Nuclear Hormone-receptor Complexes Research Articles

Steroid hormone signaling synchronizes cell migration machinery, adhesion and polarity to direct collective movement.

Migratory cells - either individually or in cohesive groups - are critical for spatiotemporally regulated processes such as embryonic development and wound healing. Their dysregulation is the underlying cause of formidable health problems such as congenital abnormalities and metastatic cancers. Border cell behavior during Drosophila oogenesis provides an effective model to study temporally regulated, collective cell migration in vivo. Developmental timing in flies is primarily controlled by the steroid hormone ecdysone, which acts through a well-conserved, nuclear hormone receptor complex. Ecdysone signaling determines the timing of border cell migration, but the molecular mechanisms governing this remain obscure. We found that border cell clusters expressing a dominant-negative form of ecdysone receptor extended ineffective protrusions. Additionally, these clusters had aberrant spatial distributions of E-cadherin (E-cad), apical domain markers and activated myosin that did not overlap. Remediating their expression or activity individually in clusters mutant for ecdysone signaling did not restore proper migration. We propose that ecdysone signaling synchronizes the functional distribution of E-cadherin, atypical protein kinase C (aPKC), Discs large (Dlg1) and activated myosin post-transcriptionally to coordinate adhesion, polarity and contractility and temporally control collective cell migration.

UTX coordinates steroid hormone-mediated autophagy and cell death

Correct spatial and temporal induction of numerous cell type-specific genes during development requires regulated removal of the repressive histone H3 lysine 27 trimethylation (H3K27me3) modification. Here we show that the H3K27me3 demethylase dUTX is required for hormone-mediated transcriptional regulation of apoptosis and autophagy genes during ecdysone-regulated programmed cell death of Drosophila salivary glands. We demonstrate that dUTX binds to the nuclear hormone receptor complex Ecdysone Receptor/Ultraspiracle, and is recruited to the promoters of key apoptosis and autophagy genes. Salivary gland cell death is delayed in dUTX mutants, with reduced caspase activity and autophagy that coincides with decreased apoptosis and autophagy gene transcripts. We further show that salivary gland degradation requires dUTX catalytic activity. Our findings provide evidence for an unanticipated role for UTX demethylase activity in regulating hormone-dependent cell death and demonstrate how a single transcriptional regulator can modulate a specific complex functional outcome during animal development.

Fertility: Role of Repressor of Estrogen Receptor Activity

Uterine adenogenesis, a unique postnatal event in mammals, refers to morphological differentiation and development of the uterus that is vulnerable to adverse effects of endocrine disrupters (1). Key morphogenetic events in uterine adenogenesis include 1) organization and stratification of stromal cells, 2) differentiation and growth of the myometrium, and 3) differentiation and morphogenesis of the uterine glands (1, 2). Progressive development of uterine luminal epithelium (LE) gives rise to buds that transition to glandular epithelium (GE) followed by tubulogenesis and coiling and branching morphogenesis to form mature uterine glands (see Fig. 1). Uterine adenogenesis is disrupted by inappropriate exposure of the neonatal lamb to either estradiol-17 or progesterone between postnatal day (PND) 0 and PND 56 (2) resulting in an aglandular uterus. Exposure of the neonatal mouse to progesterone between PND 3 and PND 9 also abrogates uterine adenogenesis (3). Disruption of uterine adenogenesis in both ewes and mice results in infertility. The effectiveness of nuclear hormone-receptor complexes depends on coregulator partner proteins, some of which affect uterine development and fertility. The importance of the coregulator designated repressor of estrogen receptor activity (REA) in uterine adenogenesis has been clarified through research of collaborating scientists at the University of Illinois and Baylor College of Medicine (4).Globaldeletionof theREAgenewas embryonic lethal; therefore, REA was conditionally deleted only in cells that express progesterone receptors to determine effects on postembryonic stages of development in mice. Mice conditionally homozygous for deletion of REA (REAd/d) developed to adulthood and had normal ovarian function but were infertile due to disruption of uterine adenogenesis resulting in the absence of uterine glands and failure of implantation and endometrial decidualization. However, mice heterozygous for REA (REAf/d) were hyperresponsive to estradiol-17 as evidenced by increased proliferation of uterine LE and enhanced fluid imbibition associated with altered expression of aquaporin genes. Park et al. (4) concluded that uterine development and regulation of estrogen receptor activities have a bimodal dependence on gene dosage of REA. There was normal uterine development and function in wild-type mice, whereas partial deletion of REA (REAf/d) resulted in uterine hyperresponsiveness and subfertility, and complete deletion of REA (REAd/d) abrogated uterine adenogenesis and fertility. Uterine adenogenesis also fails in mice lacking functional genes required for interactions between epithelium and stroma including Wnt7a (5), Wnt5a (6), Foxa2 (7), a member of the forkhead transcription factor family, and E-cadherin (Cdh1) (8). Hormonal regulation of Cdh1 in uteri of mice is not known, but expression of Wnt7a, Wnt5a, and Foxa2 genes is affected by sex steroids (7, 9). At birth, uteri of mice and rats consist of uterine LE supported by undifferentiated mesenchyme (10). On PND 5, epithelial invaginations form buds for development of uterine GE and developed GE are present on PND 7 and 9 in mice and rats, respectively (11). In rats, uterine adenogenesis proceeds from PND 9 through PND 15, resulting in development of simple, tubular, and slightly coiled uterine glands (11). Interestingly, Park et al. (4) found the altered uterine phenotype in REAd/d mice between PND 10 and 14, the period of development of uterine glands.

Study of Proliferative Processes and Nuclear Estradiol and Progesterone Receptors in Myocytes in Pregnant and Postpartum Mouse Uterus

Numerical densities of the nuclei were morhometrically evaluated in all myocytes and myocytes expressing nuclear estrogen- and progesterone-receptor complexes, which were revealed immunohistochemically with monoclonal antibodies in C57Bl/6 mice. It was shown that the above quantitative parameters of myometrial cells after the first pregnancy were similar to those in nonpregnant mice by day 10 after delivery. In the third pregnancy, especially developed after the second interrupted pregnancy, proliferation processes in the myometrium were not completed by postpartum day 10, but dramatically progressed. It was associated with a significant decrease in the fraction of myocytes carrying nuclear hormone-receptor complexes with estradiol and progesterone and their disturbed physiological relations in the myometrium during and after pregnancy probably due to dedifferentiation of a considerable part of myocytes.

Uterine Development and Fertility Are Dependent on Gene Dosage of the Nuclear Receptor Coregulator REA

Although the effectiveness of nuclear hormone-receptor complexes is known to depend on coregulator partner proteins, relatively little is known about the roles of coregulators in uterine development and early stages of pregnancy and implantation. Because conventional genetic deletion of the coregulator, repressor of estrogen receptor activity (REA), was embryonic lethal, we here study REA conditional knockout mice generated by cre-loxP recombination, in which REA function was abrogated only in progesterone receptor-expressing tissues, to define the roles of REA in postembryonic stages and in a tissue-specific manner. We find that REA has gene dose-dependent activity impacting uterine development and fertility. Conditional homozygous mutant (REA(d/d)) mice developed to adulthood and showed normal ovarian function, but females were infertile with severely compromised uterine development and function characterized by cell cycle arrest, apoptosis, and altered adenogenesis (endometrial gland morphogenesis), resulting in failure of implantation and decidualization. By contrast, mice heterozygous for REA (REA(f/d)) had a very different phenotype, with estradiol treatment resulting in hyperstimulated, large uteri showing increased proliferation of luminal epithelial cells, and enhanced fluid imbibition associated with altered regulation of aquaporins. These REA(f/d) female mice showed a subfertility phenotype with reduced numbers and sizes of litters. These findings highlight that uterine development and regulation of estrogen receptor activities show a bimodal dependence on the gene dosage of REA. Optimal uterine development and functional activities require the normal gene dosage of REA, with partial or complete deletion resulting in hyperresponsiveness or underresponsiveness to hormone and subfertility or infertility, respectively.

Detection of Nucleic Acid–Nuclear Hormone Receptor Complexes with Mass Spectrometry

Nuclear receptors, such as the retinoic acid receptor (RAR) or the 9-cis retinoic acid receptor (RXR), interact not only with their ligands but also with other types of receptors and with DNA. Here, two complementary mass spectrometry (MS) methods were used to study the interactions between retinoic receptors (RXR/RAR) and DNA: non-denaturing nano-electrospray (nanoESI MS), and high-mass matrix-assisted laser desorption ionization (MALDI MS) combined with chemical cross-linking. The RAR x RXR heterodimer was studied in the presence of a specific DNA sequence (DR5), and a specific RAR x RXR x DNA complex was detected with both MS techniques. RAR by itself showed no significant homodimerization. A complex between RAR and the double stranded DR5 was detected with nanoESI. After cross-linking, high-mass MALDI mass spectra showed that the RAR binds the single stranded DR5, and the RAR dimer binds both single and double stranded DR5. Moreover, the MALDI mass spectrum shows a larger RAR dimer signal in the presence of DNA. These results suggest that a gene-regulatory site on DNA can induce quaternary structural changes in a transcription factor such as RAR.

Genomic mapping of binding regions for the Ecdysone receptor protein complex

We determined the physical locations of the heterodimeric Ecdysone receptor/Ultraspiracle (ECR/USP) nuclear hormone receptor complex throughout the entire nonrepetitive genome of Drosophila melanogaster using a cell line (Kc167) that differentiates in response to 20-hydroxyecdysone (20-HE). 20-HE, the natural ligand of this complex, controls major aspects of insect development, including molting, metamorphosis, and reproduction. Direct gene targets of 20-HE signaling were identified by combining this physical binding-site profiling with gene expression profiling after treatment with 20-HE. We found 502 significant regions of ECR/USP binding throughout the genome. Only 42% of these regions are nearby genes that are 20-HE responsive in these cells. However, at least three quarters of the remaining ECR/USP regions are near 20-HE-regulated genes in other tissue and cell types during metamorphosis, suggesting that binding at many regulatory elements in the genome is largely noncell-type specific. The majority (21/26) of the early targets of 20-HE encode transcriptional regulatory factors. To determine whether any of these targets are required for the morphological differentiation of these cells, we used RNAi to reduce the expression of each of the 26 early genes. Accordingly, we found that three direct targets of ECR/USP--hairy, vrille, and Hr4--are required for cellular differentiation in response to the hormone. Initial mutational analysis of vrille in vivo reveals that it is required for metamorphosis.

Parent-of-origin-specific binding of nuclear hormone receptor complexes in the H19-Igf2 imprinting control region.

Parent-of-origin-specific expression of the mouse insulin-like growth factor 2 gene (Igf2) and the closely linked H19 gene located on distal chromosome 7 is regulated by a 2.4-kb imprinting control region (ICR) located upstream of the H19 gene. In somatic cells, the maternally and paternally derived ICRs are hypo- and hypermethylated, respectively, with the former binding the insulator protein CCCTC-binding factor (CTCF) and acting to block access of enhancers to the Igf2 promoter. Here we report on a detailed in vivo footprinting analysis-using ligation-mediated PCR combined with in vivo dimethyl sulfate, DNase I, or UV treatment-of ICR sequences located outside of the CTCF binding domains. In mouse primary embryo fibroblasts carrying only maternal or paternal copies of distal chromosome 7, we have identified five prominent footprints specific to the maternal ICR. Each of the five footprinted areas contains at least two nuclear hormone receptor hexad binding sites arranged with irregular spacing. When combined with fibroblast nuclear extracts, these sequences interact with complexes containing retinoic X receptor alpha and estrogen receptor beta. More significantly, the footprint sequences bind nuclear hormone receptor complexes in male, but not female, germ cell extracts purified from fetuses at a developmental stage corresponding to the time of establishment of differential ICR methylation. These data are consistent with the possibility that nuclear hormone receptor complexes participate in the establishment of differential ICR methylation imprinting in the germ line.

Regulation of neurotransmitter interactions in the ventral striatum.

Transsynaptic activation of neuronal circuits originating in the basal forebrain contributes to psychostimulant-evoked dopamine and glutamate release and consequent changes in medium spiny neuronal gene expression in the ventral striatum. New evidence from microdialysis studies indicates that amphetamine-induced dopamine and glutamate release in vivo is partially calcium dependent. The calcium-dependent component is totally blocked by a kappa opioid receptor agonist, indicating that endogenous opioids may regulate dopamine-glutamate interactions in the ventral striatum. Further, muscarinic receptor blockade increases, and muscarinic receptor stimulation decreases, dialysate glutamate levels in the striatum. Pre- and postsynaptic muscarinic receptors contribute to the ability of the muscarinic antagonist, scopolamine, to augment D1 receptor-stimulated immediate early and neuropeptide gene expression. Moreover, scopolamine prevents a D2 antagonist from blocking D1 agonist-induced gene expression, indicating that activation of cholinergic interneurons contributes to D1/D2 interactions in the striatum. Thus, transsynaptic activity and presynaptic muscarinic and kappa opioid receptors regulate dopamine and glutamate interactions that switch on and off multiple intracellular signaling cascades. Changes in immediate early and neuropeptide gene expression that result from activation of these cascades are mediated by such nuclear transcription factors as phosphorylated cyclase response element-binding protein. In addition, a novel signaling pathway involving the RAR/RXR nuclear hormone receptor complex is implicated in the control of dopamine receptor and neuropeptide gene expression in the striatum.

High level transactivation by the ecdysone receptor complex at the core recognition motif.

Ecdysteroid signaling in insects is mediated by the ecdysone receptor complex that is composed of a heterodimer of the ecdysone receptor and Ultraspiracle. The DNA binding specificity plays a critical role of defining the repertoire of target genes that respond to the hormone. We report here the determination of the preferred core recognition motif by a binding site selection procedure. The consensus sequence consists of a perfect palindrome of the heptameric half-site sequence GAGGTCA that is separated by a single A/T base pair. No binding polarity of the ecdysone receptor/Ultraspiracle heterodimer to the core recognition motif was observed. This core motif mediated the highest level of ligand-induced transactivation when compared to a series of synthetic ecdysone response elements and to the natural element of the Drosophila hsp27 gene. This is the first report of a palindromic sequence identified as the highest affinity DNA binding site for a heterodimeric nuclear hormone receptor complex. We further present evidence that the ligand of the ecdysone receptor preferentially drives Ultraspiracle from a homodimer into a heterodimer. This mechanism might contribute additionally to a tight control of target gene expression.

Binding of the glucocorticoid receptor to the rat liver nuclear matrix. The role of disulfide bond formation.

The nuclear matrix is a putative skeletal structure which has been implicated in many nuclear functions. To assess a possible role of the nuclear matrix in glucocorticoid action, purified rat liver nuclei containing glucocorticoid-receptor complexes were treated with DNase I +/- RNase A followed by 1.6 M NaCl, thus yielding salt-extractable and salt-resistant (nuclear matrix) fractions. The subnuclear distribution of hormone-receptor complexes was determined by following the fate of unmetabolized radiolabel after injection of labeled triamcinolone acetonide into adrenalectomized animals and subjecting various subfractions to immunoblotting using a monoclonal antibody which recognizes the glucocorticoid receptor. Both techniques indicated that 50-70% of the total nuclear hormone-receptor complexes were recovered in the nuclear matrix fraction. Previous results (Kaufmann, S. H., and Shaper, J. H. (1984) Exp. Cell Res. 155, 477-495) suggest that a variety of nuclear polypeptides become nuclease- and salt-resistant as a result of the formation of intermolecular disulfide bonds. The following evidence suggests that disulfide bonds mediate the association between the glucocorticoid receptor and the nuclear matrix. When nuclei were isolated in the absence of sulfhydryl-blocking and -cross-linking reagents, sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions revealed that the receptor was present as a high molecular weight disulfide-cross-linked complex. When nuclei were isolated in the presence of the irreversible sulfhydryl-blocking reagent iodoacetamide, the disulfide bonds which cross-linked the receptor into high molecular weight complexes were absent; and 85-100% of the hormone-receptor complexes were salt-extractable. When nuclei (isolated in the absence of iodoacetamide) were treated with the sulfhydryl-cross-linking reagent sodium tetrathionate, greater than 95% of the nuclear hormone-receptor complexes became resistant to extraction with nucleases and 1.6 M NaCl. The implications of these results for other matrix-associated nuclear functions are discussed.

Effects of protease inhibitors on nuclear binding of glucocorticoid hormones in C3H10T [formula omitted] cells

We have measured incorporation of the glucocorticoid hormone cortisone into nuclear hormone-receptor complexes in the C3H10T 1 2 cell line. As we had found cortisone to be capable of malignantly transforming these cells in vitro, and certain protease inhibitors have been shown to suppress transformation in this cell line, we investigated the effects of these protease inhibitors (antipain, chymostatin and the Bowman-Birk inhibitor) on the formation of nuclear cortisone-receptor complexes. All 3 inhibitors were found to suppress wholly or partially formation of nuclear cortisone-receptor complexes, suggesting that such complexes may be involved in the process of glucocorticoid-enhanced transformation.

Characterization of nuclear ecdysteroid receptor from crayfish integument

The existence of nuclear ecdysteroid receptors in isolated nuclei from crayfish integument was demonstrated. Nuclei were prepared from crayfish hypodermis by use of a modified Chauveau-method and exposed to [ 3H]-ecdysteroids. The nuclear hormone receptor complex sedimented at about 5S and the available binding sites had a high affinity to [ 3H] ecdysteroids. Treatment of the nuclei with pronase and DNase completed or partially reduced nuclear bound ecdysone, whereas RNase was without any effect. Competition of nuclear bound ecdysone by various steroids confirmed ponasterone A and kaladasterone as the best competitors followed by 20-OH-ecdysone and ecdysone, whereas compounds like poststerone, “triol” or hydrocortisone did not compete. If [ 3H]-20-OH-ecdysone was used as the radio-ligand, 20-OH-ecdysone was a better competitor than ecdysone indicating that there may be two different binding sites for ecdysone and 20-OH-ecdysone; this was further substantiated by the finding of two different number of binding sites for these steroids.

Specific binding of 1alpha,25-dihydroxycholecalciferol to nuclear components of chick intestine.

Specific binding of 1alpha,25-dihydroxycholecalciferol to macromolecular components of small intestinal mucosa nuclei is demonstrated in vitamin D-deficient chicks. The nuclear 1alpha,25-dihydroxycholecalciferol-macromolecule complex was isolated on sucrose density gradients and sediments at 3.7 S in the presence of 0.3 M KCl. Agarose gel filtration of the nuclear component indicated an apparent molecular weight of 47,000. The nuclear receptor complexes could not be distinguished from previously described cytoplasmic 1alpha,25-dihydroxycholecalciferol-binding components by the ultracentrifugation and chromatographic procedures employed. The association of the 3-H-sterol with the nuclear component is thermolabile and is destroyed by treatment with pronase, but not by nucleases; the receptor component is therefore presumed to be a protein. The macromolecular-1alpha,25-dihydroxycholecalciferol complex formed in vivo or in vitro at 25 degrees can be extracted from intestinal nuclei by 0.3 M KCl, but not by low salt buffers. Smaller amounts of the 3.7 S binding component can be detected in isolated purified chromatin or after incubation of 1alpha,25-dihydroxy[3-H]cholecalciferol with reconstituted cytosol-chromatin at 0 degrees. Following incubation of the labeled hormone with reconstituted cytosol-chromatin at 0 degrees, 1alpha,25-dihydroxy[3-H]cholecalciferol is primarily associated with the cytoplasmic receptor, After shifting the incubation temperature to 25 degrees, a progressive increase in the concentration of the nuclear receptor complex and a concomitant decrease in the concentration of the cytoplasmic binding component occur. Thus the 1alpha,25-dihydroxycholecalciferol binding molecules appear to exist primarily in the cytoplasm, where they presumably function to transport the hormone into the nucleus. Experiments employing incubation of 1alpha,25-dihydroxy[3-H]cholecalciferol with reconstituted cytosol-chromatin from nontarget tissues indicate a requirement for both intestinal cytosol and chromatin for maximal formation of the nuclear hormone-receptor complex. These results suggest that the nuclear-binding component arises from hormone-dependent transfer of the cytoplasmic 1alpha,25-dihydroxycholecalciferol receptor to intestinal chromatin acceptor sites.

Oestrogenic stimulation of macromolecular synthesis is correlated with nuclear hormone-receptor complex in neonatal rat uterus.

THE ability of oestrogen to elicit pronounced physiological responses in the mammalian uterus depends on specific receptor proteins that recognize and retain the hormone1–3. The formation of cytoplasmic oestradiol-receptor complexes and their transformation to nuclear complexes seem to be an important prelude to the stimulation of RNA synthesis in the organ2,3. Since a cytoplasmic oestradiol-receptor complex is present in the uterus of the neonatal rat 1 d after birth4, we have investigated whether the uterus of the neonatal rat can transform the cytoplasmic hormone-receptor complex to the nuclear complex, as described for the immature or older animal1,3. Because relatively little is known about the growth responses of the neonatal uterus to oestrogen, we have examined quantitatively the synthesis and concentrations of DNA and protein in the control and hormone-stimulated uterus, as a function of time after birth. It has been reported5 that oestrogen administered 24 h before death to rats less than 15 d old does not stimulate uterine weight or macro-molecular synthesis.

Les recepteurs de l'oestradiol dans l'uteurs de genisse: Stimulation de la biosynthese de RNA in vitro

Oestradiol receptors in the calf uterus. Stimulation of RNA biosynthesis in vitro 1. 1. Cytoplasmic and nuclear oestradiol receptor proteins were prepared from calf uterine endometrium. 2. 2. The cytoplasmic hormone-receptor complex was studied by analytical ultracentrifugation at different ionic strengths. A sedimentation coefficient of 7.7 S was obtained at low ionic strength. At higher ionic strengths a value of 5 S was obtained. It was shown that the bound oestradiol is associated with the 7.7-S and 5-S protein peaks. 3. 3. The incorporation of [5- 3H]UTP into RNA by nuclei isolated from calf uterine endometrium was stimulated by the cytoplasmic 5-S receptor complex and also by a nuclear hormone-receptor complex, but not by the 7.7-S complex. 4. 4. Two enzyme fractions containing RNA polymerase activity were isolated from calf uterine endometrium. The “total RNA polymerase” fraction was stimulated by the cytoplasmic 5-S receptor complex, while the “soluble RNA polymerase” fraction was slightly inhibited. 5. 5. A hypothesis is presented concerning the mechanism of action of oestrogen at the level of the uterine endometrium.