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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We report a strategy for regulating the activity of a cytoplasmic signaling molecule, the protein kinase encoded by raf-1. Retroviruses encoding a gene fusion between an oncogenic form of human p74raf-1 and the hormone-binding domain of the human estrogen receptor (hrafER) were constructed. The fusion protein was nontransforming in the absence of estradiol but could be reversibly activated by the addition or removal of estradiol from the growth media. Activation of hrafER was accompanied in C7 3T3 cells by the rapid, protein synthesis-independent activation of both mitogen-activated protein (MAP) kinase kinase and p42/p44 MAP kinase and by phosphorylation of the resident p74raf-1 protein as demonstrated by decreased electrophoretic mobility. The phosphorylation of p74raf-1 had no effect on the kinase activity of the protein, indicating that mobility shift is an unreliable indicator of p74raf-1 enzymatic activity. Removal of estradiol from the growth media led to a rapid inactivation of the MAP kinase cascade. These results demonstrate that Raf-1 can activate the MAP kinase cascade in vivo, independent of other "upstream" signaling components. Parallel experiments performed with rat1a cells conditionally transformed by hrafER demonstrated activation of MAP kinase kinase in response to estradiol but no subsequent activation of p42/p44 MAP kinases or phosphorylation of p74raf-1. This result suggests that in rat1a cells, p42/p44 MAP kinase activation is not required for Raf-1-mediated oncogenic transformation. Estradiol-dependent activation of p42/p44 MAP kinases and phosphorylation of p74raf-1 was, however, observed in rat1a cells expressing hrafER when the cells were pretreated with okadaic acid. This result suggests that the level of protein phosphatase activity may play a crucial role in the regulation of the MAP kinase cascade. Our results provide the first example of a cytosolic signal transducer being harnessed by fusion to the hormone-binding domain of the estrogen receptor. This conditional system not only will aid the elucidation of the function of Raf-1 but also may be more broadly useful for the construction of conditional forms of other kinases and signaling molecules.
Mol Cell Biol 1993 Oct
PMID:Conditional transformation of cells and rapid activation of the mitogen-activated protein kinase cascade by an estradiol-dependent human raf-1 protein kinase. 841 24

Estrogens are considered to act as promoters in a multistep process of hormonal carcinogenesis, although the molecular mechanisms by which these hormones act in tumorigenesis are unclear at present. Estradiol is known to induce expression of certain proto-oncogenes, and this led us to examine potential regulatory regions of the cellular c-fos oncogene. The 5'-flanking region of the murine c-fos contains a 13-bp palindromic sequence (GGTCTnnnAGACC) with striking homology to the consensus estrogen-responsive element (ERE) GGTCAnnnTGACC. However, the c-fos sequence did not bind the human estrogen receptor or confer hormonal responsiveness in a yeast-based transcriptional test system. Importantly, a single base change in the fifth position of the c-fos sequence (GGTCTnnnAGACC to GGTCA/GnnnAGACC) produced an element that bound the estrogen receptor and conferred estrogen-dependent transcriptional activation of a reporter gene. This suggests a specific hypothesis by which estrogens could act as tumor promoters. In this paradigm, the regulatory region of the cellular oncogenes, tumor suppressor genes, and growth-factor genes contain inactive sequences with close homologies to hormone-responsive elements. Initiation occurs when some agent (e.g., a chemical carcinogen) causes a mutation in such a sequence to create a functional hormone-responsive element. Estrogens, acting through their receptors and the mutated element, can then activate the target gene to stimulate cell proliferation and increase the population of initiated cells.
Mol Carcinog 1993
PMID:Creation of an active estrogen-responsive element by a single base change in the flanking sequence of a cellular oncogene: a possible mechanism for hormonal carcinogenesis? 845 91

The ability of the sex hormones progesterone, testosterone and estradiol-17 beta and the glucocorticoid dexamethasone to modulate expression of the interleukin-5 (IL-5) gene in T cell lines has been investigated. The T cell lines used show analogous regulation of IL-5 gene expression to that occurring in T-lymphocytes, in that IL-5 mRNA levels are undetectable unless the cells are induced with phorbol myristate acetate (PMA). Progesterone and testosterone were as effective as PMA in inducing IL-5 mRNA levels in the T cell hybrid NIMP-TH1 and induced IL-5, -3 and -2 mRNA accumulation in the T cell lymphoma EL-4. Estradiol-17 beta also induced IL-5 mRNA accumulation but less effectively than testosterone. Nuclear run-on experiments suggested that the effects of progesterone, testosterone and PMA on IL-5 gene expression were mediated at the level of transcription. The presence of the protein synthesis inhibitor cycloheximide completely prevented PMA-induced synthesis of IL-5 mRNA by both NIMP-TH1 and EL-4 cells, indicating that induction of IL-5 mRNA via PMA stimulation requires de novo synthesis of a presumptive trans-acting factor(s). PMA-, testosterone- and progesterone-induced expression of the IL-5 gene was completely blocked by the anti-inflammatory steroid dexamethasone. Stimulation of IL-5 expression by PMA was relatively resistant to the immuno- suppressive drug cyclosporin A although inhibition did occur at very high levels. Testosterone- and progesterone-induced IL-5 gene expression was not inhibited by cyclosporin A. The in vivo significance of these findings are not yet clear but the results show that sex hormones have the potential to regulate cytokine gene expression in cells possessing the appropriate steroid receptors.
J Steroid Biochem Mol Biol 1993 Mar
PMID:Sex hormones and dexamethasone modulate interleukin-5 gene expression in T lymphocytes. 846 Dec 54

We describe here an in vitro technique to assess the estrogenic activity of chemicals. This technique is based on rainbow trout hepatocytes incubated in a basic medium free of any additional growth factors or estrogenic chemicals and uses the production of vitellogenin (VTG) as a marker for the estrogenic potency of the compounds tested. The system allows at least some of the metabolic transformations which are undertaken by the liver cells in vivo and could therefore be used for xenobiotic compounds which exhibit estrogenic activities after liver metabolic transformation. A dose-response curve was always consistently obtained using estradiol-17 beta (E2), with a mid point at around 100 nM E2 and a maximum response at around 1000 nM. Established estrogens such as 17 a 1 ethynylestradiol (EE2) or diethylstilboestrol (DES) were also tested. EE2 appeared to be equipotent with E2 and DES slightly less potent. E2 conjugates were, perhaps surprisingly, also very potent. Estradiol-3-sulfate was equipotent with E2 and estradiol-17 beta-glucuronide approx. 10% as potent. Other steroids such as androgens and progesterone, though active in the bioassay, were 3 orders of magnitude less potent than E2. Of the various steroids tested, only cortisol, at concentrations up to 50 microM, was completely inactive. Six different phytoestrogens were tested in the assay. All were weakly estrogenic, possessing approximately one thousandth the potency of E2 (they were as potent as the androgens and progesterone). All six phytoestrogens, as well as the androgens and progesterone, were tested in the presence of tamoxifen. In all cases tamoxifen reduced the production of VTG significantly, demonstrating that the estrogenic action of all of these compounds was most likely mediated by the E2 receptor. The potencies determined here may not reflect the situation in vivo but can provide complementary results about the activity of chemicals which need an hepatic metabolization to be estrogenic. Hepatocyte cultures would profitably be developed in other species to sustain these results.
J Steroid Biochem Mol Biol 1993 Mar
PMID:Vitellogenin synthesis in cultured hepatocytes; an in vitro test for the estrogenic potency of chemicals. 846 Dec 58

Estradiol-treated, rat pituitary cells were studied to examine the effects of progesterone (P) on follicle-stimulating hormone (FSH) synthesis and secretion. Progesterone was administered prior to or concurrent with 3 h secretory challenges with either gonadotropin-releasing hormone (GnRH), the iontophore A23187, the protein kinase C activator phorbol 12,13-myristate (PMA), or no secretagogue. Medium FSH levels and cell FSH stores were quantified by radioimmunoassay and bioassay. Acute (< 6 h) exposures to P increased medium levels of immunoreactive and bioactive FSH following GnRH challenge without influencing total (cell + medium) values whereas chronic (9-24 h) treatments increased both parameters. Chronic P elevated total FSH levels even when no secretagogue was present. Studies with antiprogestins, 5 alpha-dihydroprogesterone and 5 alpha-reductase inhibitors revealed that this direct action of P depended on progestin receptor occupation but not on 5 alpha-reduction. These studies indicate that P selectively increases bioactive and immunoactive FSH levels, presumably by increasing FSH synthesis, and characterize the time course and cellular mechanisms of this response. To accommodate for P modulation of total FSH levels, FSH secretion was standardized as the percentage of cellular stores available for release. Progesterone modulation of GnRH-stimulated FSH secretion was multiphasic, i.e. increased at 0-6 h, unchanged at 9 h and suppressed at 24 h. Acute and chronic exposures to P similarly modulated A23187-stimulated FSH release, whereas both P treatments increased PMA-stimulated FSH secretion. In these experiments P modulated luteinizing hormone secretion in parallel fashion, suggesting that common cellular mechanisms underlie peptidergic and steroidal regulation of the secretion of both gonadotropins.
Mol Cell Endocrinol 1993 Feb
PMID:Progesterone modulation of gonadotropin secretion by dispersed rat pituitary cells in culture. IV. Follicle-stimulating hormone synthesis and release. 847 44

Estradiol-17 beta (E2) is a mitogen in vivo for the proliferation of granulosa cells in the rat ovary. E2 is synthesized by the preovulatory follicle through a series of gonadotrophin-dependent events: LH stimulates thecal cells to synthesize androgens (androstenedione and testosterone) which are substrates for FSH-induced aromatization to estrogens in granulosa cells. More recently, we have found that transforming growth factor-beta (TGF-beta) stimulates DNA synthesis in rat granulosa cells in vitro and this effect is augmented by FSH. Since E2 is a mitogen in vivo and TGF-beta is the only known growth factor to stimulate proliferation in vitro, the possible link between the actions of E2 and TGF-beta were examined. E2 stimulated the secretion of a TGF-beta-like factor by rat granulosa cells in culture, and with time DNA synthesis was stimulated. The mitogenic action of E2 was enhanced in the presence of FSH, and attenuated by a neutralizing antibody to TGF-beta. The latter observations have identified TGF-beta as the "missing-link" in the mitogenic actions of E2 on rat granulosa cells. In addition to the growth-promoting actions of TGF-beta plus FSH, TGF-beta enhanced FSH-induced aromatase activity. Consequently, FSH plus TGF-beta stimulates both the proliferation and aromatization capacity of rat granulosa cells. We propose that interactions between FSH, E2 and TGF-beta lead to the exponential increase in serum E2 levels that occurs during the follicular phase of the cycle. Similarly, FSH stimulates the aromatization of exogenous androgens to estrogen by Sertoli cells isolated from immature rat testes, and there is a correlation between FSH-induced aromatization and mitotic activity. We have shown that FSH plus TGF-beta stimulates DNA synthesis in Sertoli cells. Since E2 increases the secretion of TGF-beta by Sertoli cells, interactions between FSH, E2 and TGF-beta may provide the mitogenic stimulus for Sertoli cells during the prepubertal period. In summary, our findings suggest that the estrogen-induced growth of rat granulosa cells is mediated through the production of TGF-beta, which acts as an autocrine regulator of proliferation. We also propose that the growth-promoting actions of FSH on Sertoli cells may depend upon a cascade series of events involving estrogens and TGF-beta.
J Steroid Biochem Mol Biol 1993 Mar
PMID:Interactions between FSH, estradiol-17 beta and transforming growth factor-beta regulate growth and differentiation in the rat gonad. 847 58

The intracellular conversion of testosterone to estradiol by the aromatase enzyme complex is an important step in many of the central actions of testosterone. In rats, estrogen given alone, or in combination with dihydrotestosterone, mimics most of the behavioral effects of testosterone, whereas treatment with antiestrogens or aromatase inhibitors block facilitation of copulatory behavior by testosterone. We used a highly sensitive in vitro radiometric assay to analyze the distribution and regulation of brain aromatase activity. Studies using micropunch dissections revealed that the highest levels of aromatase activity are found in an interconnected group of sexually dimorphic nuclei which constitutes a neural circuit important in the control of male sexual behavior. Androgen regulated aromatase activity in many diencephalic nuclei, including the medial preoptic nucleus, but not in the medial and cortical nuclei of the amygdala. Additional genetic evidence for both androgen-dependent and -independent control of brain AA was obtained by studies of androgen-insensitive testicular-feminized rats. These observations suggest that critical differences in enzyme responsiveness are present in different brain areas. Within several nuclei, sex differences in aromatase induction correlated with differences in nuclear androgen receptor concentrations suggesting that neural responsiveness to testosterone is sexually differentiated. Estradiol and dihydrotestosterone acted synergistically to regulate aromatase activity in the preoptic area. In addition, time-course studies showed that estrogen treatment increased the duration of nuclear androgen receptor occupation in the preoptic area of male rats treated with dihydrotestosterone. These results suggest possible ways that estrogens and androgens may interact at the cellular level to regulate neural function and behavior.
J Steroid Biochem Mol Biol 1993 Mar
PMID:Aromatase activity in the rat brain: hormonal regulation and sex differences. 847 64

Androgens down-regulate the levels of androgen receptors (AR) and AR mRNA in the penis and prostate of castrated rats, and are assumed to cause their decrease during sexual maturation in the penile smooth muscle of intact rats. In order to determine whether these effects occur directly at the target cell level, and to what extent they are due to testosterone (T) or to their metabolites, we have measured AR mRNA in cultures of smooth muscle cells from the adult rat corpora cavernosa treated in vitro with sex steroids. T at high concentrations (100 nM) acted like dihydrotestosterone (DHT) in increasing moderately the levels of AR mRNA in both proliferating and contact-inhibited cells. However, when conversion of T to DHT was blocked by the 5-alpha reductase inhibitor finasteride, the levels of AR mRNA were considerably down-regulated by T (10-500 nM), particularly in the contact-inhibited cells. Finasteride by itself was inactive. These effects in both types of cultures were inhibited by platelet derived growth factor (PDGF) (20 ng/ml), a growth factor that up-regulates AR mRNA levels, and by fadrozole (100 nM), an aromatase inhibitor of the T/estrogen conversion. Estradiol (50 nM) was even more potent than T in decreasing AR mRNA levels. With the exception of PDGF none of the treatments affected significantly cell growth, as measured by DNA synthesis and content. Our results indicate that it is possible to modulate in vitro AR mRNA levels in the penile smooth muscle cells, and that under normal conditions DHT and T act as moderate up-regulators. When DHT formation is inhibited, the aromatization pathway of T to estradiol will prevail and induce a pronounced down-regulation of AR mRNA levels. We assume that the in vivo AR down-regulation in the penile smooth muscle by androgens is an indirect effect mediated by a paracrine or endocrine mechanism elicited in another tissue.
J Steroid Biochem Mol Biol 1993 May
PMID:Testosterone down-regulates the levels of androgen receptor mRNA in smooth muscle cells from the rat corpora cavernosa via aromatization to estrogens. 849 43

In England, pharmacologists and a biochemist at the University of Liverpool used an established human endometrial cancer cell line (HEC-1A) and human endometrial tissue in vitro to confirm that HEC-1A and tissue metabolize oral contraceptive (OC) steroids. They used on-line radiometric high-performance liquid chromatography to analyze metabolic activity. Surgeons obtained the endometrial tissue from women undergoing dilatation and curettage or hysterectomy at the Royal Liverpool University Hospital. Earlier research showed that HEC-1A cells interconvert estrone (E1) and 17 beta-estradiol (E2), with E2 predominating in the equilibrium. In this in vitro study, healthy endometrial tissue extensively changed E2 to E1, while malignant tissue caused this conversion to a much lesser extent. The healthy endometrial tissue of some patients formed sulphate conjugates. Both HEC-1A and endometrial tissue hydrolyzed E1 3-sulphate. They did not bring about phase I metabolism when ethinyl estradiol (EE2) was the substrate. Yet, incubation with healthy tissue from some women did lead to conversion of the presumed 3-sulphate conjugate. Incubation with HEC-1A cells completely removed the acetyl group from norgestimate, resulting in mainly norgestrel oxime (55.1% of metabolites) within 24 hours. It also resulted in some norgestrel (16.3%). Incubation with endometrial tissue also brought about complete metabolism of norgestimate within 24 hours. The tissue from different women brought about qualitative and quantitative differences. HEC-1A and endometrial tissue did not metabolize much of 3-ketodesogestrel (3-KDG). In fact, the major metabolite formed by HEC-1A was 3 alpha-hydroxydesogestrel, which made up 3.3% of total added radiolabeled steroid. Healthy endometrial tissue did not produce any phase I metabolites of 3-ketodesogestrel, while tumor tissue may have produced a small amount of radiometabolites. These findings indicate that the endometrium does metabolize the OC EE2, 3-KDG, and norgestimate.
J Steroid Biochem Mol Biol 1993 May
PMID:Metabolism of the oral contraceptive steroids ethynylestradiol, norgestimate and 3-ketodesogestrel by a human endometrial cancer cell line (HEC-1A) and endometrial tissue in vitro. 849 48

Oral therapy with natural or synthetic estrogens, like ethinylestradiol, suffers from low, suboptimally defined bioavailability and excess hepatic estrogen actions. N,N-alkylated and non-alkylated sulfamates of ethinylestradiol, estradiol and estrone overcome these deficiencies. Ovariectomized Wistar rats (n = 6-7/group) were orally treated for 7 days, and killed on day 8, plasma was gained on days 0, 4, and 8. Systemic estrogenicity was quantified by assessment of uterine weight, vaginal cornification, and measurement of gonadotropins by homologous RIA. Estrogenicity in the liver was analysed. Angiotensinogen was estimated by RIA of angiotensin-1 after incubation of EDTA-plasma with porcine renin. Total and high-density cholesterol were measured by enzymatic methods. Preliminary biotransformation studies were performed after oral administration of 10 micrograms, 5 microCi [2,4,6,7-3H]estradiol sulfamate. Ethinylestradiol led to distinct elevation of angiotensin-1 and dramatic depression of cholesterol fractions, reflecting hepatic estrogen effects, already at doses with marginal systemic effects. Estradiol and estrone had systemic and hepatic estrogenic activity at much higher doses only. Estrogen sulfamates had systemic estrogen activity 10-90-fold above that of their parent estrogen. Non-alkylated sulfamates of given estrogens were more active than N-alkylated ones. Elevation of systemic estrogen activity was always combined with a dramatic reduction of hepatic estrogenicity. Estradiol sulfamate had a 90-fold elevated systemic estrogen activity vs estradiol, but lacked hepatic activity including the 30-fold dose inducing vaginal response. Three hours after administration no unchanged estradiol sulfamate was detectable in plasma. Rather peaks, probably representing estradiol and estrone, were found. Estrogen sulfamates are considered prodrugs of their parent estrogen, which do not interact with any liver function during the first-pass. They represent a new strategy of oral hormone administration. Their main potential seems to be the systemic generation of natural estrogens when used in oral contraceptives.
J Steroid Biochem Mol Biol 1995 Dec
PMID:Sulfamates of various estrogens are prodrugs with increased systemic and reduced hepatic estrogenicity at oral application. 854 Dec 36


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