The dual role of ultraspiracle, the Drosophila retinoid X receptor, in the ecdysone response

The Drosophila homolog of the retinoid X receptor, ultraspiracle (USP), heterodimerizes with the ecdysone receptor (EcR) to form a functional complex that mediates the effects of the steroid molting hormone ecdysone by activating and repressing expression of ecdysone response genes. As with other retinoid X receptor heterodimers, EcR/USP affects gene transcription in a ligand-modulated manner. We used in vivo, cell culture, and biochemical approaches to analyze the functions of two usp alleles, usp3 and usp4, which encode stable proteins with defective DNA-binding domains. We observed that USP is able to activate as well as repress the Z1 isoform of the ecdysone-responsive broad complex (BrC-Z1). Activation of BrC-Z1 as well as EcR, itself an ecdysone response gene, can be mediated by both the USP3 and USP4 mutant proteins. USP3 and USP4 also activate an ecdysone-responsive element, hsp27EcRE, in cultured cells. These results differ from the protein null allele, usp2, which is unable to mediate activation [Schubiger, M. & Truman, J. W. (2000) Development 127, 1151–1159]. BrC-Z1 repression is compromised in all three usp alleles, suggesting that repression involves the association of USP with DNA. Our results distinguish two mechanisms by which USP modulates the properties of EcR: one that involves the USP DNA-binding domain and one that can be achieved solely through the ligand-binding domain. These newly revealed properties of USP might implicate similar properties for retinoid X receptor.

Cloning and characterization of a hormonally regulated rat long chain acyl-CoA synthetase

A previously unidentified gonadotropin-regulated long chain acyl-CoA synthetase (GR-LACS) was cloned and characterized as a 79-kDa cytoplasmic protein expressed in Leydig cells of the rat testis. GR-LACS shares sequence identity with two conserved regions of the LACS and luciferase families, including the ATP/AMP binding domain and the 25-aa fatty acyl-CoA synthetase signature motif, but displays low overall amino acid similarities (23–28%). GR-LACS mRNA is expressed abundantly in Leydig cells of the adult testis and to a lesser degree in the seminiferous tubules in spermatogonia and Sertoli cells. It is also observed in ovary and brain. Immunoreactive protein expression was observed mainly in Leydig cells and minimally in the tubules but was not detected in other tissues. In vivo, treatment with a desensitizing dose of human chorionic gonadotropin caused transcriptional down-regulation of GR-LACS expression in Leydig cells. The expressed protein present in the cytoplasm of transfected cells displayed acyl-CoA synthetase activity for long chain fatty acid substrates. GR-LACS may contribute to the provision of energy requirements and to the biosynthesis of steroid precursors and could participate through acyl-CoA's multiple functions in the regulation of the male gonad.

High-Molecular-Weight FK506-Binding Proteins Are Components of Heat-Shock Protein 90 Heterocomplexes in Wheat Germ Lysate1

In animal cell lysates the multiprotein heat-shock protein 90 (hsp90)-based chaperone complexes consist of hsp70, hsp40, and p60. These complexes act to convert steroid hormone receptors to their steroid-binding state by assembling them into heterocomplexes with hsp90, p23, and one of several immunophilins. Wheat germ lysate also contains a hsp90-based chaperone system that can assemble the glucocorticoid receptor into a functional heterocomplex with hsp90. However, only two components of the heterocomplex-assembly system, hsp90 and hsp70, have thus far been identified. Recently, purified mammalian p23 preadsorbed with JJ3 antibody-protein A-Sepharose pellets was used to isolate a mammalian p23-wheat hsp90 heterocomplex from wheat germ lysate (J.K. Owens-Grillo, L.F. Stancato, K. Hoffmann, W.B. Pratt, and P. Krishna [1996] Biochemistry 35: 15249–15255). This heterocomplex was found to contain an immunophilin(s) of the FK506-binding class, as judged by binding of the radiolabeled immunosuppressant drug [3H]FK506 to the immune pellets in a specific manner. In the present study we identified the immunophilin components of this heterocomplex as FKBP73 and FKBP77, the two recently described high-molecular-weight FKBPs of wheat. In addition, we present evidence that the two FKBPs bind hsp90 via tetratricopeptide repeat domains. Our results demonstrate that binding of immunophilins to hsp90 via tetratricopeptide repeat domains is a conserved protein interaction in plants. Conservation of this protein-to-protein interaction in both plant and animal cells suggests that it is important for the biological action of the high-molecular-weight immunophilins.

4-Hydroxytamoxifen binds to and deactivates the estrogen-related receptor γ

The estrogen-related receptors (ERRα, ERRβ, and ERRγ) form a family of orphan nuclear receptors that share significant amino acid identity with the estrogen receptors, but for which physiologic roles remain largely unknown. By using a peptide sensor assay, we have identified the stilbenes diethylstilbestrol (DES), tamoxifen (TAM), and 4-hydroxytamoxifen (4-OHT) as high-affinity ligands for ERRγ. In direct binding assays, 4-OHT had a Kd value of 35 nM, and both DES and TAM displaced radiolabeled 4-OHT with Ki values of 870 nM. In cell-based assays, 4-OHT binding caused a dissociation of the complex between ERRγ and the steroid receptor coactivator-1, and led to an inhibition of the constitutive transcriptional activity of ERRγ. ERRα did not bind 4-OHT, but replacing a single amino acid predicted to be in the ERRα ligand-binding pocket with the corresponding ERRγ residue allowed high-affinity 4-OHT binding. These results demonstrate the existence of high-affinity ligands for the ERR family of orphan receptors, and identify 4-OHT as a molecule that can regulate the transcriptional activity of ERRγ.

From seed germination to flowering, light controls plant development via the pigment phytochrome.

Plant growth and development are regulated by interactions between the environment and endogenous developmental programs. Of the various environmental factors controlling plant development, light plays an especially important role, in photosynthesis, in seasonal and diurnal time sensing, and as a cue for altering developmental pattern. Recently, several laboratories have devised a variety of genetic screens using Arabidopsis thaliana to dissect the signal transduction pathways of the various photoreceptor systems. Genetic analysis demonstrates that light responses are not simply endpoints of linear signal transduction pathways but are the result of the integration of information from a variety of photoreceptors through a complex network of interacting signaling components. These signaling components include the red/far-red light receptors, phytochromes, at least one blue light receptor, and negative regulatory genes (DET, COP, and FUS) that act downstream from the photoreceptors in the nucleus. In addition, a steroid hormone, brassinolide, also plays a role in light-regulated development and gene expression in Arabidopsis. These molecular and genetic data are allowing us to construct models of the mechanisms by which light controls development and gene expression in Arabidopsis. In the future, this knowledge can be used as a framework for understanding how all land plants respond to changes in their environment.

The extreme C terminus of progesterone receptor contains a transcriptional repressor domain that functions through a putative corepressor.

Binding of a hormone agonist to a steroid receptor leads to the dissociation of heat shock proteins, dimerization, specific DNA binding, and target gene activation. Although the progesterone antagonist RU486 can induce most of these events, it fails to activate human progesterone receptor (hPR)-dependent transcription. We have previously demonstrated that a conformational change is a key event leading to receptor activation. The major conformational distinction between hormone- and antihormone-bound receptors occurs within the C-terminal portion of the molecule. Furthermore, hPR mutants lacking the C terminus become transcriptionally active in the presence of RU486. These results suggest that the C terminus contains a repressor domain that inhibits the transcriptional activity of the RU486-bound hPR. In this study, we have defined a 12 amino acid (12AA) region in the C terminus of hPR that is necessary and sufficient for the repressor function when fused to the C-terminal truncated hPR or to the GAL4 DNA-binding domain. Mutations in the 12AA domain (aa 917-928) generate an hPR that is active in the presence of RU486. Furthermore, overexpression of the 12AA peptide activates the RU486-bound wild-type hPR without affecting progesterone-dependent activation. These results suggest that association of the 12AA repressor region with a corepressor might inactivate hPR activity when it is bound to RU486. We propose that binding of a hormone agonist to the receptor changes its conformation in the ligand-binding domain so that association with coactivator is promoted and activation of target gene occurs.

Role for the class A macrophage scavenger receptor in the phagocytosis of apoptotic thymocytes in vitro.

Numerous immature thymocytes undergo apoptosis and are rapidly engulfed by phagocytic thymic macrophages. The macrophage surface receptors involved in apoptotic thymocyte recognition are unknown. We have examined the role of the class A macrophage scavenger receptor (SR-A) in the engulfment of apoptotic thymocytes. Uptake of steroid-treated apoptotic thymocytes by thymic and inflammatory-elicited SR-A positive macrophages is partially inhibited by an anti-SR-A mAb and more completely by a range of scavenger receptor ligands. Thymic macrophages from mice with targeted disruption of the SR-A gene show a 50% reduction in phagocytosis of apoptotic thymocytes in vitro. These data suggest that SR-A may play a role in the clearance of dying cells in the thymus.

p300 is a component of an estrogen receptor coactivator complex.

The estrogen receptor (ER) is a ligand-dependent transcription factor that regulates expression of target genes in response to estrogen in concert with other cellular signaling pathways. This suggests that the mechanism by which ER transmits an activating signal to the general transcription machinery may include factors that integrate these diverse signals. We have previously characterized the estrogen receptor-associated protein, ERAP160, as a factor that complexes with ER in an agonist-dependent manner. We have now found that the transcriptional coactivator p300 associates with agonist bound ER and augments ligand-dependent activation by ER. Our studies show that an ER coactivator complex involves a direct hormone-dependent interaction between ER and ERAP160, resulting in the recruitment of p300. In addition, antibodies directed against the cloned steroid receptor coactivator 1 (SRC1) recognize ERAP160. The known role of p300 in multiple signal transduction pathways, including those involving the second messenger cAMP, suggests p300 functions as a point of integration between ER and these other pathways.

Estradiol stimulates tyrosine phosphorylation of the insulin-like growth factor-1 receptor and insulin receptor substrate-1 in the uterus.

The signaling pathways associated with estrogen-induced proliferation of epithelial cells in the reproductive tract have not been defined. To identify receptor tyrosine kinases that are activated in vivo by 17 beta-estradiol (E2), uteri from ovariectomized mice were examined for enhanced tyrosine phosphorylation of various receptors and a receptor substrate following treatment with this hormone. Within 4 hr after hormone exposure, extracts showed increased phosphotyrosine (P-Tyr) immunoreactivity at several bands, including 170- and 180-kDa; these bands were still apparent at 24 hr after E2. Analysis of immunoprecipitates from uterine extracts revealed that E2 enhanced tyrosine phosphorylation of the insulin-like growth factor-1 receptor (IGF-1R) and insulin receptor substrate-1 (IRS-1) by 6 hr. Comparison of supernatants from IRS-1 and control rabbit IgG immunoprecipitates indicated that the 170-kDa P-Tyr band in extracts was equivalent to IRS-1. The receptors for epidermal growth factor, platelet-derived growth factor, and basic fibroblast growth factor did not exhibit an E2-induced increase in P-Tyr content. The nonestrogenic steroid hormones examined did not stimulate the P-Tyr content of IGF-1R or IRS-1. Immunolocalization of P-Tyr and IRS-1 revealed strong reactivity in the epithelial layer of the uterus from E2-treated mice, suggesting that the majority of P-Tyr bands observed in immunoblots originate in the epithelium. Since hormonal activation of IRS-1 is epithelial, estrogen-specific, and initiated before maximal DNA synthesis occurs following treatment with hormone, this protein, as part of the IGF-1R pathway, may be important in mediating estrogen-stimulated proliferation in the uterus.

Moderate increase in histone acetylation activates the mouse mammary tumor virus promoter and remodels its nucleosome structure.

The mouse mammary tumor virus (MMTV) promoter is regulated by steroid hormones through a hormone-responsive region that is organized in a positioned nucleosome. Hormone induction leads to a structural change of this nucleosome which makes its DNA more sensitive to cleavage by DNase I and enables simultaneous binding of all relevant transcription factors. In cells carrying either episomal or chromosomally integrated MMTV promoters, moderate acetylation of core histones, generated by treatment with low concentrations of the histone deacetylase inhibitors sodium butyrate or trichostatin A, enhances transcription from the MMTV promoter in the absence of hormone and potentiates transactivation by either glucocorticoids or progestins. At higher concentrations, histone deacetylase inhibitors reduce basal and hormone induced MMTV transcription. Inducing inhibitor concentrations lead to the same type of nucleosomal DNase I hypersensitivity as hormone treatment, suggesting that moderate acetylation of core histone activates the MMTV promoter by mechanisms involving chromatin remodeling similar to that generated by the inducing hormones.

Analysis of estrogen receptor transcriptional enhancement by a nuclear hormone receptor coactivator.

The estrogen receptor (ER), a member of a large superfamily of nuclear hormone receptors, is a ligand-inducible transcription factor that regulates the expression of estrogen-responsive genes. The ER, in common with other members of this superfamily, contains two transcription activation functions (AFs)--one located in the amino-terminal region (AF-1) and the second located in the carboxyl-terminal region (AF-2). In most cell contexts, the synergistic activity of AF-1 and AF-2 is required for full estradiol (E2)-stimulated activity. We have previously shown that a ligand-dependent interaction between the two AF-containing regions of ER was promoted by E2 and the antiestrogen trans-hydroxytamoxifen (TOT). This interaction, however, was transcriptionally productive only in the presence of E2. To explore a possible role of steroid receptor coactivators in transcriptional synergism between AF-1 and AF-2, we expressed the amino terminal (AF-1-containing) and carboxyl-terminal (AF-2-containing) regions of ER as separate polypeptides in mammalian cells, along with the steroid receptor coactivator-1 protein (SRC-1). We demonstrate that SRC-1, which has been shown to significantly increase ER transcriptional activity, enhanced the interaction, mediated by either E2 or TOT, between the AF-1-containing and AF-2-containing regions of the ER. However, this enhanced interaction resulted in increased transcriptional effectiveness only with E2 and not with TOT, consistent with the effects of SRC-1 on the full-length receptor. Our results suggest that after ligand binding, SRC-1 may act, in part, as an adapter protein that promotes the integration of amino- and carboxyl-terminal receptor functions, allowing for full receptor activation. Potentially, SRC-1 may be capable of enhancing the transcriptional activity of related nuclear receptor superfamily members by facilitating the productive association of the two AF-containing regions in these receptors.

Cholesterol homeostasis in human brain: evidence for an age-dependent flux of 24S-hydroxycholesterol from the brain into the circulation.

We have investigated whether side chain-hydroxylated cholesterol species are important for elimination of cholesterol from the brain. Plasma concentrations of 24-hydroxycholesterol (24-OH-Chol) in the internal jugular vein and the brachial artery in healthy volunteers were consistent with a net flux of this steroid from the brain into the circulation, corresponding to elimination of approximately 4 mg cholesterol during a 24-h period in adults. Results of experiments with rats exposed to 18O2 were also consistent with a flux of 24-OH-Chol from the brain into the circulation. No other oxysterol measured showed a similar behavior as 24-OH-Chol. These results and the finding that the concentration of 24-OH-Chol was 30- to 1500-fold higher in the brain than in any other organ except the adrenals indicate that the major part of 24-OH-Chol present in the circulation originates from the brain. Both the 24-OH-Chol present in the brain and in the circulation were the 24S-stereoisomer. In contrast to other oxysterols, levels of plasma 24-OH-Chol were found to be markedly dependent upon age. The ratio between 24-OH-Chol and cholesterol in plasma was approximately 5 times higher during the first decade of life than during the sixth decade. There was a high correlation between levels of 24-OH-Chol in plasma and cerebrospinal fluid. It is suggested that the flux of 24-OH-Chol from the brain is important for cholesterol homeostasis in this organ.

Menstrual breakdown of human endometrium can be mimicked in vitro and is selectively and reversibly blocked by inhibitors of matrix metalloproteinases.

The mechanisms underlying the menstrual lysis leading to shedding of the human endometrium and its accompanying bleeding are still largely unknown. In particular, whether breakdown of the endometrial fibrillar extra-cellular matrix that precedes bleeding depends on aspartic-, cysteine-, serine-, or metalloproteinases remains unclear. In the present study, menstrual regression of the human endometrium was mimicked in organ culture. Whereas sex steroids could preserve tissue integrity only in nonperimenstrual explants, matrix breakdown upon sex steroid deprivation was completely and reversibly inhibited at all stages of the menstrual cycle by specific inhibitors of matrix metalloproteinases, but not by inhibitors of the other classes of proteinases. Matrix metalloproteinases are thus identified as the key class of proteinases involved in the initiation of menstruation.

NMR evidence for the participation of a low-barrier hydrogen bond in the mechanism of delta 5-3-ketosteroid isomerase.

Delta 5-3-Ketosteroid isomerase (EC 5.3.3.1) promotes an allylic rearrangement involving intramolecular proton transfer via a dienolic intermediate. This enzyme enhances the catalytic rate by a factor of 10(10). Two residues, Tyr-14, the general acid that polarizes the steroid 3-carbonyl group and facilitates enolization, and Asp-38 the general base that abstracts and transfers the 4 beta-proton to the 6 beta-position, contribute 10(4.7) and 10(5.6) to the rate increase, respectively. A major mechanistic enigma is the huge disparity between the pKa values of the catalytic groups and their targets. Upon binding of an analog of the dienolate intermediate to isomerase, proton NMR detects a highly deshielded resonance at 18.15 ppm in proximity to aromatic protons, and with a 3-fold preference for protium over deuterium (fractionation factor, phi = 0.34), consistent with formation of a short, strong (low-barrier) hydrogen bond to Tyr-14. The strength of this hydrogen bond is estimated to be at least 7.1 kcal/mol. This bond is relatively inaccessible to bulk solvent and is pH insensitive. Low-barrier hydrogen bonding of Tyr-14 to the intermediate, in conjunction with the previously demonstrated tunneling contribution to the proton transfer by Asp-38, provide a plausible and quantitative explanation for the high catalytic power of this isomerase.

16 alpha-substituted analogs of the antiprogestin RU486 induce a unique conformation in the human progesterone receptor resulting in mixed agonist activity.

Previously, we have shown that agonists and antagonists interact with distinct, though overlapping regions within the human progesterone receptor (hPR) resulting in the formation of structurally different complexes. Thus, a link was established between the structure of a ligand-receptor complex and biological activity. In this study, we have utilized a series of in vitro assays with which to study hPR pharmacology and have identified a third class of hPR ligands that induce a receptor conformation which is distinct from that induced by agonists or antagonists. Importantly, when assayed on PR-responsive target genes these compounds were shown to exhibit partial agonist activity; an activity that was influenced by cell context. Thus, as has been shown previously for estrogen receptor, the overall structure of the ligand-receptor complex is influenced by the nature of the ligand. It appears, therefore, that the observed differences in the activity of some PR and estrogen receptor ligands reflect the ability of the cellular transcription machinery to discriminate between the structurally different complexes that result following ligand interaction. These data support the increasingly favored hypothesis that different ligands can interact with different regions within the hormone binding domains of steroid hormone receptors resulting in different biologies.