Regulation of SBP synthesis in human cancer cell lines by steroid and thyroid hormones

The presence of human sex steroid binding protein (SBP) in liver cells, the supposed site of SBP synthesis, and in other target cells for sex steroid hormones such as breast, endometrium and prostate epithelium, have been demonstrated by indirect immunofluorescence. It is not known whether SBP enters endometrial and prostate cells by endocytosis, possibly mediated by a cell membrane receptor process, or if SBP is synthesized in these cells. SBP mRNA has been searched in human cancer cell lines originated from liver (Hep G2/H5A), breast (MCF-7), endometrium (RL95-2) and prostate (LNCaP). It was only found in hepatoma cells where it is regulated by estradiol, antiestrogen tamoxifen and triiodothyronine, in a similar way as secreted SBP. This work provides evidence that human SBP is synthesized in the liver, and it also suggests that its regulation may involve non-steroidal factors.     

Estrogen regulates the IFN-gamma promoter

The greater immune reactivity of females has been attributed in part to the influence of sex steroid hormones, but the underlying mechanisms are unknown. Here we report evidence that expression of the IFN-gamma gene may be subject to direct hormonal control. In a transient expression assay, the sex steroid 17 beta-estradiol markedly increases activity of the IFN-gamma promoter in lymphoid cells that express the appropriate hormone receptor. This effect is mediated by sequences in the 5'-flanking region of the gene, and can augment the effect of T cell-activating agents. Short term exposure to estradiol also increases IFN-gamma mRNA expression in Con A-treated murine spleen cells. Hormonal regulation of this pleiotropic cytokine may account in part for the ability of estrogen to potentiate many types of immune responses, and for the disproportionate susceptibility of females to autoimmune disease.     

Hormonal control of SBP in human hepatoma cells

Since it is currently believed that the biosynthesis of human sex steroid binding plasma protein (SBP) takes place in the liver, the secretion of this protein and its hormonal control were studied in a human hepatoma cell line. The human hepatoma-derived cell line, Hep G2, and a clone, H5A, isolated from Hep G2, were both found to secrete SBP-like protein. This protein had the same dihydrotestosterone binding parameters as plasma SBP, with a Kd ranging from 0.3 to 1 nM at 4 degrees C, and it cross-reacted with a monospecific goat anti-human SBP antiserum. In a chemically defined medium, SBP-like protein secretion was stimulated approx 2-fold by estradiol (1 microM) whereas a smaller concentration of estrogen (100 nM) has only a slight effect. A combined incubation with estradiol (100 nM) and triiodothyronine (10 nM) increased SBP-like protein secretion more than estradiol (1 microM) alone. In response to dexamethasone (100 nM) or tamoxifen (100 nM) treatment, a 3-fold increase is obtained. Therefore, these human parenchymal cells should provide a potent material for investigation of the hormonal regulation of SBP gene.     

Interaction of blood sex steroid-binding globulin-steroid complexes with plasma membranes of human decidual endometrium cells

Plasma membranes of decidual tissue cells specifically bind the sex steroid-binding globulin (SBP) complexes with estrogen (estradiol, estriol, estrone) and with the pharmacological agent danazol but do not interact with the SBP-testosterone or SBP-dihydrotestosterone complexes. The selectivity of interaction of the SBP-steroid complexes with decidual tissue cellular membranes provide evidence for the active role of SBP in the realization of steroid effects on the target tissue.     

Androgens and bone

Testosterone is the major gonadal sex steroid produced by the testes in men. Testosterone is also produced in smaller amounts by the ovaries in women. The adrenal glands produce the weaker androgens dehydroepiandrosterone, dehydroepiandrosterone sulfate, and androstenedione. These androgens collectively affect skeletal homeostasis throughout life in both men and women, particularly at puberty and during adult life. Because testosterone can be metabolized to estradiol by the aromatase enzyme, there has been controversy as to which gonadal sex steroid has the greater skeletal effect. The current evidence suggests that estradiol plays a greater role in maintenance of skeletal health than testosterone, but that androgens also have direct beneficial effects on bone. Supraphysiological levels of testosterone likely have similar effects on bone as lower levels via direct interaction with androgen receptors, as well as effects mediated by estrogen receptors after aromatization to estradiol. Whether high doses of synthetic, non-aromatizable androgens may, in fact, be detrimental to bone due to suppression of endogenous testosterone (and estrogen) levels is a potential concern that warrants further study.     

Hormonally mediated epigenetic changes to steroid receptors in the developing brain: Implications for sexual differentiation

The establishment of sex-specific neural morphology, which underlies sex-specific behaviors, occurs during a perinatal sensitive window in which brief exposure to gonadal steroid hormones produces permanent masculinization of the brain. In the rodent, estradiol derived from testicular androgens is a principal organizational hormone. The mechanism by which transient estradiol exposure induces permanent differences in neuronal anatomy has been widely investigated, but remains elusive. Epigenetic changes, such as DNA methylation, allow environmental influences to alter long-term gene expression patterns and therefore may be a potential mediator of estradiol-induced organization of the neonatal brain. Here we review data that demonstrate sex and estradiol-induced differences in DNA methylation on the estrogen receptor α (ERα), estrogen receptor β (ERβ), and progesterone receptor (PR) promoters in sexually dimorphic brain regions across development. Contrary to the overarching view of DNA methylation as a permanent modification directly tied to gene expression, these data demonstrate that methylation patterns on steroid hormone receptors change across the life span and do not necessarily predict expression. Although further exploration into the mechanism and significance of estradiol-induced alterations in DNA methylation patterns in the neonatal brain is necessary, these results provide preliminary evidence that epigenetic alterations can occur in response to early hormone exposure and may mediate estradiol-induced organization of sex differences in the neonatal brain.     

Sex steroid binding protein (SBP) receptors in estrogen sensitive tissues

Since the discovery of a specific membrane binding site for sex steroid binding protein (SBP) in human decidual endometrium and in hyperplastic prostate numerous speculations have been raised on the existence of an additional non-receptor-mediated system for steroid hormone action. In the present work SBP cell membrane binding was investigated in human estrogen target tissues other than those previously studied either in the absence of steroids or in the presence of varying amounts (10⁻¹⁰−10⁻⁶M) of estradiol, testosterone and dihydrotestosterone, respectively. Plasma membranes obtained by differential centrifugation from homogenized samples of pre-menopausal endometrium, endometrium adenocarcinoma, normal liver and post-menopausal breast showed a specific binding of highly purified [¹²⁵I]SBP: a major displacement of labeled SBP was elicited by radioinert SBP, while no significant displacement occurred when other human plasma proteins were used as cold competitors (molar excess ranging 500–10,000-fold). A specific, time-dependent binding of [¹²⁵I]SBP was also observed in MCF-7 and in Hep-G2 cell lines. The different patterns of specific binding, observed in membranes from different tissues when SBP was liganded with different sex steroid molecules, leads us to consider the tissue individuality of the receptor as a further entity in the membrane recognition system for SBP.     

Comparative contents of mRNAs of sex steroid receptors and enzymes of their metabolism in arterial walls of men

The potential role of estrogens in regulation of metabolism in arteries of men was studied. Contents of mRNAs of sex hormone receptors, of some enzymes of their metabolism, and of some potential markers of the hormone effects were determined by real-time polymerase chain reaction in fragments of 18-54-year-old men's large arteries with and without atherosclerotic lesions. Contents of estrogen receptor alpha (ERalpha) and transferrin receptor mRNAs were significantly different in undamaged fragments of the aorta and of the carotid and coronary arteries. Contents of some mRNAs in the carotid artery and aorta were found to correlate, which suggested a similarly directed regulation of their expressions. The levels of ERalpha and aromatase mRNAs negatively correlated with the blood plasma concentration of estradiol. Levels of steroid sulfatase and aromatase mRNAs were lower and the level of estrogen sulfotransferase mRNA was higher in blood vessel fragments with atherosclerotic lesions than in undamaged fragments. It is suggested that large arteries should be different in sensitivity to estrogens and that atherosclerotic lesions could lead to local suppression of the effect of estrogen on the cells of arteries.     

Sex steroid hormone metabolism takes place in human ocular cells

Steroids are potentially important mediators in the pathophysiology of ocular diseases. In this study, we report on the gene expression in the human eye of a group of enzymes, the 17beta-hydroxysteroid dehydrogenases (17HSDs), involved in the biosynthesis and inactivation of sex steroid hormones. In the eye, the ciliary epithelium, a neuroendocrine secretory epithelium, co-expresses the highest levels of 17HSD2 and 5 mRNAs, and in lesser level 17HSD7 mRNA. The regulation of gene expression of these enzymes was investigated in vitro in cell lines, ODM-C4 and chronic open glaucoma (GCE), used as cell models of the human ciliary epithelium. The estrogen, 17beta-estradiol (10(-7) M) and androgen agonist, R1881 (10(-8) M) elicited in ODM-C4 and GCE cells over a 24 h time course a robust up-regulation of 17HSD7 mRNA expression. 17HSD2 was up-regulated by estradiol in ODM-C4 cells, but not in GCE cells. Under steady-state conditions, ODM-C4 cells exhibited a predominant 17HSD2 oxidative enzymatic activity. In contrast, 17HSD2 activity was low or absent in GCE cells. Our collective data suggest that cultured human ciliary epithelial cells are able to metabolize estrogen, androgen and progesterone, and that 17HSD2 and 7 in these cells are sex steroid hormone-responsive genes and 17HSD7 is responsible to keep on intra/paracrine estrogenic milieu.     

IL-1beta-mediated proinflammatory responses are inhibited by estradiol via down-regulation of IL-1 receptor type I in uterine epithelial cells

The objective of this study was to examine the effects of sex hormones on IL-1beta-mediated responses by uterine epithelial cells. The mRNA expression and secretion of human beta-defensin-2 and CXCL8 by uterine epithelial cells was examined following stimulation with IL-1beta in the presence of estradiol or progesterone. Estradiol inhibited the IL-1beta-mediated mRNA expression and secretion of human beta-defensin-2 and CXCL8 by uterine epithelial cells while progesterone had no effect. Inhibition of the IL-1beta-mediated response by estradiol was dose dependent, with maximal inhibition observed using 10(-7) to 10(-10) M, and was shown to be mediated through the estrogen receptor because addition of a pure estrogen receptor antagonist abrogated this effect. The mechanism by which estradiol inhibits IL-1beta-mediated responses by uterine epithelial cells appears to be the down-modulation of the IL-1R type I, thereby reducing the uterine epithelial cell's ability to respond to IL-1beta. These results suggest that the inhibitory effect of estradiol on IL-1beta-mediated inflammatory responses by uterine epithelial cells indicates a link between the endocrine and immune systems and may be crucial for dampening proinflammatory responses during the time of ovulation or pregnancy.     

Smoking effects on the hormonal balance of fertile women.

We evaluated serum pituitary hormones (prolactin, follicle-stimulating hormone, luteinizing hormone), gonadal hormones (estrone, estradiol, progesterone), sex steroid binding protein (SBP) and urine estrogens in 684 healthy fertile women, subdivided into smokers (n = 237) and nonsmokers (n = 447). The aim of the work was to elucidate whether smoking habits can affect hormonal balance. Smoking interference of estrogen metabolism has been postulated, but no unequivocal data have been reported. A protective role against breast cancer has even been suggested on the basis of a reduced estrogenic activity found in smokers. Our data showed a considerable interference of smoking on PRL secretion, probably related to a direct inhibiting activity of nicotine. Estrogen catabolism could also be involved, and a catabolic shift of 16 alpha-hydroxylation in favour of 2 alpha-hydroxylated catabolites, via the hepatic cytochrome P-450 system could be hypothesized.     

Expression of steroidogenic enzymes and synthesis of sex steroid hormones from DHEA in skeletal muscle of rats

The functional importance of sex steroid hormones (testosterone and estrogens), derived from extragonadal tissues, has recently gained significant appreciation. Circulating dehydroepiandrosterone (DHEA) is peripherally taken up and converted to testosterone by 3beta-hydroxysteroid dehydrogenase (HSD) and 17beta-HSD, and testosterone in turn is irreversibly converted to estrogens by aromatase cytochrome P-450 (P450arom). Although sex steroid hormones have been implicated in skeletal muscle regulation and adaptation, it is unclear whether skeletal muscles have a local steroidogenic enzymatic machinery capable of metabolizing circulating DHEA. Thus, here, we investigate whether the three key steroidogenic enzymes (3beta-HSD, 17beta-HSD, and P450arom) are present in the skeletal muscle and are capable of generating sex steroid hormones. Consistent with our hypothesis, the present study demonstrates mRNA and protein expression of these enzymes in the skeletal muscle cells of rats both in vivo and in culture (in vitro). Importantly, we also show an intracellular formation of testosterone and estradiol from DHEA or testosterone in cultured muscle cells in a dose-dependent manner. These findings are novel and important in that they provide the first evidence showing that skeletal muscles are capable of locally synthesizing sex steroid hormones from circulating DHEA or testosterone.     

Estrogen receptors and androgen receptors in the mammalian liver

An estrogen receptor and an androgen receptor are present in the mammalian liver. In the liver of the rat, the estrogen receptor concentration increases markedly at puberty and this change correlates with enhanced estrogen stimulation of plasma renin substrate synthesis. High doses of estrogen are required for nuclear binding in liver when compared to doses for the uterus. The high dose requirement appears to be predominantly due to extensive metabolism in the hepatocyte of the estrogen to inactive derivatives. Furthermore, estradiol is much weaker than ethinyl estradiol for promoting nuclear binding in the liver. This is due to extremely rapid and extensive metabolism of estradiol. In human liver the concentration of estrogen receptor is low. An androgen receptor is present in high concentration in rabbit liver and is located predominantly in the nucleus after androgen administration. High concentrations of a putative androgen receptor are also present in human liver cytosol. Preliminary studies indicate that synthetic progestins can attach to the human liver androgen receptor. To date, a progesterone receptor has not been found in the mammalian liver. Thus, it appears that extensive steroid metabolism in liver preferentially diminishes sex steroid interaction with liver receptors and that androgen receptors may mediate progestin effects in liver. These observations provide a scientific basis for improved safety of oral contraceptives. Lowering the estrogen and progestin doses in oral contraceptives will decrease the major side-effects, which are liver mediated, and still maintain the desired effects at the hypothalamic-pituitary axis and uterus. Furthermore, it is likely that by selecting which estrogen, progestin or androgen is administered as well as by utilizing a parenteral route of administration that sex steroid effects on the liver could be minimized.     

Steroid sulfatase activity and expression in mammary myoepithelial cells

PURPOSE: This investigation examined mRNA expression and enzymatic activity of steroid sulfatase (STS) in human mammary myoepithelial cells (MMECs) and MCF-7 cells and assessed the effects of 17-beta estradiol on the activity of STS. METHODS: The mRNA level of STS in MMECs was determined by RT-PCR analysis using specific primers for STS. STS enzymatic activity prior to and after treatment with 17-beta estradiol was determined by measuring 3H-metabolites formed after exposure to [3H]estrone 3-sulfate (E1S) and [3H]dehydroepiandrosterone-sulfate (DHEA-S). RESULTS: Our data demonstrate the presence of STS in the MMECs. Based on RT-PCR analysis, MMECs had slightly lower levels of STS compared to MCF-7 cells. However, sulfatase activity was about 120 times greater in the MMECs than the MCF-7 cells (E1S V(max)=2640nmol/(mg DNAh) compared to 20.9nmol/(mg DNAh)). Exposure to 17-beta estradiol was associated with 70% reduction in E1S sulfatase activity in the MCF-7 cells and 9% increase in the MMECs after 6 days. DISCUSSION: Our studies indicate for the first time the presence of STS in MMECs. This is suggestive of a previously undetermined role for MMECs in converting precursor hormones into active steroid hormones within mammary tissue. In addition, differential response of the MMECs and the MCF-7 cells to estrogen demonstrates differences in hormone metabolism between these two cell types, perhaps related to the absence of estrogen receptors in the MMECs and their presence in the MCF-7 cells. The MMECs may have an important role in hormonal regulation within mammary tissue.     

Binding of xenoestrogens to the sex steroid-binding protein in plasma from Arctic charr (Salvelinus alpinus L.)

A specific sex steroid-binding protein (SBP) is believed to be involved in regulation of circulating sex steroids, steroid delivery to target cells and intracellular signalling in sex steroid-sensitive tissues. In the present work, interactions between xenoestrogens and the plasma SBP in Arctic charr (Salvelinus alpinus L.) were determined using ligand-protein binding studies. The test compounds were all able to displace tritiated 17 beta-estradiol (E2) from the Arctic charr SBP (acSBP) in a competitive and dose-dependent manner. The acSBP affinities for the xenoestrogens ranged over several orders of magnitude (17 beta-estradiol>ethynylestradiol (EE2)>zearalenone (ZEA)>diethylstilbestrol (DES)>genistein (GEN)>bisphenol A (BPA), 4-t-octylphenol (OP)>o,p'-DDT, and dieldrin (DIN)), but were consistently lower than that of 17 beta-estradiol (about 4 x 10(2) -10(6)-fold less potent). The relative binding affinity (RBA) for selected chemicals were independent of both gender, age and maturation status, as well as variations of acSBP binding affinity. The affinity of endogenous steroids and estrogen mimics for the acSBP shows a high correlation to the affinity for the rainbow trout SBP, thus suggesting a phylogenetically conserved ligand-binding site between closely related species. Furthermore, it is argued that interaction with the acSBP- and SBP-mediated processes may introduce novel pathways for endocrine disruption, which may work in concert with the classical receptor-mediated effects.     

Regucalcin is under‐expressed in human breast and prostate cancers: Effect of sex steroid hormones

Regucalcin plays an important role in maintenance of intracellular Ca²⁺ homeostasis, suppresses cell proliferation, inhibits expression of oncogenes, and increases the expression of tumour suppressor genes. This suggests that regucalcin functions may be altered in cancer tissues. In this study the regucalcin expression in breast and prostate cancer cases was analysed by RT‐PCR and immunohistochemistry showing that the mRNA and/or protein are under‐expressed in these tumors. The effect of sex steroid hormones on regucalcin expression in breast and prostate cancer cells was determined by real‐time PCR. MCF‐7 and LNCaP cells were stimulated with 0, 1, and 10 nM of 17β‐estradiol (E₂) or 5α‐dihydrotestosterone (DHT), respectively, for 0, 6, 12, 24, and 48 h. MCF‐7 cells were also stimulated with E₂ conjugated to BSA (E₂‐BSA). To explore the mechanisms underlying the sex steroid regulation of regucalcin expression, control treatments with ICI 182,780, flutamide and cyclohexamide were carried out. E₂ effects regulating regucalcin expression were not abrogated in the presence of ICI 182,780, and were similar to those observed with E₂‐BSA, which suggests the involvement of a membrane‐bound estrogen receptor. In LNCaP cells, DHT down‐regulated regucalcin expression, an effect inhibited by the presence of both flutamide and cyclohexamide, suggesting the involvement of androgen receptor and de novo protein synthesis. The loss of regucalcin expression in breast and prostate cancer cases and the regulation of its expression by sex steroid hormones suggest that it may be associated with development and progression of these human tumors. J. Cell. Biochem. 107: 667–676, 2009. © 2009 Wiley‐Liss, Inc.