PubMed Health⌕ Search

Biomedical subjects

R M Evans

Publications and source records attributed to R M Evans.

At least 109 records · Page 6Linked to original sources
PubMed1997

Nuclear receptor repression mediated by a complex containing SMRT, mSin3A, and histone deacetylase.

The transcriptional corepressors SMRT and N-CoR function as silencing mediators for retinoid and thyroid hormone receptors. Here we show that SMRT and N-CoR directly interact with mSin3A, a corepressor for the Mad-Max heterodimer and a homolog of the yeast global-transcriptional repressor Sin3p. In addition, we demonstrate that the recently characterized histone deacetylase 1 (HDAC1) interacts with Sin3A and SMRT to form a multisubunit repressor complex. Consistent with this model, we find that HDAC inhibitors synergize with retinoic acid to stimulate hormone-responsive genes and differentiation of myeloid leukemia (HL-60) cells. This work establishes a convergence of repression pathways for bHLH-Zip proteins and nuclear receptors and suggests this type of regulation may be more widely conserved than previously suspected.

Antineoplastic Agents↗
PubMed1997

Hypolipidemic drugs, polyunsaturated fatty acids, and eicosanoids are ligands for peroxisome proliferator-activated receptors alpha and delta.

Fatty acids (FAs) and their derivatives are essential cellular metabolites whose concentrations must be closely regulated. This implies that regulatory circuits exist which can sense changes in FA levels. Indeed, the peroxisome proliferator-activated receptor alpha (PPARalpha) regulates lipid homeostasis and is transcriptionally activated by a variety of lipid-like compounds. It remains unclear as to how these structurally diverse compounds can activate a single receptor. We have developed a novel conformation-based assay that screens activators for their ability to bind to PPARalpha/delta and induce DNA binding. We show here that specific FAs, eicosanoids, and hypolipidemic drugs are ligands for PPARalpha or PPARdelta. Because altered FA levels are associated with obesity, atherosclerosis, hypertension, and diabetes, PPARs may serve as molecular sensors that are central to the development and treatment of these metabolic disorders.

Eicosanoids↗
PubMed1997

Coexpression of nuclear receptor partners increases their solubility and biological activities.

The biological activities of the retinoids are mediated by two nuclear hormone receptors: the retinoic acid receptor (RAR) and the retinoid-X receptor (RXR). RXR (and its insect homologue ultraspiracle) is a common heterodimeric partner for many other nuclear receptors, including the insect ecdysone receptor. As part of a continuing analysis of nuclear receptor function, we noticed that, whereas RXR can be readily expressed in Escherichia coli to produce soluble protein, many of its heterodimeric partners cannot. For example, overexpression of RAR results mostly in inclusion bodies with the residual soluble component unable to interact with RXR or ligand efficiently. Similar results are seen with other RXR/ultraspiracle partners. To overcome these problems, we designed a novel double cistronic vector to coexpress RXR and its partner ligand-binding domains in the same bacterial cell. This resulted in a dramatic increase in production of soluble and apparently stable heterodimer. Hormone-binding studies using the purified RXR-RAR heterodimer reveal increased ligand-binding capacity of both components of 5- to 10-fold, resulting in virtually complete functionality. Based on these studies we find that bacterially expressed receptors can exist in one of three distinct states: insoluble, soluble but unable to bind ligand, or soluble with full ligand-binding capacity. These results suggest that coexpression may represent a general strategy for biophysical and structural analysis of receptor complexes.

Binding Sites↗
PubMed1997

The phantom ligand effect: allosteric control of transcription by the retinoid X receptor.

Regulation of gene expression via allosteric control of transcription is one of the fundamental concepts of molecular biology. Studies in prokaryotes have illustrated that binding of small molecules or ligands to sequence-specific transcription factors can produce conformational changes at a distance from the binding site. These ligand-induced changes can dramatically alter the DNA binding and/or trans-activation abilities of the target transcription factors. In this work, analysis of trans-activation by members of the steroid and thyroid hormone receptor superfamily identifies a unique form of allosteric control, the phantom ligand effect. Binding of a novel ligand (LG100754) to one subunit (RXR) of a heterodimeric transcription factor results in a linked conformational change in the second noncovalently bound subunit of the heterodimer (RAR). This conformational change results in both the dissociation of corepressors and association of coactivators in a fashion mediated by the activation function of the non-liganded subunit. Without occupying the RAR hormone binding pocket, binding of LG100754 to RXR mimics exactly the effects observed when hormone is bound to RAR. Thus, LG100754 behaves as a phantom ligand.

Allosteric Regulation↗
PubMed1997

Terminal differentiation of human liposarcoma cells induced by ligands for peroxisome proliferator-activated receptor gamma and the retinoid X receptor.

Induction of terminal differentiation represents a promising therapeutic approach to certain human malignancies. The peroxisome proliferator-activated receptor gamma (PPAR gamma) and the retinoid X receptor alpha (RXR alpha) form a heterodimeric complex that functions as a central regulator of adipocyte differentiation. Natural and synthetic ligands for both receptors have been identified. We demonstrate here that PPAR gamma is expressed at high levels in each of the major histologic types of human liposarcoma. Moreover, primary human liposarcoma cells can be induced to undergo terminal differentiation by treatment with the PPAR gamma ligand pioglitazone, suggesting that the differentiation block in these cells can be overcome by maximal activation of the PPAR pathway. We further demonstrate that RXR-specific ligands are also potent adipogenic agents in cells expressing the PPAR gamma/RXR alpha heterodimer, and that simultaneous treatment of liposarcoma cells with both PPAR gamma- and RXR-specific ligands results in an additive stimulation of differentiation. Liposarcoma cell differentiation is characterized by accumulation of intracellular lipid, induction of adipocyte-specific genes, and withdrawal from the cell cycle. These results suggest that PPAR gamma ligands such as thiazolidinediones and RXR-specific retinoids may be useful therapeutic agents for the treatment of liposarcoma.

Adipocytes↗
PubMed1997

Inducible gene expression in mammalian cells and transgenic mice.

Advances in biomedicine have accentuated the need to develop methods to deliberately modulate gene activity. In addition to improved versions of the system based on components of the tetracycline resistance operon, several strategies have recently emerged to control gene function at the transcriptional level. Particularly promising are approaches based on non-mammalian steroid hormones, and on small molecules that bind immunophilins.

Animals↗
PubMed1997

The germ cell nuclear factor mGCNF is expressed in the developing nervous system.

Using a reinoic acid receptor hybridization probe, we have isolated a mouse embryonic cDNA that encodes the germ cell nuclear factor (mGCNF). The in vitro translated protein binds specifically to the direct repeat of the sequence-AGGTCA-which is characteristic of a subclass of nuclear receptors, albeit with a spacing of zero that is unique among the receptors. Northern analysis shows embryonic expression after mouse gastrulation and during early organogenesis, as well as expression in the adult testis. The message level decreases during embryonic development. Whereas there are two transcripts in the testis, only the larger one is found in embryos. In situ analysis of embryos at days 6.5-10.5 of gestation shows that, in the early postimplantation period, high transcript levels are found in ectodermal cells and in the primitive streak. During further development the expression becomes more restricted. Mainly cells of the developing nervous system are expressing the receptor. Our findings imply that GCNF is involved in two apparently disparate developmental events.

Amino Acid Sequence↗
PubMed1997

Compartment-selective sensitivity of cardiovascular morphogenesis to combinations of retinoic acid receptor gene mutations.

Several aspects of normal cardiovascular development require signaling by the vitamin A metabolite retinoic acid. We have previously established germ-line mutations in mice in the genes that encode the RAR alpha 1, RAR beta, and RXR alpha retinoic acid receptors as a means of studying the function of these receptors in vivo. Although mutation of RXR alpha results in fetal ventricular defects, the RAR alpha 1 and RAR beta mutations are apparently nonphenotypic in the heart and elsewhere. In this study, we have established and analyzed combinations of these receptor gene mutations. Malformations of the ventricular chamber (chamber hypoplasia and muscular ventricular septal defects), conotruncus (double-outlet right ventricle, transposition, and membranous ventricular septal defects), aortic sac (persistent truncus arteriosus and aorticopulmonary window), and aortic arch-derived arteries were recovered in various combinations of the RAR alpha 1, RAR beta, and RXR alpha gene mutations. Depending on the combination of receptor mutations, selective defects were obtained in specific cardiovascular compartments, suggestive of differential expression or function of each receptor within domains of the developing heart.

Animals↗
PubMed1997

A role for ultraspiracle, the Drosophila RXR, in morphogenetic furrow movement and photoreceptor cluster formation.

Many of the same genes needed for proper eye and limb development in vertebrates, such as hairy, hedgehog, patched and cyclic AMP-dependent protein kinase A, are responsible for patterning Drosophila imaginal discs, the tissues that will give rise to the adult cuticle structures. This is well demonstrated in the control of morphogenetic furrow movement and differentiation in the eye imaginal disc. We report that ultraspiracle, the gene encoding the Drosophila cognate of the Retinoid X Receptor, is required for normal morphogenetic furrow movement and ommatidial cluster formation. Examination of the expression of genes involved in regulating the furrow suggests that ultraspiracle defines a novel regulatory pathway in eye differentiation.

Animals↗
PubMed1997

An essential role for retinoid signaling in anteroposterior neural patterning.

The vertebrate central nervous system (CNS) is induced by signals emanating from the dorsal mesoderm, or organizer, that divert the ectoderm away from an epidermal and towards a neural fate. Additional signals from the organizer pattern the neural ectoderm along the anteroposterior axis. We devised highly specific methods utilizing constitutively active or dominant negative receptors to evaluate the role of retinoids in neural patterning. Microinjection of these reagents either augments or reduces retinoid signaling in specific regions of the embryo. We show that increased receptor activity suppresses anterior neural structures while dominant negative receptors lead to anterior enhancement. Similarly, microinjection of the dominant negative receptor leads to the loss of posterior marker genes. We demonstrate that retinoid receptors comprise a critical component in neural posteriorization and are required for proper neuronal differentiation. These results support a quantitative role for retinoid signaling in regionalization of the CNS.

Animals↗
PubMed1997

Tetracycline regulated expression of vimentin in fibroblasts derived from vimentin null mice.

Fibroblast cell lines were derived from vim-/- mice that express a mouse vimentin transgene in a tetracycline regulatable manner. Vimentin null mouse primary embryo fibroblasts were transformed with SV-40 early genes and vim- cell lines were isolated. A vim- cell line was then serially transfected with an expression plasmid encoding the tetracycline regulatable transactivator (tTA) and a mouse vimentin cDNA expression plasmid under the regulation of Escherichia coli tet operator and minimal CMV promoter sequences. Two stable cell lines were obtained that contained little or no vimentin in the presence of low concentrations of tetracycline but rapidly expressed abundant vimentin filaments after removal of tetracycline. The vimentin content of one cell line was similar to that of control vim+/+ fibroblasts. The level of transgene expression could be regulated by the concentration of tetracycline in a dose dependent fashion. Induction of vimentin expression in these cells did not observably affect cell growth, the distribution of microfilaments or microtubules, or the shape of the nucleus. Enucleation studies indicated that while disassembly of microfilaments significantly increased the sensitivity of the cells to enucleation, the presence or absence of vimentin had no detectable effect on the degree of enucleation with increasing sedimentation force. Monolayer wounding experiments demonstrated that vimentin expression did not alter the mobility of polarized cells at the edge of the wound. Experiments to more directly test the effect of vimentin expression on the capacity of these fibroblasts to survive mechanical trauma indicated that vimentin expression had no obvious effect on the survival of suspension cells subjected to nitrogen cavitation or the fraction of cells that survived the mechanical scraping of monolayer culture. These studies indicate that vimentin expression in a single population of cells does not have an obvious effect on cytoplasmic organization and provides a useful system to study the effects of IFs on the capacity of individual cells to resist mechanical injury.

Animals↗
PubMed1997

Retinoid receptors in development and disease.

Nuclear receptors comprise a large family of ligand-dependent transcription factors that display considerable specificity in and selectivity in regulating the genetic programs they ultimately influence. The response to retinoic acid (RA) is mediated by two families of transcription factors which include the retinoic acids receptors (RARs) and the retinoid X receptors (RXRs). In human acute promyelocytic leukemia (APL), RAR alpha becomes an activated oncogene as a consequence of its fusion to the PML locus. Because patients with APL can be induced into remission with high dose RA therapy, we propose that PML-RAR is a new class of dominant negative oncogene that disrupts a structure that includes at least five other proteins. This mega-complex, referred to as a "POD", is disrupted in leukemic cells expressing the oncoprotein and is reassembled following high dose RA therapy in both cell culture an in patients.

Aging↗
PubMed1996

Activation of specific RXR heterodimers by an antagonist of RXR homodimers.

Retinoid X receptor (RXR) plays a central role in the regulation of many intracellular receptor signalling pathways and can mediate ligand-dependent transcription, acting as a homodimer or as a heterodimer. Here we identify an antagonist towards RXR homodimers which also functions as an agonist when RXR is paired as a heterodimer to specific partners, including peroxisome proliferator-activated receptor and retinoic acid receptor. This dimer-selective ligand confers differential interactions on the transcription machinery: the antagonist promotes association with TAF110 (TATA-binding protein (TBP)-associated factor 110) and the co-repressor SMRT, but not with TBP, and these properties are distinct from pure RXR agonists. This unique class of RXR ligands will provide a means to control distinct target genes at the level of transcription and allow the development of retinoids with a new pharmacological action.

DNA-Binding Proteins↗
PubMed1996

RXR alpha deficiency confers genetic susceptibility for aortic sac, conotruncal, atrioventricular cushion, and ventricular muscle defects in mice.

Retinoid-dependent pathways play a central role in regulating cardiac morphogenesis. Recently, we characterized gene-targeted RXR alpha -/- embryos, which display an atrial-like ventricular phenotype with the development of heart failure and lethality at embryonic day 14.5. To quantitate the frequency and complexity of cardiac morphogenic defects, we now use microdissection and scanning electron microscopy to examine 107 wild-type, heterozygous, and homozygous embryos at embryonic day 13.5, 14.5, and 15.5. RXR alpha -/- embryos display complex defects, including ventricular septal, atrioventricular cushion, and conotruncal ridge defects, with double outlet right ventricle, aorticopulmonary window, and persistent truncus arteriosus. In addition, heterozygous RXR alpha embryos display a predisposition for trabecular and papillary muscle defects, ventricular septal defects, conotruncal ridge defects, atrioventricular cushion defects, and pulmonic stenosis. Lastly, we show that the intermediate anatomic phenotype displayed by heterozygous embryos is mirrored in the molecular marker MLC-2a. The intermediate phenotype of RXR alpha heterozygous embryos documents a gene dosage effect for RXR alpha in maintaining normal cardiac morphogenesis. In addition, some defects in RXR alpha mutant mice are phenocopies of human congenital heart defects, thereby suggesting that a relative deficiency in RXR alpha or molecules downstream in its signaling pathway may represent congenital heart disease-susceptibility genes.

Animals↗
PubMed1996

Role of CBP/P300 in nuclear receptor signalling.

The nuclear receptor superfamily includes receptors for steroids, retinoids, thyroid hormone and vitamin D, as well as many related proteins. An important feature of the action of the lipophilic hormones and vitamins is that the maintenance of homeostatic function requires both intrinsic positive and negative regulation. Here we provide in vitro and in vivo evidence that identifies the CREB-binding protein (CBP) and its homologue P300 (refs 6,7) as cofactors mediating nuclear-receptor-activated gene transcription. The role of CBP/P300 in the transcriptional response to cyclic AMP, phorbol esters, serum, the lipophilic hormones and as the target of the E1A oncoprotein suggests they may serve as integrators of extracellular and intracellular signalling pathways.

CREB-Binding Protein↗