Estrogen-mediated actions in female reproductive organs are tightly regulated, mainly through estrogen receptor 1 (ESR1). The mouse vaginal epithelium cyclically exhibits cell proliferation and differentiation in response to estrogen and provides a unique model for analyzing the homeostasis of stratified squamous epithelia. To address the role of ESR1-mediated tissue events during homeostasis, we analyzed mice with a vaginal epitheliumspecific knockout of Esr1 driven by keratin 5-Cre (K5-Esr1KO). We show here that loss of epithelial ESR1 in the vagina resulted in aberrant epithelial cell proliferation in the suprabasal cell layers and led to failure of keratinized differentiation. Gene expression analysis showed that several known estrogen target genes, including erbB growth factor ligands, were not induced by estrogen in the K5-Esr1KO mouse vagina. Organ culture experiments revealed that the addition of erbB growth factor ligands, such as amphiregulin, could activate keratinized differentiation in the absence of epithelial ESR1. Thus, epithelial ESR1 integrates estrogen and growth factor signaling to mediate regulation of cell proliferation in squamous differentiation, and our results provide new insights into estrogen-mediated homeostasis in female reproductive organs.estrogen receptor | vagina | keratinization | amphiregulin | epithelium E strogens play important roles in vertebrate reproductive biology, with effects principally mediated through the nuclear estrogen receptors (ESRs). ESRs are members of the nuclear receptor superfamily and typically up-regulate gene transcription upon ligand binding. Two different genes encoding the two ESR subtypes, ESR1 (formerly named ERα) and ESR2 (formerly named ERβ), have been identified from a variety of vertebrate species. ESR1 is the predominant subtype regulating estrogen-mediated proliferation and differentiation of female reproductive organs. Administration of estrogens increases organ weights and promotes cell proliferation and differentiation, whereas such events were not evoked in the Esr1-deficient mice (1-3).Estrogen actions on the uterus and mammary gland have been extensively analyzed. During the first step of estrogen action, epithelial-stromal interactions play a pivotal role in epithelial cell proliferation. In vitro tissue recombination experiments with Esr1 knockout (KO)-and wild-type-derived epithelium and stroma revealed that the effects of estrogen on uterine and mammary epithelial cell proliferation are mediated primarily via stromally expressed ESR1 (4-6). Estrogen-induced growth factors secreted from the stroma subsequently promote epithelial cell proliferation (7-10). Experiments using epithelial cell-specific KO of Esr1 revealed that estrogen can induce proliferation of uterine epithelial cells despite the absence of epithelial ESR1 (11). In contrast, similar experiments using a mammary epithelium-specific Esr1 KO showed that ESR1 is required in the epithelial cells for mammary gland growth and ductal epithelial cell proliferation (12). It is u...