Effect of 17β-estradiol on the human osteosarcoma cell line MG-63

We present evidence that 17β-estradiol (17β-E₂) regulates 1,25(OH)₂D₃-induced alkaline phosphatase synthesis and osteocalcin secretion by the human osteosarcoma cell line MG-63. When cells were pre-treated with 17β-E₂ for 48 h prior to treatment with 1,25(OH)₂D₃ (50 nM) for another 48 h, alkaline phosphatase activity increased by 40% (P < 0.025) with 2 nM 17β-E₂ and plateaued at levels of 20 and 200 nM 17β-E₂. Under the same experimental conditions, osteocalcin secretion was enhanced by 37% (P < 0.005) with 2 nM E₂. However, 17β-E₂ had no effect on basal alkaline phosphatase or on osteocalcin secretion. Moreover, simultaneous addition of 17β-E₂ and 1,25(OH)₂D₃ to cells did not result in any additional effect over l,25(OH)₂D₃ treatment alone. Tamoxifen (10 nM) inhibited 17β-E₂-induced activities in l,25(OH)₂D₃-treated cells while not affecting control cells. Dexamethasone pretreat-ment (100 nM, 48 h) also stimulated alkaline phosphatase activity in MG-63 cells. Moreover, dexamethasone pretreatment followed by treatment with 17β-E₂ and l,25(OH)₂D₃ gave an additive effect for alkaline phosphatase activity. 17α-Estradiol (17α-E₂), a less active form of estrogen, failed to modify, at low concentrations, control or l,25(OH)₂D₃-induced alkaline phosphatase synthesis and osteocalcin secretion. In fact, a 100–1000-fold higher concentration of 17α-E₂ was necessary to reproduce the effects of 17β-E₂ on osteocalcin secretion. The addition of insulin-like growth factor I (IGF-I) for 24 h (1–50 ng/ml) to MG-63 cells did not modify 1,25(OH)₂D₃-induced osteocalcin release from these cells. However, longer incubations with 50 ng/ml IGF-I did reproduce some of the effects observed with 17β-E₂. Thus, the effects of 17β-E₂ are probably not related to IGF-I production in MG-63 cells since under these conditions the addition of IGF-I alone should have produced a response at shorter incubation times and in the presence of lower concentrations of IGF-I. Since 17β-E₂ pretreatment was necessary to observe any effects on l,25(OH)₂D₃-induced activities, we hypothesized that 17β-E₂ regulated 1,25(OH)₂D₃ receptors in MG-63 cells. When cells were treated with 100 nM 17β-E₂ for 48 h, the binding affinity was unchanged: 37.3 ± 1.9 versus 35.1 ± 0.4 pM for cells whether treated or not with l7β-E₂, respectively. In contrast, a significant increase in binding capacity (B_max) was noted (15 ± 3.5%; P < 0.025). These results suggest that the estrogen analogue 17β-E₂ induces the differentiation of MG-63 cells into a more osteoblastic-like phenotype while 17α-E₂ is without physiological effect. They also suggest that estrogens may regulate bone remodeling by modulating hormonal-induction of proteins involved in bone mineralization. This effect is indirect since it does not modify basal activities, but involves a regulation of 1,25(OH)₂D₃ receptor levels in these MG-63 cells.     

Apoptotic and anti-proliferative effects of 17β-estradiol and 17β-estradiol-like compounds in the Hep3B cell line

Since there is evidence for estrogen and estrogen-like compounds to have beneficial effect on the pathogenesis of hepatocellular carcinoma (HCC), this study was designed to investigative the apoptotic and anti-proliferative effects of these compounds on the human hepatoma Hep3B cell line. The Hep3B cells were treated with 17β-estradiol (E2), diethylstilbestrol (DES), tamoxifen, and genistein. After treatments of these compounds at the concentration of 10^-6 or 10^-8 M, the Hep3B cells were demonstrated to have significant DNA fragmentation, nucleus condensation, cytochrome-c leaking from the mitochondria and caspase-3 activation by DAPI and Western blotting. The cells were also observed to have declined proliferative potential by MTT assay, arrested cell cycle by flow-cytometry measurements. However, the cytochrome-c leaking from the mitochondria induced by E2 and E2-like compounds was blocked totally by ICI 182,780 treatment. These finding suggest that estrogen and the estrogen-like compounds may induce anti-proliferative and apoptotic effects in Hep3B cells, and the E2 and the E2-like compounds mediated apoptotic effect was estrogen receptor dependent. Among the drugs tested, E2, E2 agonists (DES and genistein) and partial antagonist (tamoxifen), all showed the stronger anti-tumor potential. The last two authors, Wei-Wen Kuo and Chih-Yang Huang, share equal contribution.     

Paradoxical effects of 17β-estradiol on glucose transport in primary cell cultures of a rat mammary tumor

Primary cell cultures of rat mammary carcinoma R3230AC exhibited a rapid, reversible and dose-related inhibition of carrier-mediated 3-0-methyl-D-glucose transport when estrone, 17β-estradiol, estriol, diethylstilbestrol or phloretin was present during the transport assay. With 17β-estradiol, maximal transport inhibition (66%) was observed at 40μM, a concentration also effective in preventing cell growth when present in the media. Cultures preincubated in growth media containing 5mM glucose plus 40μM 17β-estradiol for one day displayed enhanced rates of carrier mediated 3-0-methyl-D-glucose transport. This increase was prevented by 50mM glucose and can be explained as an adaptive response to a condition simulating glucose starvation.     

Regulation of Akt expression and phosphorylation by 17β-estradiol in the rat uterus during estrous cycle

Background

Ablation of the low-affinity receptor subunit for leukemia inhibitory factor (LIFR) causes multi-systemic defects in the late gestation fetus. Because corticosterone is known to have a broad range of effects and LIF function has been associated with the hypothalamo-pituitary-adrenal axis, this study was designed to determine the role for LIFR in the fetus when exposed to the elevated maternal glucocorticoid levels of late gestation. Uncovering a requirement for LIFR in appropriate glucocorticoid response will further understanding of control of glucocorticoid function.

Methods

Maternal adrenalectomy or RU486 administration were used to determine the impact of the maternal glucocorticoid surge on fetal development in the absence of LIFR. The mice were analyzed by a variety of histological techniques including immunolabeling and staining techniques (hematoxylin and eosin, Alizarin red S and alcian blue). Plasma corticosterone was assayed using radioimmunoassay.

Results

Maternal adrenalectomy does not improve the prognosis for LIFR null pups and exacerbates the effects of LIFR loss. RU486 noticeably improves many of the tissues affected by LIFR loss: bone density, skeletal muscle integrity and glial cell formation. LIFR null pups exposed during late gestation to RU486 in utero survive natural delivery, unlike LIFR null pups from untreated litters. But RU486 treated LIFR null pups succumb within the first day after birth, presumably due to neural deficit resulting in an inability to suckle.

Conclusion

LIFR plays an integral role in modulating the fetal response to elevated maternal glucocorticoids during late gestation. This role is likely to be mediated through the glucocorticoid receptor and has implications for adult homeostasis as a direct tie between immune, neural and hormone function.     

Mitochondrial dynamics is affected by 17β-estradiol in the MCF-7 breast cancer cell line. Effects on fusion and fission related genes

Mitochondrial dynamics, specifically fusion and fission processes, maintain mitochondria integrity and function, yet at this time, effect of estrogens on fusion and fission in breast cancer cell lines has not been studied. The aim of this study was to characterize the effect of 17β-estradiol on fusion and fission-related genes, as well as on mitochondria proliferation and function. We used MCF-7 breast cancer cell line, which is estrogen sensitive (estrogen receptor positive). Cells were grown in Dulbecco's modified Eagle medium charcoal-stripped fetal bovine serum and treated with 1nM of 17β-estradiol and with/without 100nM of ICI 182,780, a drug that caused rapid degradation of estrogen receptor. mRNA levels of fusion (mfn1, mfn2, opa1) and fission-related genes (drp1 and fis1) were examined by RT-PCR, cardiolipin content by N-acridyl-orange fluorescence and oxidative phosphorylation protein levels, as well as, the major fusion and fission related protein levels, by Western blot. mRNA expression of fusion-related genes increased after 17β-estradiol-treatment for 4h; however fis1 fission-related gene expression decreased. All these effects were not found in cells pre-treated with ICI 182,780, save for the changes in mfn-1, conferring them the effects of 17β-estradiol to estrogen receptor. The changes in protein levels were less prominent, but in the same way, than in mRNA levels, showing an increase in Mfn1 and Mfn2, as well as in Drp1, but there was no change in Fis1 protein levels. Mitochondrial biogenesis was also affected by 17β-estradiol, showing an increase in mtDNA but with no change in N-acridyl-orange fluorescence. On the whole, our results suggest an imbalance in the fusion/fission ratio, with a high fusion by 17β-estradiol-estrogen receptor action, which can affect to mitochondrial biogenesis, concretely in mitochondria proliferation. According to this information, 17β-estradiol would modify mitochondrial dynamics, biogenesis and metabolism, and thus compromise the normal development and function of mitochondria in cancer affected tissues.     

Opposite regulation of XIAP and Smac/DIABLO in the rat endometrium in response to 17β-estradiol at estrus

During rat estrous cycle, the endometrium proliferates in response to sex steroids and specific endometrial epithelial cells undergo apoptosis in absence of embryonic factors. The central executioner of apoptosis is a family of aspartic acid-specific cysteine proteases known as caspases. Smac/DIABLO is released from the mitochondria during apoptosis and its stimulation promotes caspases activation by neutralizing members of the inhibitor of apoptosis proteins (IAPs) family, such as X-linked inhibitor of apoptosis protein (XIAP). The aim of this study was to investigate the involvement of Smac/DIABLO and XIAP in the control of caspases activation in endometrium of cycling rats. Polyoestrus female rats were sacrificed at each stage of estrous cycle (diestrus, proestrus, estrus, and metestrus). Endometrial protein extracts were collected to perform Western Blot analysis. Alternatively, uterine horns were sectioned for immunohistochemistry (IHC). We and others showed previously the presence of apoptosis at estrus in rat uterine epithelium. In the present study, cleaved caspase-3, -6, and -7 fragments were detected at estrus. IHC confirmed that caspase-3 was present only in luminal and glandular epithelium at estrus. XIAP was highly expressed at estrus in both epithelial and stromal cells. In contrast, expression of Smac/DIABLO was elevated at diestrus, proestrus and metestrus but was minimal at estrus. Treatment of ovariectomized rats with 17β-estradiol induced XIAP expression and inhibited Smac/DIABLO protein expression in the endometrium. Cleaved caspase-3, -6, and -7 fragments increased in endometrial protein extracts following 17β-estradiol treatment. Expression of NF-κB and IκB proteins, and IκB phosphorylation status were detected in the endometrium but were not influenced by the estrous cycle. These findings suggest that Smac/DIABLO and XIAP are regulated differently and may play important roles in the regulation of endometrial cell fate. Moreover, this study confirms a key role for executioner caspases in the control of apoptotic processes at estrus in the rat uterus.     

Effects of 17β-estradiol replacement and treadmill exercise on vertebral and femoral bones of the ovariectomized rat

To evaluate the effect of 17β-estradiol replacement (10 μg, twice a week) (E₂) and treadmill exercise (18 m/min, 45 min/day) (EX) on long bone and vertebral bone mass and density, 10-month-old rats were ovariectomized (OV) and divided into four groups: OV, OV + E₂, OV + EX, OV + EX + E₂ 2 months after surgery. After 7 weeks intervention, the calcium content and the density of lumbar-5 were higher in both OV + E₂ and OV + EX + E₂ groups than in the OV group, but, only the OV + EX + E₂ group had a significantly higher femoral bone weight and density than the OV group. After 16 weeks intervention, the bone-conserving effects of E₂ and EX were significant on lumbar-5 and femoral dry weight and density. The effect of E₂ on both two sides of bones was due to the suppression of the bone turnover rate, while EX suppressed bone turnover rate primarily on the femur. We conclude that the effect of the two interventions on lumbar-5 and femoral bone mass were additive and independent.     

Effects of 17β-estradiol replacement on the apoptotic effects caused by ovariectomy in the rat hippocampus

AimsThe aim of the present study was to investigate the effects of different periods of ovariectomy and 17β-estradiol replacement on apoptotic cell death and expression of members of the Bcl-2 family in the rat hippocampus.Main methodsHippocampi were obtained from rats in proestrus, ovariectomized (15 days, 21 days and 36 days), ovariectomized for 15 days and then treated with 17β-estradiol for 7 or 21 days, and rats ovariectomized and immediately treated with 17β-estradiol for 21 days.The expression of Bcl-2 and Bax and the number of apoptotic cells were determined.Key findingsOvariectomy decreased Bcl-2 expression and increased Bax expression and the number of apoptotic cells. Replacement with 17β-estradiol (21 days) throughout the post-ovariectomy period reduced the number of apoptotic cells to the control levels, and prevented the effects of ovariectomy on Bax expression, but only partially restored the Bcl-2 expression. After 15 days of ovariectomy, the replacement with 17β-estradiol for 21 days, but not for 7 days, restored the Bcl-2 and Bax expression and the percentage of apoptotic cells to the levels found in the proestrus control.SignificanceThe present results show that a physiological concentration of 17β-estradiol may help maintain long-term neuronal viability by regulating the expression of members of the Bcl-2 family. Even after a period of hormonal deprivation, treatment with 17β-estradiol is able to restore the expression of Bax and Bcl-2 to control levels, but the duration of the treatment is a key factor to obtain the desired effect. These data provide new understanding into the mechanisms contributing to the neuroprotective action of estrogen.     

Covalent binding of 17β-estradiol and retinoic acid to proteins in the human breast cancer cell line MCF-7

Summary Both retinoic acid and 17β-estradiol formed covalent bonds with proteins of the human breast cancer cell line MCF-7. Two-dimensional gel patterns of the labeled proteins were unique for each ligand. There were four major retinoylated proteins in MCF-7 consisting of two doublets with molecular masses of 37 kDa and 20 kDa. These proteins were designated 37a, 37b, 37c, and 20d. The extent of retinoylation was very low in a 55 kDa protein that we previously identified in the human myeloid leukemia cell line HL60 [Takahashi, N. and Breitman, T. R. (1989) J. Biol. Chem. 264, 5159–5163]. These results indicated that the protein substrates for retinoylation may vary among cell-types. About 10 proteins were labeled from 17β-estradiol. Two of these proteins had mobilities that were identitied to the retinoylated proteins 37a and 20c. These results indicate that in MCF-7 cells there are two proteins that can be retinoylated and labeled from estradiol. The demonstration that some ligands of the steroid/thyroid receptor family are covalently linked to cellular proteins suggests new mechanisms for the many effects of these agents on cells. This study is the first report showing that estradiol or one of its metabolic products covalently binds to proteins in the human breast cancer cell line MCF-7. Two of the proteins labeled from radioactive estradiol comigrate with proteins labeled from radioactive retinoic acid. These results suggest new mechanisms of action for the steroid and thyroid hormones. EDITOR’S STATEMENT This study is the first report showing that estradiol or one of its metabolic products covalently binds to proteins in the human breast cancer cellline MCF-7. Two of the proteins labeled from radioactive estradiol comigrate with proteins labeled from radioactive retinoic acid. These results suggest new mechanisms of action for the steroid and thyroid hormones.     

Effect of 17β-estradiol on calcium response to phytohaemagglutinin in human lymphocytes

Pre-treatment of human lymphocytes with 17-estradiol diminishes the increase in concentration of cytosolic free calcium after stimulation with phytohaemagglutinin. The effect is dependent on 17-estradiol concentration and on the preincubation time. The effect is not due to an interaction between 17-estradiol and phytohaemagglutinin, but appears to be a consequence of the binding of the hormone to the cell surface. The effect is specific for 17-estradiol, since the isomer and other steroid hormones (progesterone, testosterone, diethylstilbestrol and 5-androstan), have no effect. Since the effect of the 17-estradiol can be suppressed by treatment of lymphocytes with ouabain, it appears that the effect of estradiol on the rise of cytosolic calcium induced by phytohaemagglutinin is mediated by the (Na, K)-ATPase.     

Effects of decorin on the expression of α-smooth muscle actin in a human myofibroblast cell line

Myofibroblasts are metabolically and morphologically distinctive fibroblasts expressing α-smooth muscle actin (α-SMA), and their activation plays a key role in development of the fibrotic response. In an activated state, myofibroblasts cease to proliferate and start to synthesize large amounts of extracellular component proteins. The expression of α-SMA correlates with the activation of myofibroblasts. Decorin, a member of the small leucine-rich proteoglycan gene family, has been implicated in the negative control of cell proliferation primarily by upregulating the expression of p21, a potent inhibitor of cyclin-dependent kinase. In order to examine the effect of decorin on myofibroblast cell growth, we rendered a human lung myofibroblast cell line, MRC-5, quiescent by either cell–cell contact or serum starvation, and examined the relationship between decorin and α-SMA expression in these cells. The expression of decorin in cells made quiescent by serum starvation was lower than that in cells made quiescent by cell–cell contact. In contrast, the expression of α-SMA in cells made quiescent by cell–cell contact was lower than that in cells made quiescent by serum starvation. Furthermore, forced expression of decorin was accompanied by a suppression of α-SMA expression, whereas knocking down of decorin expression by RNA interference increased the expression of α-SMA.     

Methylation of adenovirus genes in transformed cells and in vitro: influence on the regulation of gene expression?

An inverse correlation has been described between the levels of DNA methylation in specific segments of adenovirus DNA integrated into the genomes of transformed and tumor cells and the extent to which these segments are expressed as messenger RNA. In the adenovirus type 2 (Ad2)-transformed hamster cell lines HE2 and HE3, the virus-specific DNA binding protein (DBP) is not expressed, and the DNA in the DBP gene is completely methylated in all 5'-CCGG-3' sites. At least part of the late promoter/leader sequence of the DBP gene is present in cell lines HE2 and HE3. In line HE1, on the other hand, the DBP is expressed, and the DNA in the DBP gene is unmethylated at the 5'-CCGG-3' (HpaII) sites. The late promotor/leader sequence of the DBP gene is expressed in cytoplasmic RNA isolated from line HE1. The effect of DNA methylation has also been tested in vitro in a microinjection system using Xenopus laevis oocytes. Unmethylated DNA fragments of Ad2 (E2a region) have been found to serve as active templates. When the same fragments are methylated at the 5'-CCGG-3' sites by the HpaII DNA-methyltransferase, viral RNA synthesis is inhibited upon microinjection into oocyte nuclei. These results provide direct evidence for the notion that DNA methylated at highly specific sites is somehow involved in the regulation of gene expression.     

Effects of 17β-estradiol and bisphenol A on the formation of reproductive organs in planarians

Planarians have a remarkable capacity for regeneration after ablation, and they reproduce asexually by fission. However, some planarians can also reproduce and maintain their sexual organs. During the regenerative process, their existing sexual organs degenerate and new ones develop. However, little is known about hormonal regulation during the development of reproductive organs in planarians. In this study, we investigated the effects of 17β-estradiol (a steroid) and bisphenol A (an endocrine disrupter) on the formation of sexual organs in the hermaphroditic planarian Dugesia ryukyuensis. Under control conditions, all worm tissues regenerated into sexual planarians with sexual organs within 4 weeks after ablation. However, in the presence of bisphenol A or 17β-estradiol, although they apparently regenerated into sexual planarians, the yolk glands, which are one of the female sexual organs, failed to regenerate even 7 weeks after ablation. These data suggest that planarians have a steroid hormone system, which plays a key role in the formation and maturation of sexual organs.