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Query: UNIPROT:P06889 (Mol)
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The biological actions of glucocorticoids in target organs are determined at least in part by the local expression of 11beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD2), which is responsible for the inactivation of glucocorticoids. The human endometrium is a glucocorticoid target tissue, and is known to express 11beta-HSD2. However, little is known about the function and regulation of 11beta-HSD2 in the endometrium, probably owing to the lack of in vitro model systems (i.e., cell lines) that express 11beta-HSD2. Here, we describe the characterization of 11beta-HSD expression in Ishikawa cells, a well-differentiated human endometrial adenocarcinoma cell line. The 11beta-HSD activity in intact Ishikawa cells was characteristic of 11beta-HSD2 in that it only possessed dehydrogenase activity (cortisol to cortisone) and had a high affinity for cortisol (apparent Km of 34 nM). The exclusive expression of 11beta-HSD2 in Ishikawa cells was confirmed by RT-PCR which demonstrated the presence of the mRNA for 11beta-HSD2 but not that for 11beta-HSD1. To investigate the regulation of 11beta-HSD2 in Ishikawa cells, we treated these cells with sex steroid hormones, glucocorticoids and epidermal growth factor (EGF), and determined the effects of these treatments on 11beta-HSD2 activity by an established intact cell radiometric conversion assay. Treatment with estradiol-17beta (E2, 10 nM) and medroxyprogesterone acetate (MPA, 100 nM) produced a classic sex steroid effect; the greatest increase (330% of the control) in the level of 11beta-HSD2 activity was caused by the combined treatment, followed by MPA (240% of the control) with E2 being the least effective (156% of the control). The stimulatory effect of E2 was blocked by the pure antiestrogen ICI 182,780. The synthetic glucocorticoid dexamethasone (Dex) increased 11beta-HSD2 activity in a time- and dose-dependent manner (200% of the control; 100 nM for 48 h), and the endogenous glucocorticoid cortisol was equally effective in this regard. The antiprogesterone-antiglucocorticoid RU486 did not counteract with MPA or Dex but rather acted as an agonist; increased 11beta-HSD2 activity (160% of the control; 100 nM for 72 h). By contrast, treatment with EGF caused a dose- and time-dependent decrease in 11beta-HSD2 activity (60% of the control; 10 ng/ml for 72 h). In addition, semi-quantitative RT-PCR analysis revealed that there were corresponding changes in the level of 11beta-HSD2 mRNA following the treatment of Ishikawa cells with these steroid hormones and EGF, indicating that the effects of these hormones and EGF are mediated, at least in part, at the level of 11beta-HSD2 gene transcription. In conclusion, we have demonstrated for the first time that the human Ishikawa endometrial cell line expresses exclusively the 11beta-HSD2 isozyme. Moreover, we have presented the first direct evidence that sex steroid hormones and glucocorticoids stimulate while EGF inhibit the expression of 11beta-HSD2 in Ishikawa cells, suggesting that endometrial 11beta-HSD2 is under the control of steroid hormones and EGF. Thus, the Ishikawa cell line represents an excellent model in which the function and regulation of endometrial 11beta-HSD2 may be studied.
J Steroid Biochem Mol Biol
PMID:Regulation of 11beta-hydroxysteroid dehydrogenase type 2 by steroid hormones and epidermal growth factor in the Ishikawa human endometrial cell line. 1062 9

We have previously shown that dexamethasone (DEX) stimulates rapid polymerization of actin and stabilization of microfilaments in human endometrial adenocarcinoma cells. As the content of total cellular actin and the concentration of the actin transcript did not change, we concluded that polymerization of actin by glucocorticoids involves nongenomic mechanisms. However, the signaling events by which the latter is achieved remain unknown. In the present study we evaluated whether tyrosine phosphorylation is required for the rapid, nongenomic DEX effect on actin assembly. In cells preincubated with the tyrosine kinase inhibitors, genistein or erbstatin analogue (EA), before adding DEX the G-/total actin ratio remained unchanged, whereas DEX in the absence of both inhibitors reduced the ratio by 25%. In addition, when cells were preincubated with the protein tyrosine phosphatase inhibitor pervanadate and subsequently incubated with DEX, the G-/total actin ratio was dramatically reduced by 65%. Furthermore, DEX increased transiently the levels of tyrosine phosphorylation of focal adhesion kinase (FAK) and paxillin within 2 to 15 min, without a change in their expression levels. Pervanadate mimicked this effect of DEX and enhanced tyrosine phosphorylation of both proteins. In addition, when cells were exposed to the anticytoskeletal agent cytochalasin B, the basal levels of tyrosine phosphorylation of both proteins were reduced. This effect was reversed by DEX, indicating that actin cytoskeleton integrity is required for the effect of DEX on tyrosine phosphorylation of FAK and paxillin. Finally, we documented enhanced expression of the Ras-related GTP-binding protein Rho-B after long-term (12- and 24-hr) treatment with DEX, whereas Rho-B levels remained unchanged after short-term (3- and 6-hr) treatment. Our observations demonstrate a novel mechanism through which the rapid nongenomic effect of DEX on actin assembly requires tyrosine phosphorylation of the cytoskeleton-associated proteins FAK and paxillin. We also propose that the DEX-induced actin polymerization may constitute a mechanism for transduction of signals resulting in tyrosine phosphorylation of FAK and paxillin. Moreover, the enhanced Rho-B levels observed after long-term treatment with DEX imply a mechanism for the well-described, long-term effects of glucocorticoids on actin cytoskeleton.
Mol Med 1999 Nov
PMID:Tyrosine phosphorylation of focal adhesion kinase and paxillin regulates the signaling mechanism of the rapid nongenomic action of dexamethasone on actin cytoskeleton. 1065 75

We have shown that moderately differentiated endometrial adenocarcinoma (RL95-2) cells differentiate in response to retinoic acid treatment, illustrated by their reorganization of actin filaments and cell enlargement (Carter et al., Anticancer Res. 16, 17-24, 1996). Tyrphostin, an inhibitor of epidermal growth factor receptor (EGF-R)-associated protein tyrosine kinases, caused a dramatic reorganization of actin filaments in RL95-2 cells, similar to retinoic-acid-treated cells (Carter and Bellido, J. Cell. Physiol. 178, 320-332, 1999). We evaluated the possibility that the differentiating effects of retinoids are due to retinoic-acid-induced decreases in phosphorylation of EGF-R and changes in downstream effector proteins. Retinoic acid caused a decrease in tyrosine phosphorylation of EGF-R. Retinoic acid treatment induced a dramatic actin filament reorganization and cell enlargement. Treatment with EGF reversed this effect, because cells treated with retinoic acid followed by EGF only possessed disrupted actin aggregates and appeared small, thus resembling medium controls. Retinoic acid induced a relocalization and decrease in the amount of Shc protein, another actin-binding protein which is an adaptor protein for EGF-R signaling. In addition, retinoic acid induced a relocalization of gelsolin from the plasma membrane to the cytoplasm. Retinoic acid decreased cell detachment in detachment assays; one-half as many retinoic-acid-treated cells detached as in controls. These results are consistent with the idea that retinoic acid induces differentiation of RL95-2 cells by interfering with the EGF-R signaling pathway.
Exp Mol Pathol 2000 Jun
PMID:Retinoic acid affects the EGF-R signaling pathway during differentiation induction of human endometrial adenocarcinoma cells. 1081 85

The transforming growth factor beta1 (TGFbeta1) is a major regulator of human endometrial function. Human endometrium possesses specific opioid binding sites, the majority of which belong to the kappa type, for which the prodynorphin-derived opioids are the endogenous ligands. Since these two systems interact in several other tissues we postulated that opioids may affect the production of TGFbeta1 in human endometrium. We have found that kappa opioids exerted a time- and dose-dependent inhibitory effect on TGFbeta1 production from endometrial stromal and epithelial cells and from the Ishikawa human endometrial adenocarcinoma cell line. This effect was reversible by the specific opioid antagonist diprenorphine. To examine if this effect represents a paracrine endometrial response to locally produced kappa opioids we searched for the presence of the endogenous kappa opioid receptor ligands. Indeed, the prodynorphin transcript was detectable on Northern blots from normal and tumoral human endometrial cells; its size was that of the pituitary transcript, i.e. approximately 2.4 kb long. Most immunoreactive dynorphin from human endometrium had a molecular weight of 8 kDa. Finally, immunofluorescence staining of normal and tumoral human endometrial cells revealed the presence of dynorphin-positive cytoplasmic secretory granules. Taken together, our data suggest that in human endometrium, kappa opioids and the TGFbeta1 form a paracrine network which appears to be retained by the Ishikawa human endometrial adenocarcinoma cell line.
Mol Hum Reprod 2000 Jul
PMID:Kappa opioids and TGFbeta1 interact in human endometrial cells. 1087 47

Insulin-like growth factor I (IGF-I) is a peptidic growth factor implicated in the proliferation of a wide variety of cell types, and especially endometrial epithelial cells. Its action is modulated by the presence of IGF-binding proteins (IGFBPs) which are secreted by IGF-I target cells. The partition of IGFBPs between cell-associated and soluble form determines the potentiation or the inhibition of IGF-I action. It is commonly accepted that cell-associated IGFBPs potentiate the IGF-I action while the soluble form of IGFBPs has an inhibitory effect. In endometrial adenocarcinoma, IGF-I is involved in tumoral progression and IGFBPs may be key modulators of the IGF-I-induced cell proliferation. Here we showed that the responsiveness of human endometrial adenocarcinoma cells (HEC-IA cell line) to the mitogenic activity of IGF-I was dependent on the pre-incubation conditions. This responsiveness to IGF-I was conditioned by a differential expression of the IGF system components (IGFBPs and IGF-I receptor) and particularly of the IGFBPs. Indeed, the IGF-I-induced proliferation of the HEC-1A cells was attenuated by the presence of cell-associated IGFBPs. Moreover, the IGF-I incubation induced a release of IGFBP-3 in the culture media as the consequence of an interaction between IGF-I and the cell-associated IGFBP-3. This effect was dose-dependent and was associated with the attenuation of the IGF-I action on cellular proliferation. Thus, IGFBP-3 might be initially expressed as a cell-associated form and then released in the interstitial fluid after a direct interaction with IGF-I. Therefore, in HEC-IA endometrial adenocarcinoma cells responsive to IGF-I, the IGFBP-3 is the main binding protein expressed and both soluble and cell-associated forms act as inhibitors of IGF-I-induced cellular proliferation.
Cell Mol Biol (Noisy-le-grand) 2000 Nov
PMID:Cell-associated insulin-like growth factor-binding proteins inhibit insulin-like growth factor-I-induced endometrial cancer cell proliferation. 1107 47

The possible adverse effects of the so-called environmental estrogens have raised considerable concern. Developmental, endocrine and reproductive disorders in wildlife animals have been linked to high exposure to persistent environmental chemicals with estrogen-like activity (xenoestrogens); yet, the potential impact of environmental estrogens on human health is currently under debate also due to lack of data. A battery of in vitro assays exist for identifying compounds with estrogenic activity, but only a few models are available to assess estrogenic potency in a multiparametric analysis. We have recently established the endometrial adenocarcinoma cell line RUCA-I; it enables us to compare estrogenic effects both in vitro and in vivo as these cells are estrogen responsive in vitro and grow estrogen sensitive tumors if inoculated in syngeneic animals in vivo. Here we report in vitro data concerning (a) the relative binding affinity of the selected synthetic chemicals Bisphenol A, nonylphenol, p-tert-octylphenol, and o,p-DDT to the estrogen receptor of RUCA-I cells and (b) the relative potency of these compounds in inducing increased production of complement C3, an endogenous estrogen-responsive gene. Competitive Scatchard analysis revealed that xenoestrogens bound with an at least 1000-fold lower affinity to the estrogen receptor of RUCA-I cells than estradiol itself, thereby exhibiting the following affinity ranking, estradiol>>>nonylphenol>bisphenol A approximately p-tert-octylphenol>o,p-DDT. Despite these low binding affinities, bisphenol A, nonylphenol and p-tert-octylphenol increased production of complement C3 in a dose dependent manner. Compared with estradiol, only 100-fold higher concentrations were needed for all the compounds to achieve similar levels of induction, except o,p-DDT which was by far less potent. Northern blot analyses demonstrated that the increased production of complement C3 was mediated by an increased transcription. In summary, cultured RUCA-I cells represent a valuable endometrial derived model system to assess the relative potencies and the molecular mode of action of environmental estrogens in vitro. Our results further show that no intimate correlation exists between the relative binding affinity and the biological response of these compounds. Therefore, data obtained from single-parametric analyses may result in misleading conclusions. On the other hand, the presented in vitro data will provide us with tools to study the activity of xenoestrogens in vivo and thus carry risk assessment one step further.
J Steroid Biochem Mol Biol 2000 Oct
PMID:Relative binding affinity does not predict biological response to xenoestrogens in rat endometrial adenocarcinoma cells. 1108 26

A new cell line of poorly differentiated human endometrial adenocarcinoma cells termed "CAC-1" cells has been established. These cells are epithelial, as indicated by positive cytokeratin and negative vimentin staining. They are rounded and possess a high nuclear-to-cytoplasmic ratio, desmosomes, surface microvilli, intercelular lumens, and pleomorphic nuclei containing multiple nucleoli. These cells have been in long-term culture for 2 years. Our previous studies demonstrated that moderately differentiated (RL95-2) cells differentiated in response to retinoic acid treatment, illustrated by their reorganization of actin filaments and cell enlargement (Carter et al., 1996; Anticancer Res. 16, 17-24). CAC-1 cells exhibited a similar response because they also organized actin filaments and enlarged in response to retinoic acid treatment. Concurrently, retinoic acid treatment caused a 40% decrease in cell detachment in an in vitro detachment assay compared to controls. A slight lag in cell growth was observed when CAC-1 cells were treated with 1 microM 13-cis or all-trans retinoic acid during a 12-day growth curve. In addition, we examined the effects of retinoic acid on protein kinase C-alpha (PKC-alpha) and myristoylated alanine-rich C-kinase substrate (MARCKS). Treatment with retinoic acid caused cytoplasmic PKC-alpha to increase concomitant with a decrease in PKC-alpha in the membrane. In contrast, MARCKS increased in the membrane in response to retinoic acid treatment. These data indicate that retinoid treatment causes inactivation of PKC-alpha, allowing MARCKS to relocalize to the membrane, where it can cross-link actin filaments. CAC-1 cells represent an ideal model for investigating the effects of retinoids on differentiation induction concomitant with actin reorganization.
Exp Mol Pathol 2000 Dec
PMID:A newly characterized human endometrial adenocarcinoma cell line (CAC-1) differentiates in response to retinoic acid treatment. 1111 59

Androgen receptors (AR) have been identified in the human endometrium, but their role in endometrial function and development towards endometrial receptivity remains poorly understood. In an effort to study the regulation and possible function in endometrial epithelium, we utilized the well-differentiated endometrial adenocarcinoma cell line, Ishikawa, as a model system. This cell line has proven to be stable, hormonally responsive, contains both estrogen and progesterone receptors, and has been shown to express endometrial proteins in a hormone responsive manner. In the present study, we demonstrate that Ishikawa cells also express AR, based on immunohistochemical staining, radioactive binding studies, RT-PCR and Northern blot analysis. The expression of AR is induced in Ishikawa cells by estrogens, similar to that reported for normal endometrium. Further, using an estrogen-responsive gene that has been characterized in this cell line, alkaline phosphatase, we show that androgens act as antiestrogens in diethylstilbestrol (DES) treated cells, inhibiting enzymatic activity in a dose-dependent manner. These data support a physiologic role for AR in the endometrium. Elevations in endometrial AR in certain clinical situations such as polycystic ovarian syndrome (PCOS) may amplify the effects of androgens on the endometrium leading to suspected defects in uterine receptivity, higher than expected infertility and high miscarriage rates observed in patients with this disorder.
J Steroid Biochem Mol Biol 2000 Nov 15
PMID:Characterization of androgen receptors in a well-differentiated endometrial adenocarcinoma cell line (Ishikawa). 1116 29

Human endometrium produces glycodelin-A (GdA). The GdA mRNA is highly expressed in progestin-sensitized human endometrial glandular epithelial cells. The mechanism of GdA gene expression, however, is not clear. To understand the cell specific GdA gene transcription, our first approach was to identify the cis-element in the GdA promoter using transfection assay in a human endometrial adenocarcinoma cell line (HEC-1B, a cell line originally derived from the glandular component of the endometrium). The GdA promoter (-1900 to +20 bp) was linked to the luciferase reporter gene to construct p1900Luc, along with two shorter promoter constructs, p1100Luc and p304Luc. Deletion analysis showed that the basal promoter activity was derived from the region between -304 to +20 bp. This region contains three putative Sp1 binding sites (Sp1-1, -243 to -238 bp; Sp1-2, -207 to -202 bp; and Sp1-3, -56 to -49 bp). Mutation analysis at the Sp1 sites showed that p304Spm2Luc and p304Spm3Luc reduced the activity by 80%, while p304Spm1-2-3Luc reduced the activity by 95%. Sp1-1 mutation, however, had no effect. These results showed that two of the three Sp1 cis-elements mediate the basal promoter activity of the GdA gene. Electrophoretic gel mobility shift showed that at least two specific binding proteins in the nuclear extracts of HEC-1B cells bound to the oligo containing Sp1-2 or Sp1-3 cis-element. Sp1 antibody reduced the specific binding complex by 70% suggesting that Sp1 transcription factor regulates GdA gene expression. In addition, over expression of Sp1 increased the promoter activity. To determine whether progestin would modulate the promoter activity, HEC-1B cells were transfected with p304Luc and with progesterone receptor (either hPR-A or hPR-B) expression vector. Medroxyprogesterone acetate increased the promoter activity (3-fold) derived from p304Luc but not from the mutant, p304Spm1-2-3Luc. In contrast, the promoter activity was slightly reduced in cells treated with estradiol and co-transfected with estrogen receptor expression vector. These data indicate that ligand-activated PR stimulates GdA gene expression mediated through the functional Sp1 sites.
Mol Cell Endocrinol 2001 May 15
PMID:Ligand activated hPR modulates the glycodelin promoter activity through the Sp1 sites in human endometrial adenocarcinoma cells. 1136 48

Recent studies have shown that in vitro steady-state expression of the tumor susceptibility gene TSG101 is important for maintenance of genomic stability and cell cycle regulation. To determine the contribution of TSG101 expression in neoplastic formation, expression of TSG101 protein levels were evaluated in primary ovarian and endometrial adenocarcinoma tumors. Expression of TSG101 was also examined in various tumor cell lines (PA-1, AN3CA, HeLa, HS578T, HCT116). Full-length TSG101 protein was detected in these tumors and cell lines indicating that intragenic deletions were not characteristic of TSG101. In addition, TSG101 protein levels were compared with aberrations of prominent cell cycle regulatory molecules such as cyclin D1, cyclin E, p16 and p53. Reduced TSG101 protein was observed in 36% (8/22) of ovarian and 17% (1/6) of endometrial adenocarcinoma. Aberrant levels of p53, p16, cyclin D or E were comparable to published studies indicating that the clinicopathological distribution of these cases did not favor advanced stage tumors. Altogether, these findings suggest that a down-regulation of TSG101 is associated with tumorigenesis in a subgroup of gynecological tumors.
Cell Mol Biol (Noisy-le-grand) 2001 Nov
PMID:TSG101 expression in gynecological tumors: relationship to cyclin D1, cyclin E, p53 and p16 proteins. 1183 66


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