Regulation of expression of the retinoic acid metabolizing enzyme CYP26A1 in uteri of ovariectomized mice after treatment with ovarian steroid hormones

The retinoic acid (RA) synthesizing enzymes, retinaldehyde dehydrogenases (RALDH), are expressed in specific spatial and temporal patterns in uterine tissues during estrous cycle and early pregnancy in mice. Expression of RALDH1 and 2 has been shown to be induced by estrogen treatment within the uterus. In this study, we determined the influence of progesterone and 17-ss-estradiol on the uterine expression of the RA-metabolizing enzyme CYP26A1 after specific time intervals (1, 4, 24, and 48 hr after treatment of ovariectomized mice). In a following experiment, we investigated the influence of gestagen (promegestone 0.3 mg/kg body weight), estrogen (estradiol 3 microg/kg), their combination, as well as the antagonizing anti-progesterone hormone (RU 486 10 mg/kg) on the uterine expression of CYP26A1. Expression of CYP26A1 was localized using in situ hybridization and quantified using RT-PCR. CYP26A1 mRNA expression was strongly--although transiently--induced in uterine endometrial epithelial and glandular cells after administration of gestagen or the combination of gestagen + estrogen, but not by estrogen alone. These observations were confirmed by semi-quantitative RT-PCR experiments on whole uteri. Thus, we show that the expression of CYP26A1 in endometrial epithelial cells is regulated by progesterone and not significantly influenced by co-administration of estrogen. These data indicate an additional level of hormonal control of endogenous RA levels in the mouse uterus, where its synthesis would rely on estrogen-dependent expression of RALDH enzymes, whereas its active metabolism would be triggered by progesterone-induced CYP26A1 expression.     

The effects of estradiol and selective estrogen receptor modulators on gene expression and messenger RNA stability in immortalized sheep endometrial stromal cells and human endometrial adenocarcinoma cells

The purpose of this study was to identify an endometrial cell line that maintained the E2 up-regulation of estrogen receptor (ER) mRNA by enhanced message stability and to assess its dependence on ER protein. Estradiol (E2) effects on gene expression were measured in three cell lines: one immortalized from sheep endometrial stroma (ST) and two from human endometrial adenocarcinomas (Ishikawa and ECC-1). E2 up-regulated ER mRNA levels in ST and Ishikawa cells, but down-regulated ER mRNA levels in ECC-1 cells. E2 up-regulated progesterone receptor (PR), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and transforming growth factor-alpha (TGF-alpha) in both Ishikawa and ECC-1 cells. The selective estrogen receptor modulator ICI 182,780 antagonized the E2-induced up-regulation of ER and/or PR mRNA levels in all three cells, while another, GW 5638, antagonized the up-regulation of PR mRNA in Ishikawa and ECC-1 cells. In mechanistic studies, E2 had no effect on ER mRNA stability in ST cells and it destabilized ER mRNA in ECC-1 cells. Thus, Ishikawa cells appear to be the most physiologically relevant cell line in which to study the up-regulation of ER mRNA levels by enhanced mRNA stability. Its antagonism by ICI 182,780 reveals that ER protein is involved in this E2 response.     

Benzo(a)pyrene exposure induces CYP1A1 activity and expression in human endometrial cells

Estrogen is a major risk factor for endometrial cancer and it has been well-established that smokers have a significantly reduced risk of endometrial cancer. Localized levels of estrogen within the uterus may determine the estrogenic response. The objective of this research was to investigate effects of cigarette smoke related hydrocarbons (benzo(a)pyrene, BP) on uterine CYP1A1/2 and 1B1, enzymes involved in estrogen metabolism. Human endometrium epithelial cells (RL95-2) were incubated with various concentrations (0.05, 0.1, 0.5, 1, and 10mM) of BP for 48h. CYP1 catalytic activity, protein and mRNA levels were determined. Selective chemical and immuno-inhibitors were used to determine the contribution of individual CYP1 isoenzymes. Cells expressing CYP1A1, CYP1A2 and CYP1B1 were used for comparisons. CYP1A1/2 protein and mRNA levels were significantly elevated by BP. Low level of constitutive CYP1 activity was observed in RL95-2 cells, which was significantly induced by BP exposure (12-fold at 1mM). CYP1 activity in BP-induced cells was significantly inhibited by specific anti-CYP1A1 and high concentration of alpha-naphthoflavone (ANF, 100nM), but not by selective CYP1A2 (furafylline) and CYP1B1 (homoeriodictoyl) inhibitors and low concentration of ANF (5nM). These studies suggest that CYP1A2 and CYP1B1 are not induced by BP in the endometrial cells. It also appears that CYP1A1 is one of the major CYP450 enzymes induced by BP.     

Estrogen-induced uterine abnormalities in TIMP-1 deficient mice are associated with elevated plasmin activity and reduced expression of the novel uterine plasmin protease inhibitor serpinb7

Tissue inhibitor of metalloproteinase-1 (TIMP-1) is a multifunctional protein capable of regulating a variety of biological processes in a wide array of tissue and cell types. We have previously demonstrated that TIMP-1 deficient mice exhibit alterations in normal uterine morphology and physiology. Most notably, absence of TIMP-1 is associated with an altered uterine phenotype characterized by profound branching of the uterine lumen and altered adenogenesis. To begin to assess the mechanism by which TIMP-1 may control these uterine events, we utilized steroid-treated ovariectomized wild-type and TIMP-1 null mice exposed to estrogen for 72 hr. Administration of estrogen to TIMP-1 deficient mice resulted in development of an abnormal uterine histo-architecture characterized by increased endometrial gland density, luminal epithelial cell height, and abnormal lumen structure. To determine the mediators which may contribute to the abnormal uterine morphology in the TIMP-1 deficient mice, cDNA microarray analysis was performed. Analysis revealed that expression of two plasmin inhibitors (serpbinb2 and serbinb7) was significantly reduced in the TIMP-1 null mice. Associated with the reduction in expression of these inhibitors was a significant increase in plasmin activity. Localization of the novel uterine serpinb7 revealed that expression was confined to the luminal and glandular epithelial cells. Further, expression of uterine serpinb7 was decreased by estrogen and showed an inverse relationship with plasmin activity. We conclude from these studies that in addition to controlling MMP activity, TIMP-1 may also control activity of serine proteases through modulation of serine protease inhibitors such as serpinb7.     

ECC-1 cells: a well-differentiated steroid-responsive endometrial cell line with characteristics of luminal epithelium

Endometrial cancer cell lines have provided a valuable model to study endometrial epithelial cells in vitro. Since the first development of HEC1B over 35 yr ago, many different cell lines have been isolated and described. One valuable cell line that maintains hormone responsiveness and unique stability over time is the ECC-1 cell line, developed originally by the late P.G. Satyaswaroop. In this study, we investigated some of the properties of these cells and present their salient characteristics. Like Ishikawa cells, ECC-1 cells maintain both estrogen receptors (ESR1 [ER alpha] and ESR2 [ER beta]), progesterone receptors (PR A and B; PGRs), and androgen receptors (ARs), along with the p160 steroid receptor coactivators NCOA1 (formerly SRC1), NCOA2 (formerly TIF2), and NCOA3 (formerly AIB1). The karyotype of these cells is abnormal, with multiple structural rearrangements in all cells analyzed. Unlike Ishikawa cells that express glandular epithelial antigens, ECC-1 cells maintain a luminal phenotype, with expression of KRT13 (cytokeratin 13) and KRT18 (cytokeratin 18). Apparent differences in the regulation of ESR2 also were evident in ECC-1 cells compared to Ishikawa cells. Like other endometrial cell lines, ECC-1 cells express the steroid receptor coactivators and exhibit epidermal growth factor-stimulated expression of known luminal proteins thought to be involved in implantation, including the hyaluronate receptor CD44 and SPP1 (formerly osteopontin) and CD55 (decay-accelerating factor). These characteristics appear to be stable and persistent over multiple cell passages, making this well-differentiated cell line an excellent choice to study endocrine and paracrine regulation of endometrial epithelium in vitro.     

Fenofibrate,a ligand for PPARalpha,inhibits aromatase cytochrome P450 expression in the ovary of mouse

Peroxisome proliferator-activated receptors (PPARs) play important roles in the metabolic regulation of lipids including steroids. In this study, we investigated whether fenofibrate, a ligand for PPARalpha, could influence estrogen synthesis in vivo in the ovary of mice. As reported, chronic treatment of C57BL6/J female mice with various amounts of fenofibrate as a diet reduced the serum triglycerides level and induced hepatomegaly in a dose-dependent manner. Northern blot analyses using hepatic RNA confirmed the induction of classical PPARalpha-target genes including acyl-CoA oxidase and lipoprotein lipase. The analyses using ovarian RNA revealed the suppression of gene expression for enzymes involved in steroidogenesis including CYP11A, CYP19, steroidogenic acute regulatory protein, and HDL receptor, but the CYP17 expression was evidently induced. Consistent with the suppression of CYP19 mRNA expression, the aromatase activity in the ovary was dose-dependently inhibited, resulting in significant decreases in the uterine size and bone mineral density. When PPARalpha null mice were treated with dietary fenofibrate, neither hepatomegaly nor inhibition of ovarian aromatase activity was observed, rather the activity was enhanced. These results demonstrate that fenofibrate inhibits ovarian estrogen synthesis by suppressing the mRNA expressions and that functional PPARalpha is indispensable for the inhibitory action of the agent in vivo.     

Absence of correlation between antiestrogenic activity and binding affinity for the estrogen receptor.

We have compared the binding to the estrogen receptor (R) of different androgens and antiestrogens with their antiestrogenic activities on uterine growth. We found that estradiol (E₂)¹ and hydroxytamoxifen, a potent antiestrogen, displayed the same affinity for R. Conversely, androgens which have a much lower affinity for R and a much higher dissociation rate than E₂, behave at high doses as full estrogens, with no significant antiestrogenic activity. We conclude that there is no correlation between the dissociation rate from R and the antiestrogenic activity of R ligands and that one cannot discriminate between estrogen and antiestrogen ligands by simply evaluating their $\text{in vitro}$ binding to the cytosol R.     

The effects of the selective estrogen receptor modulators, methyl-piperidino-pyrazole (MPP), and raloxifene in normal and cancerous endometrial cell lines and in the murine uterus

Since estrogens have vital functions in the uterus but might also contribute to endometrial cancer, we sought to determine the in vitro effects of methyl-piperidino-pyrazole (MPP), raloxifene, and beta-estradiol on Ishikawa and RL-95 endometrial cancer, and ovine luminal endometrial (oLE) cell lines and the in vivo effects of these compounds in the rodent uterus. MPP and raloxifene (1 nM) induced significant apoptosis in the endometrial cancer and oLE cell lines compared to beta-estradiol treated and control cells (P 相似文献   

Regulation of progesterone receptors and decidualization in uterine stroma of the estrogen receptor-alpha knockout mouse

Regulation of progesterone receptor (PR) in uterine stroma (endometrial stroma plus myometrium) by estrogen was investigated in estrogen receptor-alpha (ERalpha) knockout (alphaERKO) mice. 17 beta-Estradiol (E(2)) increased PR levels in uterine stroma of ovariectomized alphaERKO mice, and ICI 182 780 (ICI) inhibited this E(2)-induced PR expression. Estrogen receptor-beta(ER beta) was detected in both uterine epithelium and stroma of wild-type and alphaERKO mice by immunohistochemistry. In organ cultures of alphaERKO uterus, both E(2) and diethylstilbestrol induced stromal PR, and ICI inhibited this induction. These findings suggest that estrogen induces stromal PR via ERbeta in alphaERKO uterus. However, this process is not mediated exclusively by ERbeta+, because in ERbeta knockout mice, which express ERalpha, PR was up-regulated by E(2) in uterine stroma. In both wild-type and alphaERKO mice, progesterone and mechanical traumatization were essential and sufficient to induce decidual cells, even though E(2) and ERalpha were also required for increase in uterine weight. Progesterone receptor was strongly expressed in decidual cells in alphaERKO mice, and ICI did not inhibit decidualization or PR expression. This study suggests that up-regulation of PR in endometrial stroma is mediated through at least three mechanisms: 1) classical estrogen signaling through ERalpha, 2) estrogen signaling through ERbeta, and 3) as a result of mechanical stimulation plus progesterone, which induces stromal cells to differentiate into decidual cells. Each of these pathways can function independently of the others.     

Antiestrogenic and antifertility actions of anordrin (2α,17ga-diethynyl-A-nor-5α-androstane-2β,17β-diol 2,17-dipropionate)

Anordrin, administered in a single s.c. dose of 62.5 μg in sesame oil, stimulated sustained uterine growth (wet weight) when measured at 24 and 72 hr, but total soluble protein and total DNA per uterus was not increased. By comparison, 3 μg of estradiol-17β under the same conditions significantly increased all three parameters of uterine growth. Both of the above steroid treatments significantly increased nuclear estrogen receptor content of the uterus, but only the estradi-ol-17β treatment resulted in significantly elevated cytosol receptor content per uterus. Anordrin binds to the 8S estrogen receptor with an affinity of about 2 × 10⁵ M^-1 as determined by competition with [³H]estradiol-17β. The abortifacient activity of Anordrin when given orally (8 mg/kg b.w.) to mice on the 7th day of pregnancy was almost completely blocked by simultaneous oral administration of estradiol-17β (0.8 mg/kg b.w.). It is concluded that the actions of Anordrin on the uterus can be attributed to its antiestrogenic activities.