Epithelial 1 intrinsically mediates squamous differentiation in the mouse

Shinichi Miyagawaa,b and Taisen Iguchia,b,1

aOkazaki Institute for Integrative Bioscience, National Institute for Basic Biology, National Institutes of Natural Sciences, Okazaki, Aichi 444-8787, Japan; and bDepartment of Basic Biology, Faculty of Life Science, SOKENDAI (The Graduate University for Advanced Studies), Okazaki, Aichi 444-8787, Japan

Edited by Jan-Åke Gustafsson, University of Houston, Houston, Texas, and approved September 10, 2015 (received for review July 10, 2015) Estrogen-mediated actions in female reproductive organs are are definite requirements for epithelial ESR1 expression in uterine tightly regulated, mainly through 1 (ESR1). The epithelial functionalization (e.g., lactoferrin expression and mouse vaginal cyclically exhibits cell proliferation and prevention of cell death in the ), as well as in ductal mor- differentiation in response to estrogen and provides a unique phogenesis, alveologenesis, and lactation in the mammary gland model for analyzing the homeostasis of stratified squamous ep- (11, 12). ithelia. To address the role of ESR1-mediated tissue events dur- Another female reproductive organ, the mouse vagina, exhibits a ing homeostasis, we analyzed mice with a - unique estrogen-responsive feature. The vaginal epithelium exhibits specific knockout of Esr1 driven by 5-Cre (K5-Esr1KO). cyclical, estrogen-dependent cell proliferation and squamous dif- We show here that loss of epithelial ESR1 in the vagina resulted ferentiation during the . The vaginae of ovariectomized in aberrant epithelial cell proliferation in the suprabasal cell layers (OVX) mice show an atrophied epithelium with two to three cell and led to failure of keratinized differentiation. layers, whereas estrogen administration rapidly induces epithe- analysis showed that several known estrogen target , includ- lial cell proliferation in the basal layer. The suprabasal cells are ing erbB growth factor ligands, were not induced by estrogen in no longer mitogenic but differentiate while moving up through the K5-Esr1KO mouse vagina. Organ culture experiments revealed the epithelium, resulting in keratinization of apical cells. The fully that the addition of erbB growth factor ligands, such as amphir- stratified and keratinized vaginal epithelium resembles the typi- egulin, could activate keratinized differentiation in the absence cal stratified and keratinized epidermis found in the skin and of epithelial ESR1. Thus, epithelial ESR1 integrates estrogen and other organs. growth factor signaling to mediate regulation of cell prolifera- The formation and maintenance of such epithelia relies on a tion in squamous differentiation, and our results provide new tightly balanced process of epithelial cell proliferation and squamous insights into estrogen-mediated homeostasis in female repro- differentiation. Classical tissue recombination experiments suggest ductive organs. that epithelial ESR1 in the vagina is dispensable for cell proliferation but is indispensable for squamous differentiation (5, 14). Until now, estrogen receptor | vagina | keratinization | amphiregulin | epithelium no mouse model for tissue-specific KO of Esr1 inthevaginahas been developed. In this study, we used genetic analysis of ESR1 strogens play important roles in vertebrate reproductive biology, function in the vagina by creating an epithelium-specific Esr1 KO to Ewith effects principally mediated through the nuclear estrogen provide new insights into the potential tissue-specific function of receptors (ESRs). ESRs are members of the su- ESR1. We found that estrogen can induce cell proliferation in the perfamily and typically up-regulate gene transcription upon ligand absence of epithelial ESR1, but that the epithelial cells showed binding. Two different genes encoding the two ESR subtypes, ESR1 severe defects in keratinocyte commitment. We also found that (formerly named ERα) and ESR2 (formerly named ERβ), have amphiregulin (Areg) is required downstream of epithelial ESR1 been identified from a variety of vertebrate species. ESR1 is the for terminal differentiation in the epithelium. Thus, epithelial predominant subtype regulating estrogen-mediated proliferation and ESR1 integrates estrogen and growth factor signaling to mediate differentiation of female reproductive organs. Administration of es- the switch between cell proliferation and squamous cell differ- trogens increases organ weights and promotes cell proliferation and entiation in the mouse vagina. differentiation, whereas such events were not evoked in the Esr1- – deficient mice (1 3). Significance Estrogen actions on the uterus and mammary gland have been extensively analyzed. During the first step of estrogen action, – Estrogen regulation of tissue homeostasis, particularly the reg- epithelial stromal interactions play a pivotal role in epithelial ulation of cell proliferation and differentiation in female re- cell proliferation. In vitro tissue recombination experiments with productive organs, is interesting both in its basic biology and in Esr1 knockout (KO)- and wild-type-derived epithelium and several disease models and cancer biology. Using an epithelial stroma revealed that the effects of estrogen on uterine and estrogen receptor 1 (Esr1) conditional knockout mouse model, mammary epithelial cell proliferation are mediated primarily via we showed that vaginal epithelial cells lacking ESR1 failed to stromally expressed ESR1 (4-6). Estrogen-induced growth factors activate growth factor signaling and remained undifferentiated secreted from the stroma subsequently promote epithelial cell and in a proliferative state throughout the epithelium. Thus, – proliferation (7 10). Experiments using epithelial cell-specific KO epithelial ESR1 mediates the switch between cell proliferation of Esr1 revealed that estrogen can induce proliferation of uterine and squamous cell differentiation, demonstrating unique roles epithelial cells despite the absence of epithelial ESR1 (11). In of ESR1 in the female reproductive tract. contrast, similar experiments using a mammary epithelium-specific Esr1 KO showed that ESR1 is required in the epithelial cells for Author contributions: S.M. designed research; S.M. performed research; S.M. analyzed mammary gland growth and ductal epithelial cell proliferation (12). data; and S.M. and T.I. wrote the paper. It is uncertain whether these differences result from different The authors declare no conflict of interest. requirements for epithelial ESR1 in the uterus versus the mam- This article is a PNAS Direct Submission. mary gland or whether they result from methodological differences 1To whom correspondence should be addressed. Email: [email protected]. in tissue recombination experiments (e.g., age of donor, This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. treatment protocol) (13). Irrespective of these differences, there 1073/pnas.1513550112/-/DCSupplemental.

12986–12991 | PNAS | October 20, 2015 | vol. 112 | no. 42 www.pnas.org/cgi/doi/10.1073/pnas.1513550112 Downloaded by guest on September 27, 2021 Results layers of the vaginal epithelium (Fig. 1 G and I). KRT14 was Phenotypes of Epithelial-Specific Esr1 KO Mice. ESR1 is expressed in basal and suprabasal cells but was absent in the apical expressed in both the vaginal epithelium and the stroma in the wild- cell layer in OVX and estrogen-treated K5-Esr1KO vaginae (Fig. 1 type mouse vagina (Fig. 1A). We generated a vaginal epithelial H and J). KRT8, a marker for undifferentiated cells, was expressed cell-specific Esr1 KO mouse model (K5-Esr1KO) by crossing in a few suprabasal cells in the control OVX mouse vagina, but its Esr1-floxed mice with K5-Cre transgenic lines that express Cre expression was augmented in theOVXK5-Esr1KOmice(Fig.1L). recombinase in the vaginal epithelial cells (15). ESR1 protein KRT8-positive cells were persistent in the apical layer of cells, even was detected in the stroma, but not in the epithelium, demon- after estrogen treatment in K5-EsR1KO mice (Fig. 1 M and N). strating the epithelium-specificity of Esr1 loss of function in the The stratified and squamous differentiation marker, KRT1, was K5-Esr1KO mouse vagina (Fig. 1B). We first tested whether expressed in the differentiating suprabasal cells upon estrogen- estrogen induced cell proliferation and/or differentiation in the treatment in the control mouse vagina. Intriguingly, it was not K5-Esr1KO mouse vagina, using OVX mice to avoid any con- detected in the K5-Esr1KO mice, although these animals did ex- founding effects of the hypothalamus-pituitary-gonadal axis and hibit stratified epithelium (Fig. 1 O and P). Expression of the to simplify analysis of estrogen effects. The vaginal epithelium of terminal differentiation marker, filaggrin (FLG), was absent in the both 8-wk-old OVX control and K5-Esr1KO mice was composed K5-Esr1KO mice (Fig. 1 Q and R). of two to three layers of cuboidal cells (Fig. 1 C and D). 17β- TRP63 is a crucial regulator of squamous differentiation and is Estradiol (E2) administration induced epithelial stratification no longer expressed once cells are committed to squamous epi- and full keratinization in control mice (Fig. 1E), whereas the K5- thelial lineages (18, 19). TRP63 was mainly expressed in the Esr1KO mice exhibited stratified epithelia, but not keratinized basal cells and in the two to three upper layers of cells adjacent differentiation (Fig. 1F). The epithelia of K5-Esr1KO mice to the basement membrane in the vagina of control mice (Fig. treated with E2 were thinner than those of controls (58.4 ± 8.7 μm 1S) (20). Intriguingly, TRP63 expression was augmented and vs. 95.6 ± 8.4 μm; P < 0.05). These phenotypes were not a expanded into the superficial layer of the K5-Esr1KO mouse result of an insufficiency of E2 exposure (see the following long- vagina (Fig. 1T). We infer from these results that epithelial cells term E2 exposure experiment). The K5-Esr1KO female mice are in the K5-Esr1KO mouse vagina exhibit impaired commitment infertile, probably because of the expression of Cre recombinase in toward squamous differentiation and, therefore, are maintained uterine epithelial cells (16). in an undifferentiated state. Next we used immunohistochemical staining to further charac- terize the phenotypes of the K5-Esr1KO mouse vagina. The tem- Loss of Epithelial ESR1 Impaired Cell Proliferation and Cell Death in poral and spatial expression profiles of specific reflected Vagina. We investigated the proliferation status of vaginal epithelial the differentiation status of epithelial cells (17). The vaginal epi- cells using BrdU incorporation. The proliferation indices were not thelial marker, keratin 14 (KRT14), was expressed throughout all significantly changed between control and K5-Esr1KO vaginae with or without E2 treatment (Fig. 2 A, B,andE). Although cell proliferation was restricted in the basal cells of E2-treated control mouse vagina, several BrdU-positive cells were detected in the

suprabasal cells in K5-Esr1KO mice (red arrowheads in Fig. 2B). BIOLOGY

Likewise, MKI67, another cell proliferation marker, was expressed DEVELOPMENTAL in the basal cells in control vaginal epithelium, but it was robustly detected in the upper layer cells of vaginal epithelium in K5- Esr1KO mice (Fig. 2 C and D). Thus, suprabasal cells in K5- Esr1KO mouse vagina show proliferative potency. Taken together, these data indicate that epithelial cells in the K5-Esr1KO mouse vagina remain in an undifferentiated state and retain proliferative potency, even after moving out from the basal cell layer. We next tested whether prolonged E2 exposure could enhance cell proliferation and result in a cancerous lesion. We found that the epithelium did not exhibit keratinization, although some abnormal epithelial pathology, including down-growth into the stroma, was observed in the K5-Esr1KO mouse vagina 2 mo after implantation of an E2 pellet (Fig. S1). In the absence of es- trogens, vaginal epithelial cells did not proliferate, and K5-Esr1KO mice exhibited atrophied vaginal epithelia, even in 2-y-old animals (Fig. S2). We also examined cell death in the K5-Esr1KO mouse vagina. Apoptotic indices in vaginal epithelial cells as measured by TUNEL staining did not differ between controls and K5-Esr1KO mice (Fig. 2 F–H). However, the apoptotic index was augmented in the stroma of estrogen-treated K5-Esr1KO mice compared Fig. 1. Effects of epithelial cell-specific ablation of Esr1 on the mouse vagina. with controls (Fig. 2 F–H), suggesting that epithelial ESR1 ESR1 protein expression was evaluated by immunohistochemistry in control (A) contributes to maintaining stromal cell survival. and K5-Esr1KO mice treated with E2 (B), indicating successful deletion of Esr1 specifically in the epithelial cells in the K5-Esr1KO mice. The vaginal epithelium Evaluation of E2-Induced Gene Expression in K5-Esr1KO Mouse Vagina. of 8-wk-old OVX control (C) and K5-Esr1 (D)mice.E2 treatment induces vaginal We next evaluated expression of receptor (PGR) epithelial stratification and keratinized differentiation in the control (E), but and CCAAT/enhancer-binding protein β (CEBPB) (14). Both fails to such differentiation in the vaginal epithelium of K5-Esr1KO mice (F). genes were induced by E administration in the control mouse Expression pattern of cell differentiation markers in vaginal epithelium of OVX 2 – vagina, and their were localized in the epithelium (G, H, K,andL)andE2-treated (I, J, M T) mice. Immunohistochemical staining – for KRT14 (G–J), KRT8 (K–N), KRT1 (O and P), FLG (Q and R), and TRP63 (S and (Fig. 3 A I). Expression of these genes was not induced by E2 T). In K5-Esr1KO mice, differentiating cell-specific markers KRT1 and FLG are not administration in the K5-Esr1KO mouse vagina. It has been

expressed, even after E2 treatment (C–F). (Scale bar, 100 μm.) reported that PGR-mediated signaling inhibits vaginal epithelial

Miyagawa and Iguchi PNAS | October 20, 2015 | vol. 112 | no. 42 | 12987 Downloaded by guest on September 27, 2021 (Fig. 5 C and D). These data indicate that CEBPB is likely to be a downstream target of erbB signaling, rather than a direct target of epithelial ESR1. Organ-cultured vaginae from wild-type mice treated with E2 and anti-AREG antibody showed epithelial keratinization and ex- pressed KRT1 and FLG (Fig. S4). These results suggest that the amphiregulin effects might be redundant. We further examined the selectivity of erbB ligands for vaginal epithelial cell differentiation, including heparin-binding EGF-like protein (HBEGF) and trans- forming growth factor α (TGFA). We also tested insulin-like growth factor-I (IGF1), which is assumed to be a stromal factor that activates epithelial cell proliferation (10, 26). HBEGF induced expression of KRT1, but not FLG (Fig. S4). TGFA had a minimal effect on KRT1 and FLG expression. IGF1 did not induce KRT1 or FLG expression, and the epithelium was atrophied (Fig. S4). Thus, although there is some redundancy among erbB ligands, AREG is the most active inducer of squamous cell differentiation in the mouse vaginal epithelium. Discussion Organ growth, development, and function are regulated by complex Fig. 2. Ectopic cell proliferation in the epithelium and cell death in the stroma of interactions among cells and tissues. Female reproductive organs the K5-Esr1KO vagina. BrdU and MKI67 immunostaining in control (A and C)and K5-Esr1KO (B and D) mouse vagina. Note BrdU-positive cells in the upper layer of are excellent models to analyze such interactions because epithelial the K5-Esr1KO mice (red arrowheads in B). BrdU-labeling indices were not altered cell proliferation and differentiation can be completely controlled

between control and K5-Esr1KO in the OVX and E2-treated group (E). TUNEL by estrogen. Classical tissue recombination experiments have sug- assay indicates loss of ESR1 has not influenced cell apoptosis in the vaginal epi- gested that epithelial cell proliferation in female reproductive organs thelium, but increase in vaginal stromal cell death in the E2-treated K5-Esr1KO (e.g., uterus, vagina, and mammary gland) is mediated by stromal mice (F–H). Black arrowheads in G indicate apoptotic cells in the stroma. Dotted ESR1 in a paracrine manner (4–6). In contrast, genetic studies in μ lines indicate the basal layer of the epithelium. (Scale bar, 100 m.) mouse models have shown that epithelial ESR1 is required for cell proliferation in the mammary gland, but not in the uterus (11, 12). cell proliferation (21) and that CEBPB is indispensable for epi- dermal cell differentiation (22). Therefore, the failure of E2 to induce expression of these genes in the K5-Esr1KO mouse vagina may be associated with the observed phenotypes. To further analyze genes involved in vaginal epithelial cell dif- ferentiation, we conducted DNA microarray analysis of vaginae from control and K5-Esr1KO mice treated with E2 (Dataset S1). We found that several secreted growth factors were differentially expressed (Fig. S3). Of those, we focused on the erbB ligand, amphiregulin (Areg), because erbB ligands have been implicated as effectors of ESR1 in mammary gland development (23). Areg is also involved in keratinocyte growth and is associated with psori- asis, a hyperproliferative skin disorder (24, 25). Expression of Areg was increased after E2 administration in the control mouse vagina, but its expression was persistently low in the K5-Esr1KO vagina (Fig. 3J). AREG was exclusively expressed in the vaginal epithelium in controls, but expression was not observed in the K5-Esr1KO mice (Fig. 3 K and L), suggesting an autocrine mechanism for ep- ithelial ESR1 signaling.

Areg Is a Mediator of Vaginal Epithelial Cell Differentiation. We next evaluated the effects of AREG on vaginal epithelial cell differ- entiation, using an organ culture system. Epithelial cells exhibited keratinized cell morphology and expressed KRT1 and FLG in organ-cultured vaginae from control mice treated with E2 (Fig. 4 A–C). In contrast, neither KRT1 nor FLG was expressed in vagi- naefromK5-Esr1KOmicetreatedwithE2 (Fig. 4 D–F). However, the addition of AREG and E2 induced KRT1 and FLG expression, together with keratinized morphology (Fig. 4 G–I). In the absence of E2 supplementation, AREG alone could not induce KRT1 or FLG (Fig. 4 J–L), suggesting AREG could not bypass the whole Fig. 3. Expression patterns of cell differentiation markers in the mouse vagina. Immunohistochemistry of PGR (A–D) and CEBPB (E–H). Both proteins effects of E2 in the mouse vagina. To investigate potential epistatic relationships among Areg, Pgr,andCebpb, immunohistochemistry are detected in the suprabasal layer of differentiating vaginal epithelial cells of the control mice treated with E2 (C and G), but not in the K5-Esr1KO mice was performed using organ-cultured vaginae supplemented with E2 (B and H). Expression of Pgr, Cebpb, and Areg mRNAs failed to increase in

and AREG. Both PGR and CEBPB were expressed in the control the vagina of E2-treated K5-Esr1KO mice (I and J). AREG protein is detected mousevagina(Fig.5A and B). PGR was not expressed, although in the vaginal epithelium in the control mice treated with E2 (K), but not in CEBPB was induced, in the epithelial cells of K5-Esr1KO vaginae the K5-Esr1KO mice (L). (Scale bar, 100 μm.)

12988 | www.pnas.org/cgi/doi/10.1073/pnas.1513550112 Miyagawa and Iguchi Downloaded by guest on September 27, 2021 with genetic mouse models in both organs (current study and ref. 11). In general, progesterone-PGR signaling acts antagonistically to E2-induced effects (21, 30). Progesterone signaling that would ordinarily inhibit E2-induced epithelial cell proliferation was im- paired in the of the Esr1KO mouse (27). Thus, loss of epithelial PGR expression might contribute to dysregula- tion of the proliferative response to estrogen in the K5-Esr1KO mouse vagina. CEBPB is another estrogen-regulated in female reproductive organs (31, 32). CEBPB is a key regulator of proliferation and/or differentiation in multiple tissues, including squamous epithelia. Epidermal ablation of CEBPB leads to increased basal keratinocyte proliferation and severe defects in keratinocyte commitment and differentiation, despite some redundancy with CCAAT/enhancer-binding protein α (Cebpa) (22). CEBPB also plays a crucial role in restricting TRP63 expression to basal keratinocytes (22). These phenotypes coincide with the results shown here in the K5-Esr1KO mouse vagina and support an important role for CEBPB in squamous differentiation in the vagina. ErbB signaling has long been implicated downstream of steroid hormone signaling in female reproductive organs (8, 9, 33). In the current study, we found that the erbB ligand, Fig. 4. AREG as a potential factor for vaginal epithelial cell differentiation. AREG, is expressed specifically in the epithelium, dependent HE staining (A, D, G, and J) and immunohistochemistry for KRT1 (B, E, H, and on epithelial ESR1 expression. Organ culture experiments re- K) and FLG (C, F, I, and L) in organ-cultured vagina. Organ-cultured vaginae vealed that AREG can compensate for the loss of epithelial from control mice treated with E2 express KRT1 and FLG (B and C), but the ESR1 on squamous differentiation with respect to KRT1, FLG, vaginae of K5-Esr1KO mice do not (E and F). Note that addition of AREG and CEBPB expression. Furthermore, AREG is the most active leads to KRT1 and FLG expression (H and I). (Insets) Counterstaining with μ of the erbB ligands and growth factors we examined in rescuing Heckest. (Scale bar, 100 m.) the effects of Esr1 loss of function. Therefore, AREG is likely to be a key intrinsic factor required for squamous differentiation in Therefore, to clarify and extend those observations, we con- the mouse vagina. This indirect signaling mechanism, mediated ducted Esr1KO in the mouse vagina and demonstrated that through secreted growth factors, enables the amplification and vaginal epithelial cells can proliferate in the absence of epithelial systematic coordination of estrogen stimulation within the target cells/tissues. In contrast, neither AREG nor other growth factors

ESR1 in vivo. BIOLOGY The vaginal epithelium is a stratified keratinized squamous type examined could alone induce epithelial cell growth and squa- DEVELOPMENTAL similar to that in the epidermis. Therefore, further cell and tissue mous differentiation. Hence, we propose that additional factors interactions are required for coordinated and fine-tuned mor- downstream of Esr1 in the epithelium and/or stroma are neces- phogenetic events. Stratified squamous epithelia are maintained sary for full activation of estrogen effects in the vagina and the by proliferation of basal keratinocytes and their subsequent co- coordinated crosstalk between growth factors and that is ordinated cell cycle exit, migration into the suprabasal layers, and essential for female reproductive organ homeostasis. Intriguingly, the terminal differentiation. In wild-type mice, suprabasal cells exit the stromal cells exhibit increased cell death when epithelial ESR1 is cell cycle and express the differentiation marker KRT1 and the lost. We hypothesize that secreted factors derived from the epithe- terminal keratinized cell marker FLG in the upper layers. How- lium act on the stroma to protect against stromal cell death and are ever, in the absence of epithelial ESR1, differentiation markers currently investigating the existence of these factors. The number of were not expressed, in accord with previous tissue recombination experiments (5, 14). We also found that vaginal epithelial cells in the K5-Esr1KO mouse continue to proliferate, even after basal layers are removed, indicating that the suprabasal cells remain undifferentiated and proliferative while failing to differentiate into keratinocytes. Therefore, we infer that epithelial ESR1 is in- dispensable for keratinocyte commitment and maintenance of epithelial homeostasis by controlling cell differentiation. This is in contrast to the role of epithelial ESR1 in the uterus, where epi- thelial ESR1 is involved in the maintenance of epithelial cell survival by preventing excessive apoptosis, likely as a result of inactivation of cell cycle progression-related genes (11, 27). Sub- sequently, loss of ESR1 results in enhanced apoptosis in the uterine epithelium (11); such apoptosis was not observed in the vaginal epithelium of K5-Esr1KO mice. These results suggest that loss of ESR1 in the vaginal epithe- lium impaired the action of E2-mediated secondary factors me- diating squamous differentiation. We investigated known estrogen target genes such as Pgr and Cebpb in the epithelia. Pgr regulation Fig. 5. AREG induces CEBPB in vagina of K5-Esr1KO mouse. Immunohis- tochemistry of PGR (A and C) and CEBPB (B and D) in organ-cultured va- by estrogens has been well studied in the female reproductive gina. Organ-cultured vaginae treated with E2 and AREG from control mice tracts, using tissue recombination experiments that showed that express PGR and CEBPB (A and B). In the K5-Esr1KO mouse vagina, addition Pgr expression is controlled by stromal ESR1 in the uterus and of AREG fails to express PGR (C) but rescues CEBPB expression (D). (Scale epithelial ESR1 in the vagina (28, 29). These results are consistent bar, 100 μm.)

Miyagawa and Iguchi PNAS | October 20, 2015 | vol. 112 | no. 42 | 12989 Downloaded by guest on September 27, 2021 This timing is sufficient to allow induction of a stratified and fully keratinized epithelium in the mouse vagina. For a long treatment, OVX mice were

implantedwithE2 pellets (0.01 mg per pellet, 2 mo; Innovative Research of America) into s.c. tissue for 2 mo. All procedures and protocols were ap- proved by the institutional animal care and use committee at the National Institute for Basic Biology.

Histology and Immunohistochemistry. Hematoxylin/eosin staining was performed by a standard procedure. The significance of differences in the thickness of epithelia between control and K5-Esr1KO mice was assessed by Welch’s t test. For immu- nohistochemistry, paraformaldehyde-fixed, paraffin-embedded sections were in- cubated with the following primary antibodies: ESR1, TRP63, PGR, CEBPB (Santa Cruz), KRT8 (Progen), KRT1, KRT14, FLG (Covance), MKI67 (Thermo Fisher Scien- tific), and AREG (R&D Systems). The sections were stained with the Vectastain ABC Kit (Vector Laboratories). Immunofluorescence analysis was performed with Alexa Fluor protein-conjugated secondary antibodies (Life Technologies) and counter- stained with Hoechst 33342 (Sigma). For BrdU immunostaining, mice were injected with BrdU (Sigma) at 100 mg/kg body weight. One hour after the injection, tissues were collected. BrdU-incorporated cells were detected with anti-BrdU antibody (1:20, Roche). Fig. 6. A possible scheme depicting estrogen-induced cell proliferation and TUNEL assay for the detection of apoptotic cells was performed with the in differentiation and its regulation of homeostasis in mouse vagina. situ apoptosis detection kit (Roche). Statistical analyses were performed using Student’s t test or Welch’s t test, followed by F-test; differences with P < 0.05 were considered significant. Error bars represent the SEM. More than four apoptotic cells was not altered by supplementation of growth factors (immunohistochemistry) or six (BrdU and TUNEL) animals were analyzed; examined in the current organ culture study. representative pictures are shown. Estrogen actions need to be tightly regulated, and it is conceivable that dysregulation of estrogen signaling results in Quantitative RT-PCR. Total RNA (2 μg), isolated from each group using an reproductive disorders, including tumorigenesis. Squamous cell RNeasy kit (Qiagen), was used in RT-PCR reactions carried out with SuperScript III carcinoma is the most common invasive malignancy of the , reverse transcriptase and SYBR Green Master Mix (Life Technologies), according to manufacturer’s instructions. PCR conditions and sequences of some primer vagina, and (34). Although much remains to be learned sets are given in a previous report (33). Others used are Pgr, TAT GAG AAC CCT about the underlying molecular mechanisms, loss of ESR1 has TGA CGG TGT TG, CAG GGC CTG GCT CTC GTT A; Cebpb, CGG GTT TCG GGA CTT been associated with squamous cell carcinoma progression and GAT GCA AT, CTA GAC AGT TAC ACG TGT GTT GCG. Relative RNA equivalents invasion (35). Although we showed dysregulated epithelial cell for each sample were obtained by standardization of ribosomal protein L8 proliferation and differentiation in the K5-Esr1KO mouse vagina levels. More than three pools of samples per group were run in triplicate to and found that prolonged estrogen exposure induced abnormal determine sample reproducibility. Error bars represent the SE, with all values epithelial invagination into the stroma, cancerous lesions and represented as fold change compared with the control treatment group nor- malized to an average of 1.0. Statistical analysis was performed by ANOVA indications of metastatic phenotypes (e.g., effects on basement ’ < membrane) were not observed. This suggested that loss of the ep- followed by Dunnett s test; differences with P 0.05 were considered significant. Error bars represent the SEM. ithelial ESR1 signal alone is not sufficient to cause squamous cell carcinoma. Therefore, other factors are likely to be involved in DNA Microarray. The quality of Cyanine3-labeled cRNA was analyzed using the carcinogenesis and invasive phenotypes, and future studies are Agilent 2100 Bioanalyzer (Agilent). Cyanine3-labeled cRNA was prepared using needed to further clarify tissue-specific roles of ESR1 for carcino- the Quick Amp Labeling Kit and One-color RNA Spike-in kit (Agilent) and purified genesis, invasion, and metastasis. A different mouse background using the RNeasy Micro Kit (Qiagen) following the manufacturer’s protocol. The should also be tested, because the current mouse background quality of cRNA was analyzed using the Agilent 2100 Bioanalyzer (Agilent). (C57BL6) is genetically resistant to such cancerous lesions from cRNA (1.65 μg) was hybridized to the 4 × 44K mouse gene expression microarray estrogen treatment. (G4122F), using standard Agilent protocols. After 17 h incubation with rotation, the microarrays were washed with Gene Expression Wash Buffer Kit (Agilent) In summary, our mouse model demonstrated that the vaginal ’ epithelium undergoes cell proliferation mediated by the stromal according to the manufacturer s protocols. DNA microarrays were scanned using a GenePix 4000B scanner (Molecular Devices) at 5 μm resolution. The signal ESR1, whereas differentiation is promoted by epithelial ESR1 intensity of the spots was digitized using the microarray imager software (Fig. 6). Loss of epithelial ESR1 leads to failure of commitment (Combimatrix Molecular Diagnostics). Gene expression and statistical analyses of cells to keratinized differentiation, resulting in aberrant epithelial were performed using the Subio Platform (Subio Inc). Data files generated by cell proliferation under stimulation by stromal ESR1. Therefore, the Agilent Feature Extraction Software were imported into Subio Platform. The ESR1 is required in the vaginal epithelium for appropriate response digitized raw data were normalized by the mean value of all spots of signal to estrogens and tissue homeostasis. It is intriguing that ESR1 intensity in each array. Signal intensities (gProcessedSignal) were normalized by has unique roles and differentially mediates estrogen action in the 75th percentile and transformed into log ratios (base 2). We merged duplicates of each experimental condition and applied statistical analysis on the averages. epithelia of the mammary gland, uterus, and vagina. Overall, an < improved understanding of estrogen action at the tissue level will Subsequently, these data were assessed by t test (P 0.05) to identify differ- entially expressed genes between treatment groups. shed light on the mechanisms of estrogen-mediated homeostasis and underlying disorders in female reproductive organs such as Organ Culture. Eight volumes of Cellmatrix type I-A (Nitta Gelatin) were mixed endometriosis, endometrial hyperplasia, or cancer. with 1 volume 10× Dulbecco’s Modified Eagle’s Medium/Nutrient Mixture Ham’s F-12 (DMEM/F12; Sigma) and 1 volume 200 mM Hepes buffer con- Materials and Methods taining 262 mM NaHCO3 and 0.05 N NaOH was added to the mixture. This Mouse and Treatment. C57BL/6J (CLEA), K5-Cre(36),Esr1-null, and Esr1-floxed cold gelatin mixture (200 μL) was poured into inner millicell cell culture in- mice (1) were maintained under 12 h light/12 h dark at 23–25 °C and fed labo- serts (Merck) placed into the well of a 24-well plate and allowed to gel at ratory chow (CA-1; CLEA) and tap water ad libitum. To obtain vaginal epithelial 37 °C. Vaginae of mice aged 3 wk were cut and placed into Hank’s Balanced + − cell-specific Esr1KO mice (K5Cre/+;Esr1flox/-), K5Cre/+;Esr1 / male were crossed Salt Solution (Life Technologies). Tissues were washed three times in Hank’s + with Esr1flox/flox female mice. For control mice, Cre-negative-Esr1flox/ siblings were Balanced Salt Solution and mixed with fresh 200-μL cold gelation mixture. used. In most experiments, mice were OVX at 6 wk of age and killed at 8 wk of Tissues and gelation mixture were overlaid onto a base of gelled collagen in

age. For examining effects of estrogen, a single daily injection of 0.1 μgE2 (Sigma) each cell culture insert and allowed to gel at 37 °C. Subsequently, 200 μL was given to OVX mice for 3 d; the mice were killed 24 h after the last injection. DMEM/F12 supplemented with 20% (vol/vol) charcoal/dextran-treated FBS

12990 | www.pnas.org/cgi/doi/10.1073/pnas.1513550112 Miyagawa and Iguchi Downloaded by guest on September 27, 2021 (HyClone) was added to each well, and tissues were cultured at 37 °C in a Biology) for providing K5-Cre and ERα-floxed mice, respectively. We also

humidified, 5% CO2/air atmosphere for 3 d. Recombinant AREG, HBEGF, TGFΑ thank Dr. I. Hirakawa and Dr. H. Miyakawa (National Institute for Basic (R&D systems), IGF1 (JRH Biosciences; 0.5 μg/μL), and anti-AREG antibody (5 μg/mL) Biology) and Dr. T. Nakajima and Dr. T. Sato (Yokohama City University) for critical advice on the DNA microarray and organ culture. We are grateful to were added to the medium from day 0 of culture. Tissues for each group were Dr. B. Blumberg (University of California at Irvine) for his critical readings of collected from at least 4 mice. the manuscript. This study was supported by a Grant-in-Aid for Scientific Research B from the Ministry of Education, Culture, Sports, Science and ACKNOWLEDGMENTS. We thank Dr. J. Takeda (Osaka University) and Technology of Japan and a Health Sciences Research Grant from the Min- Pierre Chambon (Institute for Genetics and Cellular and Molecular istry of Health, Labour and Welfare, Japan.

1. Dupont S, et al. (2000) Effect of single and compound knockouts of estrogen re- 18. Yang A, et al. (1999) p63 is essential for regenerative proliferation in limb, cranio- ceptors alpha (ERalpha) and beta (ERbeta) on mouse reproductive phenotypes. facial and epithelial development. Nature 398(6729):714–718. Development 127(19):4277–4291. 19. Mills AA, et al. (1999) p63 is a homologue required for limb and epidermal 2. Couse JF, Korach KS (1999) Estrogen receptor null mice: What have we learned and morphogenesis. Nature 398(6729):708–713. where will they lead us? Endocr Rev 20(3):358–417. 20. Kurita T, Mills AA, Cunha GR (2004) Roles of p63 in the -induced 3. Lubahn DB, et al. (1993) Alteration of reproductive function but not prenatal sexual cervicovaginal adenosis. Development 131(7):1639–1649. development after insertional disruption of the mouse estrogen receptor gene. Proc 21. Yoo YA, et al. (2013) Progesterone signaling inhibits cervical carcinogenesis in mice. Natl Acad Sci USA 90(23):11162–11166. Am J Pathol 183(5):1679–1687. 4. Cunha GR, et al. (1997) Elucidation of a role for stromal steroid hormone receptors in 22. Lopez RG, et al. (2009) C/EBPalpha and beta couple interfollicular keratinocyte pro- mammary gland growth and development using tissue recombinants. J Mammary liferation arrest to commitment and terminal differentiation. Nat Cell Biol 11(10): Gland Biol Neoplasia 2(4):393–402. 1181–1190. 5. Buchanan DL, et al. (1998) Role of stromal and epithelial estrogen receptors in vaginal 23. Ciarloni L, Mallepell S, Brisken C (2007) Amphiregulin is an essential mediator of es- epithelial proliferation, stratification, and cornification. Endocrinology 139(10): trogen receptor alpha function in mammary gland development. Proc Natl Acad Sci 4345–4352. USA 104(13):5455–5460. 6. Cooke PS, et al. (1997) Stromal estrogen receptors mediate mitogenic effects of es- 24. Stoll SW, Johnson JL, Li Y, Rittié L, Elder JT (2010) Amphiregulin carboxy-terminal tradiol on uterine epithelium. Proc Natl Acad Sci USA 94(12):6535–6540. domain is required for autocrine keratinocyte growth. J Invest Dermatol 130(8): 7. Nelson KG, Sakai Y, Eitzman B, Steed T, McLachlan J (1994) Exposure to di- 2031–2040. ethylstilbestrol during a critical developmental period of the mouse reproductive 25. Cook PW, et al. (1992) Amphiregulin messenger RNA is elevated in psoriatic epidermis tract leads to persistent induction of two estrogen-regulated genes. Cell Growth and gastrointestinal carcinomas. Cancer Res 52(11):3224–3227. Differ 5(6):595–606. 26. Miyagawa S, et al. (2004) Persistent gene expression in mouse vagina exposed neo- 8. Ignar-Trowbridge DM, et al. (1992) Coupling of dual signaling pathways: epidermal natally to diethylstilbestrol. J Mol Endocrinol 32(3):663–677. growth factor action involves the estrogen receptor. Proc Natl Acad Sci USA 89(10): 27. Winuthayanon W, Hewitt SC, Korach KS (2014) Uterine epithelial cell estrogen re- 4658–4662. ceptor alpha-dependent and -independent genomic profiles that underlie estrogen 9. Nelson KG, Takahashi T, Bossert NL, Walmer DK, McLachlan JA (1991) Epidermal responses in mice. Biol Reprod 91(5):110. growth factor replaces estrogen in the stimulation of female genital-tract growth and 28. Kurita T, Lee KJ, Cooke PS, Lydon JP, Cunha GR (2000) Paracrine regulation of epi- differentiation. Proc Natl Acad Sci USA 88(1):21–25. thelial and lactoferrin by progesterone in the mouse uterus. 10. Ghahary A, Chakrabarti S, Murphy LJ (1990) Localization of the sites of synthesis and Biol Reprod 62(4):831–838. action of insulin-like growth factor-I in the rat uterus. Mol Endocrinol 4(2):191–195. 29. Kurita T, et al. (2000) Paracrine regulation of epithelial progesterone receptor by 11. Winuthayanon W, Hewitt SC, Orvis GD, Behringer RR, Korach KS (2010) Uterine epi- estradiol in the mouse female reproductive tract. Biol Reprod 62(4):821–830. thelial estrogen receptor α is dispensable for proliferation but essential for complete 30. Li Q, et al. (2011) The antiproliferative action of progesterone in uterine epithelium is biological and biochemical responses. Proc Natl Acad Sci USA 107(45):19272–19277. mediated by Hand2. Science 331(6019):912–916. 12. Feng Y, Manka D, Wagner KU, Khan SA (2007) Estrogen receptor-alpha expression in 31. Mantena SR, et al. (2006) C/EBPbeta is a critical mediator of steroid hormone-regu- BIOLOGY the mammary epithelium is required for ductal and alveolar morphogenesis in mice. lated cell proliferation and differentiation in the uterine epithelium and stroma. Proc

Proc Natl Acad Sci USA 104(37):14718–14723. Natl Acad Sci USA 103(6):1870–1875. DEVELOPMENTAL 13. Mueller SO, Clark JA, Myers PH, Korach KS (2002) Mammary gland development in 32. Robinson GW, Johnson PF, Hennighausen L, Sterneck E (1998) The C/EBPbeta tran- adult mice requires epithelial and stromal . Endocrinology scription factor regulates epithelial cell proliferation and differentiation in the 143(6):2357–2365. mammary gland. Genes Dev 12(12):1907–1916. 14. Kurita T, Cooke PS, Cunha GR (2001) Epithelial-stromal tissue interaction in para- 33. Miyagawa S, Katsu Y, Watanabe H, Iguchi T (2004) Estrogen-independent activation mesonephric (Müllerian) epithelial differentiation. Dev Biol 240(1):194–211. of erbBs signaling and estrogen receptor alpha in the mouse vagina exposed neo- 15. Miyagawa S, Sato M, Sudo T, Yamada G, Iguchi T (2015) Unique roles of estrogen- natally to diethylstilbestrol. Oncogene 23(2):340–349. dependent Pten control in epithelial cell homeostasis of mouse vagina. Oncogene 34. Platz CE, Benda JA (1995) Female genital tract cancer. Cancer 75(1, Suppl):270–294. 34(8):1035–1043. 35. Zhai Y, et al. (2010) Loss of estrogen receptor 1 enhances cervical cancer invasion. Am 16. Villacorte M, et al. (2013) β-Catenin signaling regulates Foxa2 expression during en- J Pathol 177(2):884–895. dometrial hyperplasia formation. Oncogene 32(29):3477–3482. 36. Tarutani M, et al. (1997) Tissue-specific knockout of the mouse Pig-a gene reveals 17. Gimenez-Conti IB, et al. (1994) Expression of keratins in mouse vaginal epithelium. important roles for GPI-anchored proteins in skin development. Proc Natl Acad Differentiation 56(3):143–151. Sci USA 94(14):7400–7405.

Miyagawa and Iguchi PNAS | October 20, 2015 | vol. 112 | no. 42 | 12991 Downloaded by guest on September 27, 2021