Arachidonic acid as a possible modulator of estrogen, progestin, androgen, and glucocorticoid receptors in the central and peripheral tissues

In an attempt to learn whether modulation of steroid hormone receptor by arachidonate is generalized or not, the arachidonate effect was examined in cytosol estrogen (ER), progestin (PR), androgen (AR) and glucocorticoid receptors (GCR) from the central and peripheral tissues of rats by sucrose density gradient centrifugation, and gel filtration on LH20 columns or dextran-coated charcoal absorption. Arachidonate and other long-chain fatty acids appear to inhibit the specific binding of estrogen ([3H]R2858), progestin ([3H]R5020), androgen ([3H]R1881) and glucocorticoid ([3H]dexamethasone) to the respective receptors in brain (neonatal cerebral cortex and hypothalamus-preoptic area, HPOA), uterus and prostate, with the exception of the potentiating effect on the brain estrogen receptors. The potency of the unsaturated fatty acids paralleled to some degree the number of cis double bonds and carbon, in that oleate (C18:1) arachidonate (C20:4) docosahexaenoate (C22:6). The arachidonate inhibition was dose-dependent in the tissue steroid hormone receptors, except for dose-dependent potentiation of the brain cortical estrogen receptors. Inhibitory potency as expressed by the concentration for 50% maximum inhibition (Ki) was in the range of 11-18 microM for the receptors other than the uterine estrogen receptors with the value of 44 microM, suggesting lower sensitivity for the estrogen receptor to the arachidonate effect in the uterus. Analysis on kinetics and Scatchard plot revealed the non-competitive type of the inhibition. In addition, arachidonate lowered dose-dependently the peak of labelled progestin or estrogen binding to the 8S receptor proteins, which were collected from fractions in the 8S region of the cytosols from intact or diethylstibestrol-primed rat uteri. These results suggest the generalized modulatory effect of arachidonate on the steroid hormone receptors in the central and peripheral tissues. Arachidonate could affect, negatively or positively, the estrogen receptors, and negatively the progestin, androgen and glucocorticoid receptors, through a possibly direct but weak binding at sites different from steroid binding sites on the receptor molecules. A potential messenger role of arachidonate itself has been implicated in the regulation or modulation of the steroid hormone receptors.     

The stringency and magnitude of androgen-specific gene activation are combinatorial functions of receptor and nonreceptor binding site sequences.

The mechanism by which specific hormonal regulation of gene expression is attained in vivo is a paradox in that several of the steroid receptors recognize the same DNA element in vitro. We have characterized a complex enhancer of the mouse sex-limited protein (Slp) gene that is activated exclusively by androgens but not by glucocorticoids in transfection. Potent androgen induction requires both the consensus hormone response element (HRE) and auxiliary elements residing within the 120-bp DNA fragment C' delta 9. Multiple nonreceptor factors are involved in androgen specificity, with respect to both the elevation of androgen receptor activity and the inactivity of glucocorticoid receptor (GR), since clustered base changes at any of several sites reduce or abolish androgen induction and do not increase glucocorticoid response. However, moving the HRE as little as 10 bases away from the rest of the enhancer allows GR to function, suggesting that GR is repressed by juxtaposition to particular factors within the androgen-specific complex. Surprisingly, some sequence variations of the HRE itself, within the context of C' delta 9, alter the stringency of specificity, as well as the magnitude, of hormonal response. These HRE sequence effects on expression correspond in a qualitative manner with receptor binding, i.e., GR shows a threefold difference in affinities for HREs amongst which androgen receptor does not discriminate. Altering the HRE orientation within the enhancer also affects hormonal stringency, increasing glucocorticoid but not androgen response. The effect of these subtle variations suggests that they alter receptor position with respect to other factors. Thus, protein-protein interactions that elicit specific gene regulation are established by the array of DNA elements in a complex enhancer and can be modulated by sequence variations within these elements that may influence selection of precise protein contacts.     

甾体激素受体功能特异性的结构基础

甾体激素受体家族包括雌激素受体、雄激素受体等五个亚家族,在机体组织细胞的生长分化、发育生殖、内环境稳定等几乎所有生理过程中都起着重要的作用。研究甾体激素受体亚家族的特异性可以加深对该家族功能的理解,并且具有潜在的临床应用价值。采用进化踪迹方法对该家族的配体结合域（LBD）进行分析,探讨了决定亚家族功能特异性的结构基础。结果表明,甾体激素受体的各亚家族可能同相应的内源性配体存在着共进化关系;配体结合处的踪迹残基决定了受体－配体间的氢键作用和疏水相互作用模式并导致了亚家族的配体结合特异性。上述结论可用于甾体激素受体的配体结合特异性的改造以及新型组织选择性配体（如选择性雌激素受体调节剂,SERM）的设计。     

A new highly androgen specific yeast biosensor, enabling optimisation of (Q)SAR model approaches

Recently we constructed recombinant yeast cells that express the human androgen receptor (hAR) and yeast enhanced green fluorescent protein (yEGFP), the latter in response to androgens. When exposed to 17beta-testosterone, the concentration where half-maximal activation is reached (EC50) was 50 nM. Relative androgenic potencies (RAP), defined as the ratio between the EC50 of 17beta-testosterone and the EC50 of the compound, were 1.7, 1.2 and 0.008 for 19-nortestosterone, tetrahydrogestrinone and 17beta-estradiol respectively. Steroids representative for other hormone receptors, like estrone, 17alpha-ethynylestradiol, and diethylstilbestrol for the estrogen receptor and corticosterone and dexamethasone for the glucocorticoid receptor, showed no agonistic response. Only compounds known to exert androgenic effects give a response. Determined RAPs were in line with results obtained from optimised QSAR model calculations and demonstrated that Saccharomyces cerevisiae showed no metabolism of test compounds and displayed no crosstalk from endogenous hormone receptors. The suitability of this bioassay to verify the outcomes of (Q)SAR models to predict the activities of different steroids was further examined by studies with steroid isomers and a number of designer steroids, confirming that the 17beta-hydroxyl group, 3-keto group and 5alpha-steroidal framework are extremely important for the activity of the androgenic steroid.     

Medroxyprogesterone acetate: receptor binding and correlated effects on steroidogenesis in rat granulosa cells

Medroxyprogesterone acetate (MPA), a widely used synthetic steroid, was studied to determine both its effects on steroid receptors and steroidogenesis in the well-characterized rat ovarian granulosa cell model. Initial receptor binding studies showed MPA was as potent as progesterone and 10-fold less potent than R-5020 (an active synthetic progestin) in binding to progesterone cytosolic receptors in rat ovarian granulosa cells. MPA was 20-fold less potent than testosterone, and 10-fold less potent than dexamethasone in binding to the androgen and glucocorticoid cytosolic receptors, respectively. The binding of MPA to progestrone, androgen and glucocorticoid receptors predicted direct effects of MPA on FSH-stimulated estrogen (E), progesterone (P), and 20 alpha-dihydroprogesterone (DHP) production by cultured rat ovarian granulosa cells. MPA at 10(-7) to 10(-6) M significantly augmented FSH-stimulated P and DHP production (a previously documented progestin, androgen and glucocorticoid effect). This augmentation was blocked by the concurrent addition to cell culture of 10-fold excess RU-486 (a potent anti-progestin and anti-glucocorticoid). At concentrations greater than 10(-6) M, MPA inhibited the production of P and DHP (a progestin effect), and the production of E (a progestin and glucocorticoid effect). MPA, structurally a progestin, has complex steroid hormone effects predicted by its interaction with progesterone, androgen and glucocorticoid receptors.     

Structurally based, selective interaction of arsenite with steroid receptors

Steroid binding to cognate receptors is of high affinity. However, due to the appreciable homologies in the steroid-binding domains of receptors, this binding is hardly ever totally specific. We have recently obtained evidence that a vicinal dithiol group is involved in steroid binding to glucocorticoid receptors and that these vicinal dithiols are two of the three cysteines in the 16-kDa steroid-binding core. We now report that a comparison of the placement of cysteines in the comparable region of other receptors revealed a lack of similarly closely spaced thiols, which led to the prediction that arsenite would be totally selective in its interaction with glucocorticoid receptors. In fact, 100 microM arsenite inhibited all steroid binding to glucocorticoid receptors while having no effect on the binding of androgen, estrogen, mineralocorticoid, or progesterone receptors. Such total selectivity is not seen for selenite, which is another very potent inhibitor of glucocorticoid binding. This is the first report of absolute selectivity among steroid receptors that is based upon a known structural feature of the receptor protein. This selectivity of arsenite provides the easiest method to date for distinguishing between glucocorticoid and mineralocorticoid receptors and for selectively blocking steroid binding to glucocorticoid receptors in the assays of other receptors.     

MCF-7; a human breast cancer cell line with estrogen, androgen, progesterone, and glucocorticoid receptors.

We have identified receptors for glucocorticoids, progestins, and androgens in a human breast tumor cell line (MCF-7) known to have estrogen receptor. Sucrose density gradients show that MCF-7 cytosol contains approximately 100 fm/mg protein estradiol (E2-3H) receptor, more than 300 fm/mg protein progesterone receptor (measured with R5020-3H), about 40 fm/mg protein 5alpha-dihydrotestosterone (5alpha-DHT-3H) receptor, and 800 fm/mg glucocorticoid receptor (measured with dexamethasone-3H). Dissociation constants obtained by Scatchard analyses were approximately 0.6 x 10(-10)M (E2), 1 x 10(-9)M (R5020), 2.8 x 10(-10)M (5alpha-DHT) and 8 x 10(-9)M (dexamethasone). No cross competition was found for estrogen receptor, but progestins competed for androgen and glucocorticoid binding. The androgen, but not the glucocorticoid, partially competed for R5020 binding to progesterone receptor. This first demonstration of 4 classes of steroid receptors in human breast cancer means that MCF-7 may be an excellent in vitro model for studying the mechanism of tumor response to endocrine therapy as well as the complex relationships between binding and biological actions of these hormones.     

Compound profiling using a panel of steroid hormone receptor cell-based assays

A major focus in the current discovery of drugs targeting nuclear receptors (NRs) is identifying drugs with reduced side effects by improving selectivity, not only from other receptors but also by selective modulation of the NR of interest. Cellular assays not only provide valuable information on functional activity, potency, and selectivity but also are ideally suited for differentiating partial agonists and antagonists. The ability to partially activate a receptor is believed to be closely tied to the ability to selectively modulate the NR, resulting in expression of a subset of the normally regulated genes. To this end, the authors have built a complete panel of cell-based steroid hormone receptor assays for the androgen receptor, estrogen receptor alpha, estrogen receptor beta, glucocorticoid receptor, mineralocorticoid receptor, and progesterone receptor by stably engineering a Gal4 DNA-binding domain/nuclear receptor ligand-binding domain fusion protein into an upstream activation sequence beta-lactamase reporter cell line. Each assay was validated with known agonists and antagonists for correct pharmacology and high-throughput compatibility. To demonstrate the utility of these assays, the authors profiled 35 pharmacologically relevant compounds in a dose-response format against the panel in both agonist and antagonist modes. The results demonstrated that selective estrogen receptor modulators can be identified and differentiated, as well as mixed and partial agonists and antagonists easily detected in the appropriate assays. Importantly, a comparison of the chimeric assays with full-length reporter gene assay data from the literature shows a good degree of correlation in terms of selectivity and pharmacology of important ligands. Taken together, these steroid hormone receptor assays provide good selectivity, sensitivity, and appropriate pharmacology for high-throughput screening and selectivity profiling of modulators of steroid hormone receptors.