Estrogen Receptor Null Mice - Endocrine Reviews

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June, 1999 ESTROGEN RECEPTOR NULL MICE 371 supported by numerous in vitro experiments demonstrating ligand-independent activation of the nuclear signaling pathway of the ER�, possibly via altering the phosphorylation pattern of the ER� (reviewed in Ref. 55). The culmination of these and several other studies has led to the proposed model in which the mitogenic actions of estradiol in the rodent uterus appear to be at least partially mediated by EGF; however, in turn the mitogenic effects of EGF require the presence of ER�. Therefore, the �ERKO female provides an excellent in vivo model to study this cross-talk between the ER�- and EGFsignaling systems in the uterus. The uteri of �ERKO females possess wild-type levels of functional EGF and EGF-R (170). Nonetheless, the mitogenic actions and induction of estrogen-responsive genes elicited by EGF in the wild-type uterus have been ablated in the �ERKO, confirming the interaction of these two signaling systems (170). However, not all EGF responses are lacking in the uteri of �ERKO females, as this same study demonstrated that the mechanisms for EGFmediated up-regulation of the c-fos gene remained intact (170). These studies have thereby confirmed the need for functional ER� for the mitogenic actions of EGF in the uterus. Cunha et al. has extended the use of the �ERKO mouse to investigate the intersecting roles of ER�-mediated estrogen stimulation and growth factors in the uterus through a series of tissue recombination experiments. The observation of estrogenic effects in wild-type uterine epithelial cells that are apparently lacking ER� has prompted numerous investigations to illustrate a role for paracrine factors secreted by the underlying ER�-positive stromal compartment, and thereby mediating the epithelial response (143). These studies have been advanced by methods that provide for the delicate construction of tissue recombinants, in which uterine stoma and epithelium are enzymatically disassociated and recombined with similar tissue from uteri from animals of different treatments or models to ultimately regenerate a chimeric stromal-epithelial unit (reviewed in Ref. 143). These tissue recombinants are implanted under the kidney capsule of ovariectomized nude mice, which are then acutely treated with estrogen agonists or antagonists. Later removal of the recombinant grafts allows for the evaluation of certain end points of estrogen action in each portion of the recombinant. Cooke et al. (171) described experiments in which wild-type (ER��) uterine stroma were recombined with �ERKO (ER��) uterine epithelium and vice versa. The results of these studies illustrate that proliferation of the epithelial portion of the recombinant was possible only when ER�� stroma were present and did not require ER� in the epithelium (171). Interestingly, similar recombinant experiments using tissue from the EGF-R knockout mice illustrated that the estrogensignaling pathways required for stimulation of the stroma and subsequent induction of epithelial growth are intact in the absence of EGF-R (167). Aside from the proliferative effects of estradiol, previous studies suggested that estrogen stimulation of secretory products from uterine epithelium, e.g., lactoferrin, is directly mediated by the epithelial ER� (140, 162). However, Buchanan et al. (172) recently employed tissue recombinants similar to those described to demonstrate that both stomal and epithelial ER� are required for estrogen induction of the uterine epithelial secretory products, lactoferrin and complement component C3. Therefore, estrogen-induced proliferation of the uterine epithelium requires the presence of ER� in the stromal compartment only, whereas induction of certain epithelial secretory products is dependent on the presence of ER� in both uterine compartments. 3. Maintenance of selective estrogen actions in the �ERKO uterus. A distinct advantage of null receptor models, whether naturally existing or experimentally generated via molecular methodologies, is their use as an in vivo tool for discerning alternate pathways of hormone action. Recent studies by the Lubahn laboratory have indicated the preservation of a distinct estrogen-signaling pathway in the �ERKO uterus (173, 174). Das et al. (173) reported that two consecutive treatments (over a period of 12 h) with the catecholestrogen, 4-hydroxyestradiol (4-OH-E 2)at10�g/kg body weight resulted in significant increases in water imbibition in the uteri of ovariectomized �ERKO mice. Induction of lactoferrin mRNA in the uterine epithelium of wild-type mice was 97and 85-fold after treatment with 10 �g/kg body weight estradiol or 4-OH-E 2, respectively (173). In contrast, only the 4-OH-E 2 was active in the �ERKO uterus, resulting in a 60-fold increase in lactoferrin mRNA levels compared with a 1.4-fold induction by estradiol (173). A similar, yet more modest, response was reported in the wild-type and �ERKO uteri after treatment with the xenoestrogens, kepone (15 mg/kg body weight) (173) and methoxychlor (15 mg/kg body weight) (174). Most interesting was the lack of inhibition of this response by the pure estrogen antagonist, ICI-182,780, in both the wild-type and �ERKO mice, indicating the possibility of a non-ER�-mediated signaling pathway for certain compounds exhibiting estrogenic activity. Additionally, the nature of the timing and type of lactoferrin response elicited by the 4-OH-E 2 and xenoestrogens in the �ERKO is quite distinct from that of the original descriptions by Teng et al. (175) concerning estrogen regulation of this gene in the mouse uterus. Given the knowledge of the low-to-absent expression of ER� in the uterus and the ability of the ICI compounds to antagonize ER� signaling in vitro, it is not likely that this receptor is involved in this phenomenon. The catecholestrogens are naturally synthesized and proposed to play a role in steroid regulation of the hypothalamus and pituitary (176, 177), ovarian function (178), and embryo implantation (179). Furthermore, the discovery of local synthesis of these estrogens in mammary tissue has led to implications of their involvement in breast cancer (180). Therefore, further investigation into the alternate mechanisms by which these compounds may activate nuclear processes is needed. 4. Maintenance of progesterone action in the �ERKO uterus. Like estradiol, ovarian derived progesterone, is an integral steroid hormone in the physiology and function of the uterus. The PR has been localized to cells composing all three anatomical compartments of the uterus and exhibits varied levels in each during the stages of the estrous cycle (reviewed in Ref. 181). The PR also exists in two forms, PR A and PR B, which differ only in the length of the N� terminus. In contrast to the ER, PR A and PR B are encoded by a single gene but transcribed
372 COUSE AND KORACH Vol. 20, No. 3 from two distinct promoters in both the rat (160) and human (159). As previously discussed, the PR gene is strongly regulated by the estradiol-ER� complex. This is evidenced by in vitro and in vivo studies showing inhibition of estrogen stimulation of the PR gene with antiestrogens as well as the presence of a single imperfect estrogen-response element in the proximal region of the PR gene promoter (160). However, in vitro assays indicate that both the proximal as well as a distal promoter appear to be at least partially ER� dependent and may also involve ligand-independent pathways of ER� action for full expression (160, 182). The lack of estradiol-induced increases in PR mRNA levels in the �ERKO uterus confirms a regulatory dependence of the PR gene on ER� action (123). Therefore, it was hypothesized that disruption of the ER� gene may subsequently result in abnormally low levels of PR in the �ERKO uterus, and thereby render this tissue refractory to progesterone as well. However, Northern blot and ribonuclease protection assays have indicated basal levels of PR mRNA in the �ERKO uterus that are equal to those in wild-type, although estrogen-stimulated increases in these levels are absent (123). Binding assays with a radiolabeled progestin indicate levels of PR protein in the �ERKO uteri are present but reduced to approximately 60% of that in wild type (183). A notable finding was the greater proportion of PR found to be tightly associated with the nuclei of cells from the �ERKO uteri (�25%), compared with the wild type (�5%) (183). It is possible that a lack of ER� has resulted in a more plentiful pool of available coregulator proteins, allowing the PR present in the �ERKO uterus to maintain a more “active” state. Western blots indicate no difference in the relative levels of PR A and PR B between the two genotypes, with PR A consistently present in greater amounts in both (183). Therefore, a loss of ER� action in the uterus has led neither to a complete loss of PR nor to altered and preferential transcription from one of the two PR gene promoters. Curtis et al. (183) carried out a series of studies demonstrating the preservation of PR-mediated progesterone actions in the �ERKO uteri. Stimulation of the genes encoding amphiregulin (184) and calcitonin (185) in the rodent uterus has been shown to be an early and late response to progesterone, respectively. A treatment regimen of progesterone shown to be effective in inducing these two genes in the ovariectomized wild-type uterus was equally effective in the uteri of ovariectomized �ERKO females (183). Furthermore, the documented ability of estradiol to inhibit the progesterone induction of the amphiregulin gene was absent in the �ERKO (183). These studies indicate that a sufficient level of PR and an active progesterone signaling pathway are present in the uteri of �ERKO mice. A well known physiological role for progesterone is the preparation of the uterine endometrium for the forthcoming pregnancy. Implantation of the embryo is a complex process that requires a high degree of synchronicity between the blastocyst and the hormone-induced changes of the uterine endometrium (reviewed in Ref. 186). In general, ovarian synthesis of estradiol, which peaks at ovulation, serves to “prime” the uterus by eliciting increased differentiation and proliferation of the luminal and glandular epithelium, and the induction of significant increases in PR in the endometrial stroma and myometrium (162, 186). Subsequent increases in progesterone released from the ovarian corpora lutea then cause decidualization, a complex process involving massive proliferation and differentiation of the endometrial stroma along with localized increases in vascular permeability and edema (186). This process involves the synthesis of specific hormones, such as PRL, cytokines, prostaglandins, extracellular matrix components, and various enzymes within the uterine tissues (186). The result is a remarkable swelling of the uterine stroma thought to be necessary for implantation by forcing the uterus to close down on the blastocyst (186). The final stage of apposition in the mouse is the grasping of the blastocyst and ultimate attachment to the uterine wall, a process thought to be dependent on the secondary rises in ovarian derived estradiol (186). Therefore, preparation of the uterus for blastocyst implantation is dependent on the multifunctional and sometimes opposing effects of estradiol and progesterone. A laboratory model for the decidualization process has been generated for several species and generally involves exogenous steroidal treatments designed to mimic those of the ovarian cycle, coupled with a mechanical stimulus or trauma of the uterine lining to simulate implantation (186, 187). In brief, ovariectomized mice are treated with estradiol (100 ng) for 3 consecutive days, followed by 2 days of no treatment. The animals are then administered progesterone (1 mg) and a reduced dose of estradiol (7 ng) for 8 consecutive days, with mechanical stimulation of the uterus taking place on the third day. Using this treatment scheme and the progesterone receptor-knockout (PRKO) model, Lydon et al. (44) demonstrated the absolute dependence of the decidualization process on progesterone action and the PR by reporting the complete lack of decidualization in the PRKO mice. However, Curtis et al. (183) demonstrated that a progesteroneinduced decidual response is possible in the �ERKO uterus, despite its inability to respond to the estradiol priming meant to mimic estrus. Most interesting were investigations indicating that although the uterine decidualization in the wild type was dependent on estradiol, as evidenced by its inhibition with ICI-182,780, progesterone alone was sufficient to induce decidualization in the �ERKO uterus (183). The reasons for the apparent loss of estrogen dependence for successful decidualization in the �ERKO uterus can only be speculated upon at this time. One role of estrogen in uterine decidualization is thought to be the induction of significant increases in PR in the endometrial stroma (186). This is supported by recent immunohistochemical studies by Tibbetts et al. (162) demonstrating strong up-regulation of stromal PR and simultaneous down-regulation of epithelial PR in the mouse uterus after 4 days of estradiol treatment. In light of the puzzling results indicating the PR in the �ERKO uterus is strongly associated with the nucleus, and therefore possibly in a more “active” state, the ER�-independent decidualization observed in the �ERKO uterus may be due to an enhanced ability to respond to progesterone. However, a release of suprabasal levels of estradiol during the luteal phase of the ovarian cycle is synchronized with the time of implantation and thought to be critical to enhancing and maximizing the uterine decidualization process (186).
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Estrogen Receptor Null Mice - Endocrine Reviews

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