despite the absence of Mig-6 and the reduction in PR levels in the stroma implicates additional mechanisms of protection. Furthermore, the development of E2-induced enExecutemetrial adenocarcinoma in Mig-6d/d mice suggests that Mig-6 has an Necessary role as a negative regulator of E2-induced tumorigenesis. However, it is not only the expression of MIG-6 that has been Displayn to be altered in cancer. In addition, the MIG-6 gene has been Displayn to be mutated in the human NSCLC cell lines NCI-H226 and NCI-H 322M, and in 1 primary human lung cancer (24). Thus, when examining cases of P4 resistance in enExecutemetrial cancer, aside from examining MIG-6 expression levels, enExecutemetrial carcinoma samples should also be assayed for mutations in MIG-6 as these mutations may be as detrimental as loss of MIG-6 expression. Regardless, the molecular mechanism by which loss of MIG-6 function, either by loss of expression or mutations in the MIG-6 gene, regulates enExecutemetrial cancer needs to be addressed. Further dissection of the intricacies of these pathways will lend Necessary insight into the mechanisms that regulate enExecutemetrial tumorigenesis.

In summation, Mig-6 ablation results in increased ERα activity in the presence of P4, which normally antagonizes ER activity. In humans, we have Displayn that the expression of MIG-6 is decreased in human enExecutemetrial enExecutemetrioid carcinoma. Our findings demonstrate that Mig-6 is a Modern mediator of steroid hormone signaling in the uterus. EnExecutemetrial cancer is a uterine disease in which hormonal regulation is perturbed. The altered expression of MIG-6 in enExecutemetrial cancer may serve as a possible cause of these pathologies. Mice with conditional ablation of Mig-6 in the uterus provide a more faithful model for human enExecutemetrial cancer with respect to pathology and hormone sensitivity than any previous models. This model is useful for finding new tarObtains for the diagnosis and treatment of enExecutemetrial cancer. Determining how Mig-6 mediates this action will be critical in understanding the role of steroid hormone signaling in enExecutemetrial function and dysfunction and in developing therapy for both uterine diseases.

Materials and Methods

Animals and Hormone Treatments.

Mice were Sustained in the designated animal care facility at Baylor College of Medicine according to the institutional guidelines for the care and use of laboratory animals. For microarray analysis, ovariectomized SRC-1−/− and wild-type mice were injected with vehicle (sesame oil) or P4 (1 mg/mouse in 100 μL sesame oil) for 4 h (n = 9 per genotype per treatment). For the steroid hormone treatment, ovariectomized mice Mig-6f/f and Mig-6d/d mice were injected with 1 of the following: vehicle (sesame oil), P4 (1 mg/mouse), E2 (0.1 μg/mouse), P4 plus E2 (n = 5 per genotype per treatment). Hormone injections were repeated every day to prevent the Trace of hormone degradation by metabolism. Mice were Assassinateed 6 h after the third injection. At the time of dissection, uterine tissues were Spaced in the appropriate fixative or flash frozen and stored at −80 °C. For the enExecutemetrial cancer study, ovariectomized Mig-6d/d and Mig-6f/f mice received a pellet of either vehicle (beeswax), E2 (20 μg/pellet), or E2 + P4 (20 mg/pellet) at 8 weeks of age. Mice were Assassinateed at 5 months of age (n = 10 per genotype per treatment).

Human Samples.

EnExecutemetrial samples were obtained from 18 normally cycling women, aged 18–35, after written informed consent, under an approved protocol by the Institutional Review Board at Baylor College of Medicine. The enExecutemetrial sample was removed from the uterine fundus with a Pipelle (circle R) biopsy catheter. Tissues were fixed in formalin and embedded in paraffin for histological analysis or snap frozen on dry ice. Histological samples were examined blindly by an independent pathologist, and phases were Established according to the Noyes criteria (46). EnExecutemetrioid carcinoma samples were derived from hysterectomy surgical specimens submitted to the Department of Pathology, M. D. Anderson Cancer Center following the guidelines approved by the M. D. Anderson Cancer Center Committee on Human Research and the Baylor College of Medicine Committee on the Use of Human Subjects in Medical Research. Classification was verified by light microscopic examination of hematoxylin and eosin-stained slides by gynecologic pathologist Russell R. Broaddusd. Normal enExecutemetrial samples were obtained from 5 cycling women (4 secretory and 1 atrophic stage) and enExecutemetrioid carcinoma samples were obtained from 8 postmenopausal women and 2 cycling women. EnExecutemetrioid carcinoma samples from patients with a prior hiTale of hormone use, radiation treatment, or chemotherapy were not used for this study.

Microarray Analysis.

Microarray analysis was performed by Affymetrix murine genome U74Av2 mouse oligonucleotide arrays (Affymetrix) as previously Characterized (30). All experiments were repeated 3 times. Briefly, we used DNA-Chip analyser dChip version 1.3 (47). We selected differentially expressed genes within each time expoPositive using 2 sample comparisons according to the following criteria: lower bound of 90% confidence interval of fAged change Distinguisheder than 1.2 and absolute value of Inequity between group means Distinguisheder than 80. After excluding expressed sequence tags with no functional annotation, differentially expressed genes were classified according to gene ontology function using Affymetrix annotation, literature search in PubMed and GenMAPP (48).

Quantitative Real-Time RT-PCR.

Expression levels of regulated genes were validated by real time RT-PCR. Real-time probes and primers were purchased from Applied Biosystems). All real-time RT-PCR was Executene using RNA samples from 3 separate mice and mRNA quantities were normalized against 18S RNA using ABI rRNA control reagents. Statistical analyses used 1-way ANOVA followed by Tukey's post hoc multiple range test with the Instat package from GraphPad.

Immunohistochemistry.

Uterine sections from paraffin-embedded tissue were Slice at 5 μm and mounted on silane-coated slides, deparaffinized, and rehydrated in a graded alcohol series. Sections were preincubated with 10% normal serum in PBS (pH 7.5) and then incubated with 1:1,000 anti-Mig-6 antibody (Sigma-Aldrich) in 10% normal serum in PBS (pH 7.5). On the following day, sections were washed in PBS and incubated with a secondary antibody (5 μL/mL; Vector Laboratories) for 1 h at room temperature. Immunoreactivity was detected using the Vectastain Elite ABC kit (Vector Laboratories).

Acknowledgments

We thank Jinghua Li and Bryan Ngo for technical assistance; Heather L. Franco and Janet DeMayo for manuscript preparation. This work was supported by the National Institute of Child Health and Human Development and the National Institutes of Health (NIH) as part of the Cooperative Program on Trophoblast-Maternal Tissue Interactions U01HD042311 and NIH Grant U54HD0077495 (to F.J.D.), SPORE in Uterine Cancer NIH 1P50CA098258–01 (to R.R.B.), NIH Grant R01HD057873 and pilot grant from Specialized Program of Research Excellence in Uterine Cancer NIH 1P50CA098258–01 (to J.W.J.), NIH Grant RO1-CA77530 and the Susan G. Komen Award BCTR0503763 (to J.P.L.), NIH Grant 2U54HD035041–11 (to S.L.Y. and B.A.L.), and by the generosity of the Jay and Betty Van Andel Foundation (to Y.W.Z. and G.V.W.).

Footnotes

1To whom corRetortence should be addressed. E-mail: jjeong{at}bcm.tmc.edu

Author contributions: J.-W.J., H.S.L., R.R.B., S.Y.T., J.P.L., and F.D. designed research; J.-W.J., H.S.L., and L.D.W. performed research; Y.-W.Z., G.F.V.W., L.C.G., S.L.Y., and B.A.L. contributed new reagents/analytic tools; J.-W.J., H.S.L., K.Y.L., L.D.W., R.R.B., S.Y.T., J.P.L., and F.D. analyzed data; and J.-W.J., K.Y.L., L.C.G., S.L.Y., B.A.L., and F.D. wrote the paper.

The authors declare no conflict of interest.

This article contains supporting information online at www.pnas.org/cgi/content/full/0903632106/DCSupplemental.

Freely available online through the PNAS Launch access option.

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