Cognate Nuclear Receptors Research Articles

Critical Role of Desolvation in the Binding of 20-Hydroxyecdysone to the Ecdysone Receptor

The insect steroid hormone 20-hydroxyecdysone (20E) binds to its cognate nuclear receptor composed of the ecdysone receptor (EcR) and Ultraspiracle (USP) and triggers the main developmental transitions, in particular molting and metamorphosis. We present the crystal structure of the ligand-binding domains of EcR/USP in complex with 20E at 2.4A resolution and compare it with published structures of EcR/USP bound to ponasterone A (ponA). ponA is essentially identical to 20E but lacks the 25-OH group of 20E. The structure of 20E-bound EcR indicates that an additional hydrogen bond is formed compared with the ponA-bound receptor, yet, paradoxically, ponA has a significantly higher affinity for EcR than 20E. Theoretical studies based on docking and free energy methods lead to a rationale for understanding the difference in binding affinities between 20E and ponA. Results of the calculations indicate that the favorable contribution from the extra H-bond made by 25-OH of 20E is counterbalanced by its larger desolvation cost compared with that of ponA. The contribution of 25-OH to the binding affinity is further compared with those of 20- and 22-OH groups. Ligands that lack the 20- or 22-OH group are indeed known to bind less favorably to EcR than 20E, an effect opposite to that observed for ponA. The results indicate that their respective contributions to receptor-ligand complex stability reside mostly in their different contributions to solvation/desolvation. Together, the data demonstrate the critical role of ligand desolvation in determining binding affinity, with general implications for the binding of hormones to their cognate nuclear receptors.

Regulation of Gene Expression in the Bovine Mammary Gland by Ovarian Steroids

It is well established that estrogen is required for mammary epithelial cell proliferation and ductal development in the growing animal, and that lobuloalveolar development during gestation is dependent on progesterone. The effects of these steroid hormones on gene expression in the mammary gland are mediated primarily by their respective nuclear hormone receptors, which function as hormone-bound transcription factors. To gain insight into how estrogen and progesterone regulate mammary gland growth and function in cattle, we and others have characterized the expression patterns of their cognate nuclear hormone receptors in the bovine mammary gland throughout development, pregnancy, and lactation. This work has identified a lack of expression of estrogen receptor β and a greater abundance of progesterone receptor during lactation in the bovine mammary gland, compared with the rodent gland. We speculate that interactions among the estrogen receptor isoforms that regulate progesterone receptor expression may contribute to these species differences. Further, demonstrated expression of substantial quantities of estrogen receptor within the prepubertal bovine mammary fat pad, along with coordinated insulin-like growth factor-I expression, suggests that this tissue may stimulate parenchymal growth via an estrogen-responsive paracrine mechanism. In addition, the recent availability of bovine genomic sequence information and microarray technologies has permitted the study of global gene expression in the mammary gland in response to the steroid environment. We have identified more than 100 estrogen-responsive genes, of which the majority are novel estrogen gene targets. Estrogen-induced changes in gene expression were consistent with increased mammary epithelial cell proliferation, increased extracellular matrix turnover in parenchyma, and increased extracellular matrix deposition in the fat pad. A comparison of estrogen-responsive genes in the mammary glands of humans, mice, and cattle suggests considerable variation among species, as well as potential differences in regulatory elements in common estrogen receptor gene targets. Continuing studies using advanced molecular techniques should assist in elucidating the complex regulation of mammary function at the transcript level.

Opposite effects of CBP and p300 in glucocorticoid signaling in astrocytes

In the nervous system, glucocorticoid hormones play a major role during development, and they continue to affect functional and structural plasticity throughout life. Glucocorticoid actions are mediated by their cognate nuclear receptor, the glucocorticoid receptor (GR). The transcriptional activity of the GR is enhanced by the recruitment of one of the transcriptional coactivators of the p160 family (SRCs), which are a docking platform for secondary coactivators like CBP, or its close homologue p300. Here, we investigated the implication of CBP and p300 coactivators in glial cells of the central and peripheral nervous system, namely in primary cultures of astrocytes and in Schwann cells. We show that both coregulators behave differently in either cell type. CBP enhances GR transcriptional activation in astrocytes, and has no effect in Schwann cells, whereas p300 exerts an inhibitory effect in both glial cells. Studies with p300 deletion mutants show that the repressive capacity of p300 is related to its acetyltransferase activity. This work shows striking differences between CBP and p300 actions in astrocytes. Moreover, in astrocytes the opposite effects of CBP and p300 could lead to a balance in the transactivation potency of the GR, in order to fine tune the action of glucocorticoids.

Role of Retinoids and their Cognate Nuclear Receptors in Breast Cancer Chemoprevention

Retinoids are natural and synthetic compounds related to retinoic acid that act through interaction with two basic types of nuclear receptors: retinoic acid receptors (RARalpha, RARbeta and RARgamma) and retinoid X receptors (RXRalpha, RXRbeta and RXRgamma) as ligand-activated, DNA-binding, transacting, transcription-modulating proteins involved in a general molecular mechanism responsible for transcriptional responses in target genes. Function of retinoids in organisms affecting broad spectrum of various biochemical and molecular biology reactions is unimaginable without fully functional nuclear receptors--retinoid inducible transcription factors. Retinoic acids exert tumour-suppressive activity due to their antiproliferative and apoptosis-inducing effects. A number of novel retinoids and rexinoids acting through cognate nuclear receptors have been tested both in vitro and in vivo, using cell culture or animal models for breast cancer. This article briefly summarizes the role and properties of nuclear retinoid/rexinoid receptors as well as selected effects of retinoic acids or selected synthetic retinoids and rexinoids with respect to their potential use in chemoprevention of breast cancer.

Characterisation of three variants of estrogen receptor β mRNA in the common sole, Solea solea L. (Teleostei)

In all vertebrates, estrogen action is mediated by cognate nuclear receptors. In this study, we cloned the different transcripts of the estrogen receptor β ( ERβ) gene of the common sole, Solea solea. 5′-RLM-RACE (RNA Ligase-Mediated 5′-Rapid Amplification of cDNA Ends) and 3′-RACE analyses revealed three isoforms of different length, called Long, Intermediate and Short isoforms, consisting of 2212, 1531 and 1207 b, respectively. The Long isoform is characterised by an open reading frame (ORF) encoding 589 aa, with an estimated molecular weight of 65 kDa. Phylogenetic analysis established that it belongs to the teleost ERβ1 or ERβa cluster. The Intermediate isoform encodes a 490-aa protein, which lacks the first 99 aa of the Long isoform, but still retains a complete DNA-binding domain (DBD). In the Short variant (363 aa-long), all the N-terminal region, down to the two zinc fingers included, is missing, thus crippling DBD. ERβ transcription was analysed by semiquantitative RT-PCR with specific primers, common to the Long and Intermediate isoforms, in various sole tissues, such as brain, gills, muscle, stomach, intestine, spleen, head kidney, kidney, liver and gonads. This analysis revealed that ERβ displays a widespread or ubiquitous pattern of transcription, with the highest levels being found in the gonads and liver.

Vitamine D : Métabolisme, régulation et maladies associées

Vitamin D is well known as a hormone involved in mineral metabolism and bone growth. Conversion into the active metabolite 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) from the precursor is effected by cytochrome P450 enzymes in the liver (CYP27A1 and CYP2R1) and the kidney (CYP27B1). CYP27A1 has been shown to be transcriptionally regulated by nuclear receptors (PPARalpha, gamma, HNF-4alpha and SHP) which are ligand-dependent transcription factors. CYP27B1 is tightly regulated by the plasma levels of calcium, phosphate, parathyroid hormone (PTH) and 1,25(OH)2D3 itself. In vitamin D target organs, inactivation of vitamin D is attributed to CYP24A1 which is transcriptionally induced by 1,25(OH)2D3 whose action is mediated by binding to its cognate nuclear receptor, the vitamin D receptor (VDR). Diseases associated to Vitamin D deficiency (rickets in children, and osteomalacia or osteoporosis in adults) and autosomal recessive forms of inherited rickets illustrate the key role of vitamin D in calcium homeostasis and bone metabolism. Recently, discovery of 1,25(OH)2D3 new biological actions that include antiproliferative, prodifferentiating effect on many cell types and immunoregulatory properties creates a growing interest for this vitamin. In this way, a best understanding of various actors implicated in vitamin D metabolism and its regulation is of a major importance to optimise the use of vitamin D in disease prevention.

Biosynthesis, mode of action and functional significance of neurosteroids in the developing Purkinje cell

New findings over the past decade have shown that the brain has the capability of forming steroids de novo from cholesterol, the so-called “neurosteroids”. To understand neurosteroid action in the brain, data on the regio- and temporal-specific synthesis of neurosteroids are needed. Recently, we have demonstrated that the Purkinje cell, a cerebellar neuron, is a major site for neurosteroid formation in a variety of vertebrates. This is the first demonstration of de novo neuronal neurosteroidogenesis in the brain. Since this discovery, organizing actions of neurosteroids are becoming clear by the studies on mammals using the Purkinje cell as an excellent cellular model. In mammals, the Purkinje cell actively synthesizes progesterone de novo from cholesterol during neonatal life, when cerebellar neuronal circuit formation occurs. The Purkinje cell may also produces estradiol in the neonate. Interestingly, both progesterone and estradiol promote dendritic growth, spinogenesis and synaptogenesis via each cognate nuclear receptor in the developing Purkinje cell. Such organizing actions may contribute to the formation of cerebellar neuronal circuit during neonatal life. This paper summarizes the advances made in our understanding of the biosynthesis, mode of action and functional significance of neurosteroids in the developing Purkinje cell.

Dexamethasone facilitates erythropoiesis in murine embryonic stem cells differentiating into hematopoietic cells in vitro

Differentiating embryonic stem (ES) cells are increasingly emerging as an important source of hematopoietic progenitors with a potential to be useful for both basic and clinical research applications. It has been suggested that dexamethasone facilitates differentiation of ES cells towards erythrocytes but the mechanism responsible for sequential expression of genes regulating this process are not well-understood. Therefore, we in vitro induced differentiation of murine ES cells towards erythropoiesis and studied the sequential expression of a set of genes during the process. We hypothesized that dexamethasone-activates its cognate nuclear receptors inducing up-regulation of erythropoietic genes such as GATA-1, Flk-1, Epo-R, and direct ES cells towards erythropoietic differentiation. ES cells were cultured in primary hematopoietic differentiation media containing methyl-cellulose, IMDM, IL-3, IL-6, and SCF to promote embryoid body (EB) formation. Total RNA of day 3, 5, and 9-old EBs was isolated for gene expression studies using RT-PCR. Cells from day 9 EBs were subjected to secondary differentiation using three different cytokines and growth factors combinations: (1) SCF, EPO, dexamethasone, and IGF; (2) SCF, IL-3, IL-6, and TPO; and, (3) SCF IL-3, IL-6, TPO, and EPO. Total RNA from day 12 of secondary differentiated ES cells was isolated to study the gene expression pattern during this process. Our results demonstrate an up-regulation of GATA-1, Flk-1, HoxB-4, Epo-R, and globin genes (α-globin, βH-1 globin, β-major globin, ε -globin, and ζ-globin) in the 9-day-old EBs, whereas, RNA from 5-day-old EBs showed expression of HoxB-4, ε-globin, γ-globin, βH1-globin, and Flk-1. Three-day-old EBs showed only HoxB-4 and Flk-1 gene expression and lacked expression of all globin genes. These findings indicate that erythropoiesis-specific genes are activated later in the course of differentiation. Gene expression studies on the ES cells of secondary EB origin cultured in media containing dexamethasone showed a down-regulation of GATA-3 and an up-regulation of GATA-1, Flk-1, and Epo-R in comparison to the two other cytokines and growth factor combinations containing media. The secondary differentiation also showed an enhanced production of erythrocytic precursors in dexamethasone containing media in comparison to that in the control media. Our results indicate that dexamethasone can prove to be an effective agent which can be employed to enhance differentiation towards erythrocytic progenitors from ES cells.

Biosynthesis and organizing action of neurosteroids in the developing Purkinje cell

Probing undiscovered neurosubstances that play important roles in the regulation of cerebellar function is essential for the progress of our understanding of the cerebellum. New findings over the past decade have established that the cerebellum as well as other brain regions synthesizes steroids de novo from cholesterol through mechanisms at least partly independent of peripheral steroidogenic glands. Such steroids synthesized de novo in the brain are called neurosteroids. Recently the Purkinje cell, a cerebellar neuron, has been identified as a major site for neurosteroid formation in the brain. This is the first demonstration of de novo neuronal neurosteroidogenesis in the brain. In mammals, the Purkinje cell actively synthesizes progesterone de novo from cholesterol during neonatal life, when cerebellar cortical formation occurs. 3alpha,5alpha-Tetrahydroprogesterone (allopregnanolone) is metabolized from progesterone in the neonatal cerebellum. Estrogen formation in the Purkinje cell may also occur in the neonate. Subsequently, recent studies on mammals using the Purkinje cell have demonstrated organizing actions of neurosteroids. Both progesterone and estradiol promote dendritic growth, spinogenesis and synaptogenesis via each cognate nuclear receptor in Purkinje neurons. Allopregnanolone is also involved in Purkinje and granule cell survival. Thus the Purkinje cell serves as an excellent cellular model for understanding the formation of cerebellar neuronal circuit in relation to organizing actions of neurosteroids.

Convergence of vitamin D and retinoic acid signalling at a common hormone response element

Although 1,25-dihydroxyvitamin D3 (1,25D3) and retinoic acid (RA) have distinct developmental and physiological roles, both regulate the cell cycle. We provide molecular and genomic evidence that their cognate nuclear receptors regulate common genes through everted repeat TGA(C/T)TPyN8PuG(G/T)TCA (ER8) response elements. ER8 motifs were found in the promoters of several target genes of 1,25D3 and/or RA. Notably, an element was characterized in the cyclin-dependent kinase (CDK) inhibitor p19ink4d gene, and 1,25D3- or RA-induced p19INK4D) expression. P19ink4d knockdown together with depletion of p27kip1, another CDK inhibitor regulated by 1,25D3 and RA, rendered cells resistant to ligand-induced growth arrest. Remarkably, p19INK4D-deficient cells showed increased autophagic cell death, which was markedly enhanced by 1,25D3, but not RA, and attenuated by loss of p27KIP1. These results show a limited crosstalk between 1,25D3 and RA signalling by means of overlapping nuclear receptor DNA binding specificities, and uncover a role for p19INK4D in control of cell survival.

S-palmitoylation modulates estrogen receptor α localization and functions

17β-Estradiol (E2) acts as a chemical messenger in target tissues inducing both slow nuclear and rapid extra-nuclear responses. E2 binds to its cognate nuclear receptors (ER) resulting in the activation of target gene transcription in the nucleus. In addition to these genomic effects, E2 modulates cell functions through rapid non-genomic actions. Stimulation of G-proteins, Ca 2+ influx, inositol phosphate generation as well as phospholipase C, ERK/MAPK, and PI3K/AKT activation all occur within seconds to minutes after E2 binding to a small population of ERα located at the plasma membrane. The great impact of these rapid signals on cell physiology renders central the knowledge of the structural bases and mechanisms that mediate extra-nuclear signaling by E2. Several laboratories, including our own, have recently elucidated the structural requirements for localization and function of plasma membrane ERα. This review summarizes the molecular mechanisms of E2-induced rapid non-genomic actions relevant for cell functions, highlighting the role of lipid modification (i.e., palmitoylation) in the ERα localization to and residence at the plasma membrane.

Endocrine Regulation of Menstruation

In women, endometrial morphology and function undergo characteristic changes every menstrual cycle. These changes are crucial for perpetuation of the species and are orchestrated to prepare the endometrium for implantation of a conceptus. In the absence of pregnancy, the human endometrium is sloughed off at menstruation over a period of a few days. Tissue repair, growth, angiogenesis, differentiation, and receptivity ensue to prepare the endometrium for implantation in the next cycle. Ovarian sex steroids through interaction with different cognate nuclear receptors regulate the expression of a cascade of local factors within the endometrium that act in an autocrine/paracrine and even intracrine manner. Such interactions initiate complex events within the endometrium that are crucial for implantation and, in the absence thereof, normal menstruation. A clearer understanding of regulation of normal endometrial function will provide an insight into causes of menstrual dysfunction such as menorrhagia (heavy menstrual bleeding) and dysmenorrhea (painful periods). The molecular pathways that precipitate these pathologies remain largely undefined. Future research efforts to provide greater insight into these pathways will lead to the development of novel drugs that would target identified aberrations in expression and/or of local uterine factors that are crucial for normal endometrial function.

Temporal and spatial associations of oestrogen receptor alpha and progesterone receptor in the endometrium of cyclic and early pregnant mares

Uterine function is primarily controlled by the combined actions of oestrogen and progesterone working through their cognate nuclear receptors. The mechanism of establishment of pregnancy in the mare is of interest because it involves prolonged pre-attachment and conceptus migration phases, and both invasive and non-invasive placental cell types, and as such has been an important comparative model. This study characterised regulation of oestrogen (ER) and progesterone (PR) receptors in the endometrium of the mare during the oestrous cycle and early pregnancy. Endometrial tissues collected during the oestrous cycle and early pregnancy were analysed for steady-state levels of ER and PR mRNA and protein. Steady-state levels of ER and PR mRNA were highest on days 0, 17 and 20 in cyclic mares and lowest on days 11 and 14. A day-by-status interaction was detected, indicating that day 17 and day 20 pregnant mares exhibited low levels of ER and PR compared with the corresponding days of the oestrous cycle. In situ hybridisation analyses showed receptor mRNA localisation primarily in the luminal epithelium (LE), glandular epithelium (GE) and stroma around oestrus. During dioestrus and early pregnancy, receptors were not detected in the LE, and were lower in the stroma and deeper GE. Changes in hybridisation intensity in these cell types were consistent with changes in mRNA levels detected by slot-blot hybridisation. ER and PR proteins were detected in the nuclei of LE, GE and stromal cells. Consistent with results from in situ hybridisation, levels of ER and PR immunoreactivity were higher around oestrus, declined to low levels during dioestrus and remained low during early pregnancy. Results described here for temporal and spatial changes in steroid receptor gene expression in mares show the greatest similarities with those described for cattle and sheep.

N-Acylphosphatidylethanolamine-hydrolyzing Phospholipase D Is an Important Determinant of Uterine Anandamide Levels during Implantation

Implantation requires reciprocal interaction between blastocysts and a receptive uterus. In mice, one important player in this dialogue involves endocannabinoid signaling via cannabinoid receptor CB1. Anandamide is an endogenous cannabinoid ligand, and its levels are spatiotemporally regulated in the uterus during early pregnancy, showing lower levels in the receptive uterus and at the implantation site. However, the mechanism by which differential uterine anandamide gradients are established under different pregnancy status is not clearly understood. Using multiple approaches, we show here that uterine anandamide levels conducive to implantation are primarily regulated by spatiotemporal expression of Nape-Pld, the gene encoding N-acylphosphatidylethanolamine-hydrolyzing phospholipase D that generates anandamide. The expression is well correlated with its activity and anandamide levels. This study is clinically relevant, since elevated anandamide levels in peripheral circulation are associated with spontaneous pregnancy failure in women.

1,25-dihydroxyvitamin D inhibits human ANP gene promoter activity

1,25-dihydroxyvitamin D, through association with its cognate nuclear receptor, has been shown to have important effects in the cardiovascular and renal systems. We have shown previously that the liganded vitamin D receptor (VDR) inhibits hypertrophy and expression of hypertrophy-sensitive genes (i.e. those encoding atrial natriuretic peptide [ANP], brain natriuretic peptide and α skeletal actin) in neonatal cardiac myocytes. In the present study we confirm a time-, ligand- and retinoid X receptor-dependent, VDR-mediated suppression of human ANP gene promoter activity. Conventional deletion analysis demonstrated that the promoter region positioned between −217 and −104 is required for the VDR-dependent suppression of the hANP promoter. Mutation of two functional CArG elements, including one located within this critical region, failed to reverse the suppression. We found no evidence that the liganded VDR is capable of associating directly with regulatory elements positioned between −217 and −104. We conclude that the inhibition may arise from protein–protein interactions between the liganded VDR and stimulatory transcription factors that bind in this region.

Hormonal signals produced by DAF-9/cytochrome P450 regulate C. elegans dauer diapause in response to environmental cues.

In response to the environment, the nematode C. elegans must choose between arrest at a long-lived alternate third larval stage, the dauer diapause, or reproductive development. This decision may ultimately be mediated by daf-9, a cytochrome P450 related to steroidogenic hydroxylases and its cognate nuclear receptor daf-12, implying organism-wide coordination by lipophilic hormones. Accordingly, here we show that daf-9(+) works cell non-autonomously to bypass diapause, and promote gonadal outgrowth. Among daf-9-expressing cells, the hypodermis is most visibly regulated by environmental inputs, including dietary cholesterol. On in reproductive growth, off in dauer, hypodermal daf-9 expression is strictly daf-12 dependent, suggesting feedback regulation. Expressing daf-9 constitutively in hypodermis rescues dauer phenotypes of daf-9, as well as insulin/IGF receptor and TGFbeta mutants, revealing that daf-9 is an important downstream point of control within the dauer circuits. This study illuminates how endocrine networks integrate environmental cues and transduce them into adaptive life history choices.

Nuclear Receptor Coregulators Are New Players in Nervous System Development and Function

Steroid/thyroid hormones and their cognate nuclear receptors (NRs) play important roles in nervous system development and function. The spatial and temporal gene expression that is regulated by NRs in the nervous system requires transcriptional intermediary coregulators, designated as coactivators and corepressors. These coregulators enhance or repress transcriptional activity of NRs and modulate their target gene transcription. Recent progress has largely advanced our understanding of the molecular mechanisms by which NR coregulators function in the nervous system. This article summarizes our current knowledge about the molecular mechanisms, expression patterns, and biological functions of NR coactivators, such as the p160 steroid receptor coactivator family, CBP, p300, BRG1, TRAP220, PGC-1alpha, ERAP140, NIX1, and E6-AP, as well as corepressors such as NCoR and SMRT. Accumulated findings suggest that the functional spectrum of NR coregulators is much broader than was initially speculated, and these coregulators likely contribute to many physiological aspects of nervous system development and function.

Renaissance of the Biologically Active Vitamin A Derivatives: Established and Novel Directed Therapies for Cancer and Chemoprevention

Vitamin A and its biologically active derivatives are involved in a complex arrangements of physiological and developmental responses in many tissue of higher vertebrates. Retinoids are natural and synthetic compounds related to retinoic acid that act through interaction with two basic types of nuclear receptors: retinoic acid receptors (RAR alpha, RARbeta and RARgamma) and retinoid X receptors (RXRalpha, RXRbeta and RXRgamma) as retinoid-inducible transcription factors. Thus, the retinoid receptors are considered to be ligand-activated, DNA-binding, trans-acting, transcription-modulating proteins involved in a general molecular mechanism responsible for transcriptional responses in target genes. They exert both beneficial and detrimental activity; they have tumor-suppressive activity but on the other hand they are teratogenic. Retinoids inhibit carcinogenesis, suppress premalignant epithelial lesions and tumor growth and invasion in a variety of tissues. Natural and synthetic retinoids have therapeutical effects due to their antiproliferative and apoptosis-inducing effects. They are known to cause redifferentiation or to prevent further dedifferentiation of various neoplastic tissues. A number of novel chemical compounds, receptor selective retinoids and rexinoids, have been synthesized up to now and tested both in vitro and in vivo, using animal models against different cancer cells. In spite of that progress, there is still an urgent call for novel synthetic retinoids and rexinoids with greater retinoid receptor selectivity, reasonable chemotherapeutic or chemopreventive effects and reduced toxicity and side effects. This article summarizes selected effects of biologically active natural or synthetic retinoids and rexinoids, acting through their cognate nuclear receptors, and their use in chemotherapy and chemoprevention of various types of cancer.

Heterogeneous nuclear ribonucleoprotein (hnRNP) binding to hormone response elements: a cause of vitamin D resistance.

In previous studies, we have shown that steroid hormone resistance in New World primates occurs in the absence of abnormal expression of cognate nuclear receptors. Rather, these animals have elevated levels of heterogeneous nuclear ribonucleoproteins (hnRNPs) that act as hormone response element-binding proteins and attenuate target gene transactivation. Here we present evidence for a similar mechanism in humans via a patient with resistance to the active form of vitamin D [1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3))] who presented with normal vitamin D receptor (VDR) expression. Initial cotransfection studies showed that the cells of the patient suppressed basal and hormone-induced transactivation by wild-type VDR. Electrophoretic mobility-shift assays and Western/Southwestern blot analyses indicated that this suppressive effect was due to overexpression of a nuclear protein that specifically interacts with a DNA response element known to bind retinoid X receptor-VDR heterodimers. Ab blocking in electrophoretic mobility-shift assays indicated that this dominant-negative acting protein was in the hnRNPA family of nucleic acid-binding proteins. Further studies have shown that several members of this family, most notably hnRNPA1, were able to suppress basal and 1,25(OH)(2)D(3)-induced luciferase activity. We therefore propose that this case of vitamin D resistance in a human subject is similar to that previously described for New World primates in which abnormal expression of a hormone response element-binding protein can cause target cell resistance to 1,25(OH)(2)D(3). That this protein is a member of the hnRNP family capable of interacting with double-stranded DNA highlights a potentially important new component of the complex machinery required for steroid hormone signal transduction.

Proinflammatory Gene Induction by Platelet-Activating Factor Mediated Via Its Cognate Nuclear Receptor

It has been postulated that intracellular binding sites for platelet-activating factor (PAF) contribute to proinflammatory responses to PAF. Isolated nuclei from porcine cerebral microvascular endothelial cells (PCECs) produced PAF-molecular species in response to H(2)O(2). Using FACS analysis, we demonstrated the expression of PAF receptors on cell and nuclear surfaces of PCECs. Confocal microscopy studies performed on PCECs, Chinese hamster ovary cells stably overexpressing PAF receptors, and isolated nuclei from PCECs also showed a robust nuclear distribution of PAF receptors. Presence of PAF receptors at the cell nucleus was further revealed in brain endothelial cells by radioligand binding experiments, immunoblotting, and in situ in brain by immunoelectron microscopy. Stimulation of nuclei with methylcarbamate-PAF evoked a decrease in cAMP production and a pertussis toxin-sensitive rise in nuclear calcium, unlike observations in plasma membrane, which exhibited a pertussis toxin-insensitive elevation in inositol phosphates. Moreover, on isolated nuclei methylcarbamate-PAF evoked the expression of proinflammatory genes inducible nitric oxide synthase and cyclooxygenase-2 (COX-2) and was associated with augmented extracellular signal-regulated kinase 1/2 phosphorylation and NF-kappaB binding to the DNA consensus sequence. COX-2 expression was prevented by mitogen-activated protein kinase kinase/extracellular signal-regulated kinase 1/2 and NF-kappaB inhibitors. This study describes for the first time the nucleus as a putative organelle capable of generating PAF and expresses its receptor, which upon stimulation induces the expression of the proinflammatory gene COX-2.