The N-terminal transactivation domain of rat estrogen receptor is localized in a hydrophobic domain of eighty amino acids

In order to determine the transactivation domain(s) of the rat estrogen receptor (rat ER), we made a number of rat ER deletion mutants and transfected the mutant plasmids into COS7 cells together with an estrogen-responsive reporter plasmid, ERE-tk(197)-CAT, which contained the estrogen response element of Xenopus vitellogenin A2 gene. We have identified and localized the N-terminal transactivation domain of the rat ER to a hydrophobic region extending from Ala 59 to Glu 140.     

The direct effect of estrogen on cell viability and apoptosis in human gastric cancer cells

Epidemiology researches indicated that gastric cancer is a male-predominant disease; both expression level of estrogen and expression pattern of estrogen receptors (ERs) influence its carcinogenesis. But the direct effect of estrogen on gastric cancer cells is still unclear. This study aimed to explore the direct effect of β-estradiol (E2) on gastric cancer cells. SGC7901 and BGC823 were treated with a serial of concentrations of E2. The survival rates of both the cell lines were significantly reduced, and the reduction of viability was due to apoptosis triggered by E2 treatment. Caspase 3 was activated in response to the increasing E2 concentration in both SGC7901 and BGC823. Cleaved Caspase 3 fragments were detected, and the expression levels of Bcl-2 and Bcl-xL were reduced. Apoptosis was further confirmed by flow cytometry. The expression level of PEG10, an androgen receptor target gene, was reduced during E2 treatment. Both ERα and ERβ were expressed in these cell lines, and the result of bioinformatics analysis of gastric cancer from GEO datasets indicated that the expression levels of both ERα and ERβ were significantly higher in noncancerous gastric tissues than in gastric cancer tissues. Our research indicated that estrogen can reduce cell viability and promote apoptosis in gastric cancer cells directly; ERs expression level is associated with gastric cancer. Our research will help to understand the mechanism of gender disparity in gastric cancer.     

Leptin regulates estrogen receptor gene expression in ATDC5 cells through the extracellular signal regulated kinase signaling pathway

Both estrogen and leptin play an important role in the regulation of physiological processes of endochondral bone formation in linear growth. Estrogen receptors (ERα and ERβ) are known as members of the superfamily of nuclear steroid hormone receptors and are detected in all zones of growth plate chondrocytes. They can be regulated in a ligand-independent manner. Whether leptin regulates ERs in the growth plate is still not clear. To explore this issue, chondrogenic ATDC5 cells were used in the present study. Messenger RNA and protein analyses were performed by quantitative PCR and Western blotting. We found that both ERα and ERβ were dynamically expressed during the ATDC5 cell differentiation for 21 days. Leptin (50 ng/ml) significantly upregulated ERα and ERβ mRNA and protein levels 48 h after leptin stimulation (P<0.05) at day 14. The up-regulation of ERα and ERβ mRNA by leptin was shown in a dose-dependent manner, but the most effective dose of leptin was different (100 and 1,000 ng/ml, respectively). Furthermore, we confirmed that leptin augmented the phosphorylation of ERK1/2 in a time-dependent manner. A maximum eightfold change was observed at 15 min. Finally, a specific ERK1/2 inhibitor, UO126, blocked leptin-induced ERs regulation in ATDC5 cells, indicating that ERK1/2 mediates, partly, the effects of leptin on ERs. These data demonstrate, for the first time, that leptin regulates the expression of ERs in growth plate chondrocytes via ERK signaling pathway, thereby suggesting a crosstalk between leptin and estrogen receptors in the regulation of bone formation.     

Construction of cell lines that express high levels of the human estrogen receptor and are killed by estrogens

To prepare large amounts of the human estrogen receptor (ER) for biochemical and biophysical studies we have employed the cloned ER sequences to construct Chinese hamster ovary (CHO) cell line derivatives that overexpress the receptor. We have employed an efficient expression vector (SV40 enhancer, human metallothionein IIA promoter) and a new system of gene amplification based on the human metallothionein IIA gene and stepwise selection in cadmium. Cells from the initial transfected pools, before gene amplification, had as much or more ER than human MCF7 cells and responded to the subsequent stepwise cadmium selection and amplification with increases in ER levels to about 2 million receptors/cell. Cell lines isolated from these pools are stable for human ER expression and display up to 6 million receptors/cell, or about 0.4% of the total cell protein. The CHO receptor activates a transfected reporter gene in responses to estrogen, is down-regulated in response to estrogens, displays the same electrophoretic mobility as the MCF7 receptor, and is free of degradation as initially extracted. CHO cells displaying 3 million or more human ER/cell (but not cells with lower levels) flatten and stop growing within the first 24 h after exposure to physiological estrogen concentrations. After several days in estrogen the majority of the cells lyse. The antiestrogen 4-hydroxytamoxifen also causes cell death, but another antiestrogen, ICI 164,384, is without toxic effect. The basis for these phenomena are unknown, but mutants isolated for survival of estrogen treatment have lost receptor expression, thereby confirming the role of receptor in cell death.