Uterine epithelial estrogen α is dispensable for proliferation but essential for complete biological and biochemical responses

Wipawee Winuthayanona, Sylvia C. Hewitta, Grant D. Orvisb,c, Richard R. Behringerc, and Kenneth S. Koracha,1

aLaboratory of Reproductive and Developmental Toxicology, National Institute of Environmental Health Sciences, National Institutes of Health, Department of Health and Human Services, Research Triangle Park, NC 27709; bDevelopmental Biology Program, Sloan–Kettering Institute, York Avenue, New York, NY 10065; and cDepartment of Genetics, University of Texas M.D. Anderson Cancer Center, Houston, TX 77030

Edited by R. Michael Roberts, University of Missouri, Columbia, MO, and approved September 17, 2010 (received for review May 7, 2010)

Female fertility requires estrogen to specifically stimulate estrogen uterine epithelial DNA synthesis was stromal ERα-dependent via receptor α (ERα)-dependent growth of the uterine epithelium in induction of autocrine or paracrine mitogenic factors in the stro- adult mice, while immature females show proliferation in both ma that stimulated the epithelial proliferation (5). Evaluating an stroma and epithelium. To address the relative roles of ERα in epithelial-specific role for ERα in tissue proliferation was re- mediating estrogen action in uterine epithelium versus stroma, a cently reported in the mammary gland. Use of a MMTV-Cre uterine epithelial-specific ERα knockout (UtEpiαERKO) mouse line crossed with floxed ERα mice demonstrated that mammary gland was generated by crossing Esr mice with Wnt7a-Cre mice. Expres- growth and ductal epithelial cell proliferation required the mam- sion of Wnt7a directed Cre activity generated selective deletion of mary epithelial ERα (6), which contrasted with previous me- ERα in uterine epithelium, and female UtEpiαERKO are infertile. senchymal-epithelial tissue recombination studies that showed β E Herein, we demonstrate that 17 -estradiol ( 2)-induced uterine mammary epithelial ERα was dispensable for mammary epithe- epithelial proliferation was independent of uterine epithelial ERα lial cell proliferation (7). Therefore, we evaluated whether a si- because DNA synthesis and up-regulation of mitogenic mediators milar discrepancy would occur in another estrogen proliferating were sustained in UtEpiαERKO uteri after E treatment. IGF-1 treat- 2 tissue or whether uterine tissue would follow a similar pattern as BIOCHEMISTRY ment resulted in -independent ER activation in both wild- the mammary gland regarding an indispensable role of ERα in α E type (WT) and UtEpi ERKO and mimicked the 2 stimulatory effect uterine epithelial proliferation. on DNA synthesis in uterine epithelium. Uterine epithelial ERα was Several studies demonstrated that a number of growth factors, E necessary to induce lactoferrin, an 2-regulated secretory such as IGF-1, EGF, or transforming growth factor α (TGFα) selectively synthesized in the uterine epithelium. However, loss (8–10) and their receptors were induced and activated, respec- α E of uterine epithelial ER did not alter the 2-dependent progester- tively, in the uterus under the influence of E2 treatment (8–12). one receptor (PR) down-regulation in epithelium. Strikingly, the IGF-1 was shown experimentally to be a key growth factor that uterine epithelium of UtEpiαERKO had robust evidence of apopto- stimulates growth of the nearby epithelial cells as a result of their E sis after 3 d of 2 treatment. Therefore, we surmise that estrogen estrogen-dependent expression in the uterus (13). Lack of IGF-1 induced uterine hyperplasia involves a dispensable role for uterine stimulation in αERKO mice demonstrated that ERα was re- epithelial ERα in the proliferative response, but ERα is required quired for mediating the uterine IGF-1 action (14–16). The tran- subsequent to proliferation to prevent uterine epithelial apoptosis scriptional factor CCAAT enhancer binding protein beta (C/ assuring the full uterine epithelial response, illustrating the differ- EBPβ) is another estrogen-regulated in female reproductive ential cellular roles for ERα in uterine tissue and its contribution tissues that is essential for uterine functions (17) and regulation during pregnancy. of mammary epithelial cell proliferation and differentiation (18). In addition to mitogenic effects, estrogen also plays a role in conditional knockout ∣ paracrine regulation controlling apoptosis (programmed cell death) in several cell types (19). In the uterus, steroid hormones regulate apoptosis. strogens exert their actions through estrogen receptor (ER) Circulating levels of E2 are inversely correlated with apoptosis Eproteins. ER alpha and beta (α and β) are distributed through- in uterine luminal and glandular epithelium during the estrous out diverse tissues of the body. ERα is predominantly expressed cycle, with the highest level of apoptosis in the epithelium at in the uterus, mammary glands, pituitary, hypothalamus, and metestrous as E2 levels decline (20). Estrogen was shown to ovarian theca cells, whereas ERβ is primarily in ovarian granulosa suppress apoptosis in the neonatal mouse uterine epithelium cells, lung, and prostate (1). The uterus is a major target tissue for via an ERα-dependent induction of apoptosis inhibitory estrogen, and significant levels of ERα are expressed throughout (AIPs) including Birc1a (Baculoviral inhibitors of apoptosis all the major uterine compartments (luminal and glandular repeat-containing 1) and the inhibition of the activation of the epithelium, stroma, and the myometrium), and the expression le- apoptosis executioner, cysteine-aspartic protease-3 (caspase-3) vel of ERα varies during the estrous cycle and peaks during proes- (21). Herein, we evaluated the effect of E2 on adult uterine trous in accordance with the elevation of circulating 17β-estradiol epithelial apoptosis when the ERα was selectively deleted in the (E2) to elicit a proliferative response (2). Uterine proliferation is uterine epithelium. The Wnt7a-Cre recombinase and Esr1-loxP shown from a number of studies to be regulated by E2 in an ERα- dependent manner, as demonstrated by the lack of uterine stimu- α Author contributions: W.W., S.C.H., G.D.O., R.R.B., and K.S.K. designed research; lation and mitotic growth responses in ERKO uteri (3). In W.W., S.C.H., and G.D.O. performed research; W.W., S.C.H., G.D.O., and K.S.K. analyzed immature rodents, E2 stimulates the proliferation of both uterine data; G.D.O. and R.R.B. contributed new reagents/analytic tools; and W.W., S.C.H., luminal and glandular epithelium as well as stroma, whereas in G.D.O., and K.S.K. wrote the paper. adult mice, E2 stimulates the proliferation of only the luminal The authors declare no conflict of interest. and glandular epithelium (4). A prior series of studies have pro- This article is a PNAS Direct Submission. posed that estrogen acting on stromal cells initiates responses to 1To whom correspondence should be addressed. E-mail: [email protected]. mediate epithelial proliferation. Such evidence was generated This article contains supporting information online at www.pnas.org/lookup/suppl/ from neonatal tissue recombination studies in which E2-activated doi:10.1073/pnas.1013226107/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1013226107 PNAS Early Edition ∣ 1of6 Downloaded by guest on September 27, 2021 expression system was utilized in our study to address the effect of E2 on uterine epithelial responsiveness following selective loss of ERα. Results Generation of Uterine Epithelial-Specific ERα Knockout Mice. A uter- ine epithelial-specific ERα knockout (UtEpiαERKO) mouse model was generated by crossing a transgenic mouse line expres- sing Wnt7a-Cre recombinase with our new line of floxed ERα mice containing loxP sites flanking exon 3 of the mouse Esr1 gene (22). The cell type specific expression of Wnt7a-Cre resulted in complete deletion of ERα specifically in uterine luminal and glandular epithelial cells. ERα protein expression was evaluated by immunohistochemistry (IHC) and was robustly expressed throughout WT uteri (epithelium, stroma, and myometrium) (Fig. 1, Left). ERα protein was detected in stromal and myome- trial tissues but absent in the uterine luminal and glandular epithelium of UtEpiαERKO mice (Fig. 1, Right), demonstrating that the Cre-specific deletion of ERα was effective. Test matings of UtEpiαERKO females indicated that they are infertile (Table S1). Additional analysis indicates the females exhibit regular estrous cycles, and ovarian histology shows all stages of follicular development and indication of ovulation based on the presence of corpus lutea (Fig. S1). However, implantation sites were observed in 6 of 9 WT but only 0 of 6 in UtEpiαERKO after natural mating or embryo transfer into pseudopregnant fe- males, indicating that UtEpiαERKO mice have a compromised uterine embryo receptivity. Accordingly, the estrogen-regulated transcripts Lif (leukemia inhibitory factor) and Ihh (Indian Fig. 2. Uterine proliferative response to E2 and IGF-1 in WT and UtEpiαERKO hedgehog), which are essential for embryo attachment (23, 24), mice. The uterine wet weight increase after (A)24hor(B) 3-d (three conse- cutive days) treatment of vehicle, E2 in the presence or absence of ICI treat- remained unchanged in UtEpiαERKO but were up-regulated in E ment, and (C) the fluorescent signal of Hoescht 33342 and EdU incorporation/ WT after 2 treatment (Fig. S2). Alexafluor488 staining in the uterine sections from mice treated with vehicle, E2 (24 h or 3 d), or IGF-1 (24 h). (Scale bar: 20 μm.) Results are mean SEM Proliferative Effect of E2 on Uterine Epithelium Is Stromal ERα Depen- (n ¼ 7). *, and ***p < 0.05 and 0.001, significantly different from vehicle dent. E2 significantly increased the uterine wet weight in ovariec- control of the corresponding genotype, respectively. +, p < 0.05 significantly tomized (OVEX) WT mice 24 h after treatment (p < 0.001) different from E2-treated WT mice. but not in UtEpiαERKO mice (Fig. 2A). Uterine weight was sig- nificantly increased by E2 in UtEpiαERKO after a 3-d bioassay; WT and UtEpiαERKO uteri and decreased in both genotypes however, the increase was significantly lower than WT (Fig. 2B, after 3 d. p < 0.05), and the uterine proliferative response induced by E2 was fully attenuated by ICI 182,780 (ICI) in both WT and UtE- C/EBPβ and IGF-1: The Postulated Mediators of E2-Induced Prolifera- piαERKO mice, indicating the increase is an ER-dependent tion in Uterine Epithelium. Based on previous studies (5, 8, 9, 17), response. Incorporation of the deoxy-thymidine analog EdU we evaluated whether Cebpb and Igf1 could be mediators of E E (5-ethynyl-2′-deoxyuridine) demonstrated that E2 induced 2-induced uterine epithelial proliferation. 2 significantly up- DNA synthesis in luminal epithelium (Fig. 2C) at 24 h in both regulated Cebpb at 2 h (Fig. 3A) and Igf1 at 6 h (Fig. 3B) in both WT and UtEpiαERKO uteri, and the expression in both geno- types declined to the basal level at 24 h. The expression levels of Cebpb and Igf1 after E2 treatment in WT and UtEpiαERKO were not significantly different. Therefore, the uterine epithelial ERα was not required for the estrogen induction of the uterine Cebpb and Igf1. We further investigated the effects of growth factor alone on uterine epithelial proliferation. Growth factor, such as IGF-1, was known to be the mitogenic factor that stimulated rodent uter- ine epithelium proliferation (9). To bypass the effect of E2,we treated the animals with IGF-1 (50 μg∕mouse) in the absence of E2. IGF-1 induced uterine luminal epithelial DNA synthesis to a lesser extent than with E2 treatment as illustrated by EdU incorporation at 24 h in both WT and UtEpiαERKO (Fig. 2C). Therefore, IGF-1 appears to be a possible downstream effector of E2 action in the stroma that stimulates uterine epithelial prolif- eration.

Evaluation of E2-Specific Responses in Uterine Epithelium and Stroma. E Fig. 1. ERα protein expression by IHC analysis in WT (Left) and UtEpiαERKO Lactoferrin (Ltf) is a protein regulated by 2 in mammalian uter- (Right) uteri with longitudinal sections (Upper; 40× magnification) and cross- ine epithelium (3, 25). After 3 d, E2 significantly induced Ltf ections (Lower; 200× magnification). mRNA in WT uteri (Fig. 4A, p < 0.001) and also increased

2of6 ∣ www.pnas.org/cgi/doi/10.1073/pnas.1013226107 Winuthayanon et al. Downloaded by guest on September 27, 2021 Fig. 3. E2-regulated in WT and UtEpiαERKO uteri. (A) Cebpb mRNA at 2 h and 24 h, and (B) Igf1 mRNA at 6 h and 24 h after vehicle or E2 treatment (n ¼ 4). Results are mean SEM. *, **, and ***p < 0.05, 0.01, and 0.001, significantly different from vehicle control of the corresponding genotype, respec- tively. ns; not significantly different in E2 treated for 2 h (A) and 6 h (B) in WT and UtEpiαERKO, analyzed by nonparametric Mann–Whitney test.

Ltf protein in the uterine luminal and glandular epithelium in uterine epithelium caused the loss the lactoferrin production. (Fig. 4B, arrowheads), and these increases were inhibited by However, the uterine epithelial down-regulation and stromal ICI (Fig. 4A and B). Loss of ERα in UtEpiαERKO uteri resulted redistribution of PR by E2 remained intact in UtEpiαERKO in loss of the E2-stimulated lactoferrin expression (Fig. 4A and B). mice. Surprisingly, lack of functional ERα in the uterine epithe- Furthermore, the progesterone receptor (PR) is essential for lium had a pronounced effect on apoptosis. establishment and maintenance of pregnancy (26) and is also E2 is known to be a robust mitogenic ovarian hormone in regulated by E2 in the uterus (27). Therefore, we evaluated the uterine tissues that induces a pronounced hyperplastic tissue PR protein expression, which showed that PR was down-regu- response, consistent with endometrial development during the es- lated in the epithelium and increased in the stroma following trous cycle (28, 29). Staged cell labeling studies showed an effect E2 treatment in WT mice. This PR response was not mediated of E2 on recruiting uterine epithelial cells from a resting state via the uterine epithelial ERα since the response was sustained (G0) into the cell cycle and shortened both G1 and S-phases in UtEpiαERKO (Fig. 4C). Moreover, the inhibitory effect of ICI of the cell cycle to elicit proliferation (4, 30). Administration in both genotypes further indicated that stromal ERα appeared of E2 induces the proliferation in both uterine epithelium and

responsible for the E2 down-regulation of PR in the uterine stroma of immature mice but only stimulates the proliferation BIOCHEMISTRY epithelium (Fig. 4C). Other examples of uterine estrogen respon- in uterine epithelium in adult mice, and the cause of this lack sive transcripts were evaluated as well (Fig. S3) and indicate loss of stromal cell responsiveness has been linked to progesterone of some responses, such as Aqp8 (aquaporin 8) and Cdkn1a activity (4). Previous work using neonatal tissue xenograph mod- (cyclin dependent kinase inhibitor A or p21), and retention of els and extrapolation of such a model to adult tissue (5) indicate others, such as Dhcr24 (24-dehydrocholesterol reductase) and that the proliferative effect of E2 on uterine epithelium in adult Ube2c (ubiquitin-conjugating enzyme E2C). mice is independent of ERα expression in the luminal epithelium, suggesting the stromal ERα expression is crucial for the mitotic Loss of ERα in Uterine Epithelium Enhances Uterine Apoptosis. Loss of response of E2. To evaluate and extend those observations in a ERα in uterine epithelium in combination with the growth re- natural adult tissue system in vivo, our UtEpiαERKO mouse sponses following 3 d of E2 treatment altered the appearance model was generated with the specific deletion of ERα in uterine of the epithelia of UtEpiαERKO (Fig. 5A, arrowheads) in a man- epithelium. Adult UtEpiαERKO mice had a complete deletion of ner suggesting increased apoptosis in UtEpiαERKO compared to ERα protein in the uterine luminal and glandular epithelium WT. Consistent with this, E2 treatment significantly induced the throughout the uterine horn. The classical uterine bioassay was apoptosis inhibitor Birc1a expression in WT (p < 0.001) but not utilized to illustrate the estrogenic effect of E2 on uterine growth in UtEpiαERKO uteri (Fig. 5B). Cleaved caspase-3 (the active responses at both 24 h and 3 d. Absence of ERα in the uterine form of caspase-3) was slightly increased by 3 d of treatment with epithelium compromised the uterine weight increase induced by E2 in WT (Fig. 5C, yellow arrowheads) but intensively detected in E2 after 24 h or 3 d, which suggests the epithelial ERα contributes UtEpiαERKO luminal and glandular epithelium as dense, round to a full uterine growth response. Our results suggest that the in- spots on both basolateral and luminal sides of the epithelial cells itial (24 h) proliferation response is similar but that the luminal (Fig. 5C, black arrowheads). However, increased levels of the and glandular epithelial cells do not subsequently develop prop- DNA fragmentation marker, TUNEL (Terminal deoxynucleoti- erly into multilayered tissue structures secreting lactoferrin and dyltransferase-mediated dUTP nick end labeling), was found additionally show indications of apoptosis. These deficiencies only in UtEpiαERKO uteri, which further suggested increased and alterations most likely account for the hampered increase apoptosis following 3 d of E2 treatment (Fig. 5D, arrowheads). in uterine weight. However, the induction of DNA synthesis after Therefore, loss of the uterine epithelial ERα correlated with 24 h and the decline after 3 d of E2 treatment was seen in both increased uterine apoptosis subsequent to the uterine growth WTand UtEpiαERKO. Therefore, uterine epithelial ERα in vivo induced after E2 treatment for 3 d. was dispensable for appropriate mitotic proliferative response from E2, consistent with earlier xenograph experiments (5), in Discussion contrast to the requirement for epithelial ERα in mammary gland Herein, we illustrated the physiological relevance of ERα to uter- development and proliferation (6). ine epithelial proliferation and apoptosis under the influence of Next, we evaluated the possible mitogenic mediators (C/EBPβ E2 regulation. We demonstrated that uterine epithelial ERα was and IGF-1) downstream from E2. Previous studies showed that dispensable for the E2-induced uterine epithelial proliferation the transcription factor C/EBPβ is rapidly increased in both in adult OVEX mice in contrast to what has been reported for epithelial and stromal cells by E2 in the uterus (17). The essential the mammary gland epithelial proliferation (6). In the absence role of C/EBPβ in uterine epithelial proliferation has been de- of uterine epithelial ERα, potential mitogenic mediators such monstrated by the decreased E2-dependent uterine epithelial as Cebpb and Igf1 were increased with E2 treatment, and the proliferation as a result of impaired DNA replication in Cebpb growth factor IGF-1 could bypass the effect of E2 on uterine knockout mice (17, 31). IGF-1 has been demonstrated to be a epithelial proliferation. We confirmed the uterine epithelial paracrine mitogenic effector secreted from uterine stroma to sti- E2-specific loss of response by showing that deletion of ERα mulate the proliferation of uterine epithelium (5). E2 increases

Winuthayanon et al. PNAS Early Edition ∣ 3of6 Downloaded by guest on September 27, 2021 Fig. 5. Apoptosis-related mediator expressions in WT and UtEpiαERKO uteri. Fig. 4. Uterine epithelial-specific E2-regulated responses. Ltf expression by After the treatment of vehicle or E2 for three consecutive days, H&E staining using real-time PCR analysis (A), IHC analysis of lactoferrin (B), and proges- (A) indicated apoptotic appearance of epithelial cells in E2-treated UtEpiαER- terone receptor (C) protein expression in WT (Left) and UtEpiαERKO (Right) KO uteri (black arrowheads). Birc1a expression by using real-time PCR analysis uteri when treated with vehicle, E2 or E2 þ ICI for three consecutive days (B) increased only in E2-treated WT. Cleaved Caspase-3 IHC (C) increased in (n ¼ 7). (Scale bar: 40 μm.) Arrowheads indicate the positive staining of lac- both WT (yellow arrowheads) and UtEpiαERKO (black arrowheads) uteri, toferrin. Results are mean SEM. ***p < 0.001, significantly different from and TUNEL IHC (D) increased only in E2-treated UtEpiαERKO uteri (black ar- vehicle of the corresponding genotype. rowheads) (n ¼ 7). (Scale bar: 20 μm.) Results are mean SEM. ***p < 0.001, significantly different from vehicle control of the corresponding genotype, respectively. production of growth factors such as IGF-1 and EGF, and these growth factors induce uterine DNA synthesis (8–10, 16). Igf1 which implicated the essential role of IGF-1 on uterine growth knockout mice exhibited impaired uterine response to estrogen, (32). Overexpression of IGF binding protein-1 (IGFBP-1) in

4of6 ∣ www.pnas.org/cgi/doi/10.1073/pnas.1013226107 Winuthayanon et al. Downloaded by guest on September 27, 2021 the mouse uterus disrupted DNA synthesis, but the uterine wet severe apoptosis as shown by increases in active caspase-3 and weight increased in response to E2 (33). Therefore, the local pro- TUNEL staining. Moreover, in the absence of uterine epithelial duction and availability of IGF-1 appears to play a role in uterine ERα, E2-mediated increase of the apoptotic inhibitor, Birc1a,is proliferation. Moreover, evidence also suggests the proliferative dramatically attenuated. As mentioned earlier, C/EBPβ is a cri- effect of E2 on IGF-1 production in the human epithelium tical mediator of E2-stimulated proliferative response in uterine was mediated in an autocrine fashion in human uterine epithelial epithelial cells. C/EBPβ knockout mice undergo uterine epithelial cells (34). Further evidence came from the use of a growth factor apoptosis as a result of G1-S-phase arrest (31), demonstrating a inhibitor in combination with E2 administration, which partly in- need for C/EBPβ in DNA replication and damage response. In hibited the action of estrogen on the uterine weight increase (13). our study the level of C/EBPβ after E2 treatment in UtEpiαER- We previously showed that IGF-1 treatment stimulated the uter- KO is comparable to that of in WT. However, we have not eval- ine epithelial proliferation in an ERα dependent manner (16). In uated the relative levels of C/EBPβ protein in stromal and this study, the complete deletion of ERα specifically in the epithe- epithelial cells, and it is possible that there is a deficiency of this lial target cell of the uterus did not alter DNA synthesis after 24 h important factor intrinsic to the epithelial cells of UtEpiαERKO E of either 2 or IGF-1 treatment. Therefore, in the natural tissue following uterine growth. Although the uterine epithelial apop- α environment, ER residing in the stroma, not uterine epithelium, tosis in UtEpiαERKO following E2 treatment may not be due to α E was the functional ER that responded to 2 to produce the an altered C/EBPβ regulation, it may alternatively result from a growth factor(s), which was released and acted as paracrine med- defect in other mediators of DNA damage response. Therefore, iator(s) to promote DNA synthesis in the epithelium. As men- we postulate that during the adult reproductive cycle following tioned above, these findings in vivo are consistent with previous the E2 stimulated growth, the lack of ERα and E2 action in studies using neonatal tissue recombination experiments (5). the epithelial cells impairs responses initiated by unknown To substantiate the absence of ERα activity in the uterine E α 2-dependent secondary mediators resulting in apoptosis of epithelium, we evaluated gene regulation of Ltf as an ER - the epithelial cells. Thus, epithelial apoptosis occurs due to dependent estrogen-regulated response in uterine epithelium the absence of epithelial ERα. Interestingly, normal regulation (35). Expression of lactoferrin has been correlated with the cir- of apoptosis has been shown to be important for implantation culating level of E2 (36). Our result showed that the up-regulation (44). Therefore, alteration of growth and apoptosis as a result of Ltf mRNA and Ltf protein was a uterine epithelial-specific of the loss of uterine epithelial ERα leads to the infertility phe- ERα-dependent action, which was consistent with the previous notype in UtEpiαERKO. More complete fertility studies will be finding in a tissue recombinant study (37) and supports the conducted to address additional details regarding the roles of BIOCHEMISTRY absence of ERα activity in the epithelium. We found that E2 uterine epithelial ERα. redistributed the PR expression from epithelium to stroma in Overall, this model indicates that uterine epithelial prolifera- UtEpiαERKO in a similar manner as in WT. These findings again tion is mediated by ERα-dependent mitotic proliferative signal concur with the previous studies using neonatal tissue recombi- α nation models (38, 39). Therefore, the estrogen-dependent re- secreted from the stroma but that ER is needed following sponsiveness for redistribution of PR from uterine epithelium mitosis within epithelial cells to minimize apoptosis. Therefore, E ERα is required in both tissue compartments for a complete to stroma upon 2 treatment is intact and appears to be mediated α by stromal ERα. uterine growth response. It is surprising that epithelial ER is During the female reproductive cycle, estrogen stimulates the required for mammary gland proliferation but is dispensable in proliferative phase to facilitate implantation and pregnancy. In uterine response. Further studies will be required to determine rodents, the proliferate epithelial layer is eliminated by inducing how this differential cellular role of ER may change in diseases apoptosis in situ (40). The proliferation and subsequent elimina- such as endometriosis, endometrial hyperplasia, or cancer. tion of uterine epithelial cells during the estrous cycle is regulated Materials and Methods by the relative mitotic and apoptotic rates under the control of Detailed methods appear in SI Materials and Methods. These include genera- ovarian hormones, especially the circulating level of E2 (40). E tion of a Wnt7a-Cre transgenic mouse line, generating uterine epithelial- Withdrawal of 2 treatment from newborn mice results in a high specific ERα knockout mice, chemicals and compounds, uterine bioassay, apoptosis index in the uterus (41). AIPs have a protective effect RNA isolation, and real-time PCR analysis for , histological against the activation of the essential apoptosis mediator caspase- and IHC analysis of uteri, and statistical analysis. 3, which is a member of a class of cysteine-aspartyl proteases (42). Caspases are also present in normal healthy cells as inactivated ACKNOWLEDGMENTS. We thank James Clark, Page Myers, David Goulding enzymes and are activated by cleavage to initiate apoptosis and from the National Institute of Environmental Health Sciences (NIEHS) Animal induce cell death (43). In our study, the increase in active caspase- Surgery Group of Comparative Medicine branch for performing the animal 3 in WT may result from the normal regulation between growth surgeries and embryo transfer analysis, David Monroy for animal care, E Geoffrey Hurlburt and Dave Olsen (NIEHS Immunohistochemistry core) for and apoptosis after 2-induced proliferation of the uterine cleaved caspase-3 and TUNEL staining, Casey Reed for mouse genotyping epithelium. After diethylstilbestrol treatment, uteri of neonatal and Drs. April Binder and Diane Klotz for the critical reading of the manu- ERα knockout mice exhibited severe apoptosis, whereas WT script and helpful suggestions. This research was supported by the National mice sustained the apoptotic protection (21), thereby indicating Institute of Environmental Health Sciences, Division of Intramural Research (funding to W.W., S.C.H., and K.S.K.) project Z01ES70065 as well as NIH that estrogen protection against uterine epithelial apoptosis is HD30284 and CA098258 (SPORE in Uterine Cancer) to R.R.B. G.D.O. was sup- mediated via ERα. In this study, we showed that after the growth ported by the National Cancer Institute CA09299 Training Program in the induced by E2 treatment, UtEpiαERKO epithelia exhibited Molecular Genetics of Cancer.

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