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Nuclear Receptor That Identifies a Novel Retinoic Acid Response Pathway
Abstract
Molecular cloning and transcriptional activation studies have revealed a new protein similar to the steroid hormone receptors and which responds specifically to vitamin A metabolites. This protein is substantially different in primary structure and ligand specificity from the products of the previously described retinoic acid receptor gene family. By indicating the existence of an additional pathway through which retinoic acid may exert its effects, these data lead to a re-evaluation of retinoid physiology.
... A metabolites [15]. RXR differed in structure from the previously identified transcriptional regulator retinoic acid receptor (RAR) [15] but had an analogous mechanism of transcriptional regulation with that of steroid and thyroid hormone receptors (THR) [15]. ...
... A metabolites [15]. RXR differed in structure from the previously identified transcriptional regulator retinoic acid receptor (RAR) [15] but had an analogous mechanism of transcriptional regulation with that of steroid and thyroid hormone receptors (THR) [15]. Further characterisation of RXR and its activation by 9-cis retinoic acid (9-cis-RA) but not all-trans RA, the latter being an endogenous ligand of RARs, resulted in recognition of a new retinoid response pathway that not only differed from RAR in response to retinoids but was also evolutionarily conserved [15] and demonstrated ability to heterodimerise with multiple receptors [5,6]. ...
... A metabolites [15]. RXR differed in structure from the previously identified transcriptional regulator retinoic acid receptor (RAR) [15] but had an analogous mechanism of transcriptional regulation with that of steroid and thyroid hormone receptors (THR) [15]. Further characterisation of RXR and its activation by 9-cis retinoic acid (9-cis-RA) but not all-trans RA, the latter being an endogenous ligand of RARs, resulted in recognition of a new retinoid response pathway that not only differed from RAR in response to retinoids but was also evolutionarily conserved [15] and demonstrated ability to heterodimerise with multiple receptors [5,6]. ...
Retinoid X receptors (RXRs) present a subgroup of the nuclear receptor superfamily with particularly high evolutionary conservation of ligand binding domain. The receptor exists in α, β, and γ isotypes that form homo-/heterodimeric complexes with other permissive and non-permissive receptors. While research has identified the biochemical roles of several nuclear receptor family members, the roles of RXRs in various neurological disorders remain relatively under-investigated. RXR acts as ligand-regulated transcription factor, modulating the expression of genes that plays a critical role in mediating several developmental, metabolic, and biochemical processes. Cumulative evidence indicates that abnormal RXR signalling affects neuronal stress and neuroinflammatory networks in several neuropathological conditions. Protective effects of targeting RXRs through pharmacological ligands have been established in various cell and animal models of neuronal injury including Alzheimer disease, Parkinson disease, glaucoma, multiple sclerosis, and stroke. This review summarises the existing knowledge about the roles of RXR, its interacting partners, and ligands in CNS disorders. Future research will determine the importance of structural and functional heterogeneity amongst various RXR isotypes as well as elucidate functional links between RXR homo- or heterodimers and specific physiological conditions to increase drug targeting efficiency in pathological conditions.
... At the time, it was unknown whether this nuclear receptor family member was a co-regulator of multiple signaling pathways including vitamin D, thyroid hormone, and retinoic acid. David Mangelsdorf in Ron Evan's group identified RXRα as the first member of this subfamily and furthermore showed that this receptor, the ligand-binding domain of which shared little (<30%) amino acid identity with RARs, was still able to transduce an RA signal (Mangelsdorf et al. 1990), although strict binding to RA could not be demonstrated at the time. Three members of this family were eventually characterized, including that described by Hamada et al. (1989) which became RXRβ, and a third member named RXRγ (Mangelsdorf et al. 1992). ...
... The sequence and nature of the DNA-binding motifs favored by RARs and RXRs were also actively investigated at that time. A series of naturally occurring and synthetic DNA response elements was characterized for TRs (Glass et al. 1987, Umesono et al. 1991, RARs (Vasios et al. 1989, de Thé et al. 1990b, Leroy et al. 1991b, Smith et al. 1991, and RXRs (Mangelsdorf et al. 1990, Rottman et al. 1991. These response elements consisted of a sixnucleotide core motif, AGG/TTCA, occurring in tandem as either a direct or inverted 'repeat motif'. ...
For almost a century, vitamin A has been known as a nutrient critical for normal development, differentiation, and homeostasis; accordingly, there has been much interest in understanding its mechanism of action. This review is about the discovery of specific receptors for the vitamin A derivative, retinoic acid (RA), which launched extensive molecular, genetic and structural investigations into these new members of the nuclear receptor superfamily of transcriptional regulators. These included two families of receptors, the RAR isotypes (α, β, and γ) along with three RXR isotypes (α, β, and γ), which bind as RXR/RAR heterodimers to cis-acting response elements of RA target genes to generate a high degree of complexity. Such studies have provided deep molecular insight into how the widespread pleiotropic effects of RA can be generated.
... The sustained pursuit to associate ligands with newly cloned orphan nuclear receptors channeled the Evans team to the discovery of a second retinoid-responsive system. David Mangelsdorf (Mangelsdorf 1990) showed that the activity of an orphan receptor referred to as RXR could be induced by pharmacological concentrations of at-RA. In a genomic organization similar to that of the RARs, three distinct genes encoding RXR isoforms (α, β, γ) were identified in mouse and human (Hamada et al. 1989, Mangelsdorf et al. 1990, Fleischhauer et al. 1992, Leid et al. 1992. ...
... David Mangelsdorf (Mangelsdorf 1990) showed that the activity of an orphan receptor referred to as RXR could be induced by pharmacological concentrations of at-RA. In a genomic organization similar to that of the RARs, three distinct genes encoding RXR isoforms (α, β, γ) were identified in mouse and human (Hamada et al. 1989, Mangelsdorf et al. 1990, Fleischhauer et al. 1992, Leid et al. 1992. A search for a higher affinity retinoid for RXR led to the identification of the 9-cis isomer of RA (9-cis-RA) as a suitable ligand (Heyman et al. 1992, Levin et al. 1992. ...
The landmark 1987 discovery of the retinoic acid receptor (RAR) came as a surprise, uncovering a genomic kinship between the fields of vitamin A biology and steroid receptors. This stunning breakthrough triggered a cascade of studies to deconstruct the roles played by the RAR and its natural and synthetic ligands in embryonic development, skin, growth, physiology, vision and disease as well as providing a template to elucidate the molecular mechanisms by which nuclear receptors regulate gene expression. In this Review, written from historic and personal perspectives, we highlight the milestones that led to the discovery of the RAR, and the subsequent studies that enriched our knowledge of the molecular mechanisms by which a low abundant dietary compound could be so essential to the generation and maintenance of life itself.
... Receptor kwasu 9-cis-retinowego (RXR) zidentyfikowano i opisano po raz pierwszy w 1990 r. jako receptor sierocy [6]. Dopiero późniejsze badania wykazały, iż białko to wiąże retinoidy, w tym kwas 9-cis-retinowy (9cRA) i stąd też jego nazwa [7]. ...
... Rycina 4.Wzory strukturalne wybranych ligandów RXR, wg[6,46] ...
Abstrakt
Receptory jądrowe (NRs) tworzą największą nadrodzinę czynników transkrypcyjnych, które odgrywają ważną rolę w regulacji wielu procesów biologicznych. Receptor kwasu 9-cis-retinowego (RXR) wydaje się odgrywać szczególną rolę wśród tej grupy białek, a to ma związek z jego zdolnością do tworzenia dimerów z innymi NRs. Ze względu na kontrolę ekspresji wielu genów, RXR stanowi bardzo dobry cel licznych terapii. Nieprawidłowości w szlakach modulowanych przez RXR są powiązane m.in. z chorobami neurodegeneracyjnymi, otyłością, cukrzycą, a także nowotworami. Istnieje wiele związków mogących regulować aktywność transkrypcyjną RXR. Jednak obecnie dopuszczonych do użytku klinicznego jest tylko kilka z nich. Retinoidy normalizują wzrost i różnicowanie komórek skóry i błon śluzowych, ponadto działają immunomodulująco oraz przeciwzapalnie. Stąd są stosowane przede wszystkim w chorobach skóry i w terapii niektórych chorób nowotworowych. W artykule przedstawiono ogólne wiadomości na temat RXR, jego budowy, ligandów i mechanizmu działania oraz potencjalnej roli w terapii nowotworów i zespołu metabolicznego.
... RA signaling is mediated through RAR and RXR receptors and regulates gene transcription by recruiting coactivator proteins (47). RAR signaling can be inhibited by BMS493, a pan-RAR inverse agonist that allosterically binds to all three RAR isoforms and strongly enhances the interaction between RAR ligandbinding domain with nuclear corepressor, decreasing the basal transcriptional activity of RAR (48). ...
Retinoic acid (RA) is a fundamental vitamin A metabolite involved in regulating immune responses through the nuclear RA receptor (RAR) and retinoid X receptor. While performing experiments using THP-1 cells as a model for Mycobacterium tuberculosis infection, we observed that serum-supplemented cultures displayed high levels of baseline RAR activation in the presence of live, but not heat-killed, bacteria, suggesting that M. tuberculosis robustly induces the endogenous RAR pathway. Using in vitro and in vivo models, we have further explored the role of endogenous RAR activity in M. tuberculosis infection through pharmacological inhibition of RARs. We found that M. tuberculosis induces classical RA response element genes such as CD38 and DHRS3 in both THP-1 cells and human primary CD14+ monocytes via a RAR-dependent pathway. M. tuberculosis-stimulated RAR activation was observed with conditioned media and required nonproteinaceous factor(s) present in FBS. Importantly, RAR blockade by (4-[(E)-2-[5,5-dimethyl-8-(2-phenylethynyl)-6H-naphthalen-2-yl]ethenyl]benzoic acid), a specific pan-RAR inverse agonist, in a low-dose murine model of tuberculosis significantly reduced SIGLEC-F+CD64+CD11c+high alveolar macrophages in the lungs, which correlated with 2× reduction in tissue mycobacterial burden. These results suggest that the endogenous RAR activation axis contributes to M. tuberculosis infection both in vitro and in vivo and reveal an opportunity for further investigation of new antituberculosis therapies.
... Vitamin A is present in foods in the form of retinyl ester, and provitamin A carotenoid is stored as a retinyl ester derivative in the stellate cells of the liver. Vitamin A is transported to target cells in the form of retinol and acts via nuclear receptors called retinoic acid receptor (RAR) and retinoid X receptor (RXR) [3][4][5]. ...
Vitamin A ensures intestinal homeostasis, impacting acquired immunity and epithelial barrier function; however, its role in innate immunity is mostly unknown. Here, we studied the impact of vitamin A in different dextran sulfate sodium (DSS)-induced colitis animal models. Interestingly, more severe DSS-induced colitis was observed in vitamin A-deficient (VAD) mice than in vitamin A-sufficient (VAS) mice; the same was observed in VAD severe combined immunodeficient mice lacking T/B cells. Remarkably, IL-1β production, LC3B-II expression, and inflammasome activity in the lamina propria were significantly elevated in VAD mice. Electron microscopy revealed numerous swollen mitochondria with severely disrupted cristae. In vitro, non-canonical inflammasome signaling-induced pyroptosis, LC3B-II and p62 expression, and mitochondrial superoxide levels were increased in murine macrophages (RAW 264.7) pretreated with retinoic acid receptor antagonist (Ro41-5253). These findings suggest that vitamin A plays a crucial role in the efficient fusion of autophagosomes with lysosomes in colitis.
... Recently, in the studies published in a reputed journal, vitamin A was reported as a nutrient having pleiotropic effects in the immune system through the molecular effects of its metabolites. [16,17] Vitamin A was required for optimum mucosal barrier function, appropriate functioning of neutrophils, macrophages, and natural killer (NK) cells, and also for the components of the adaptive immune system including T cells and B cells. [18] Way back to 1925, researchers reported that in vitamin A deficiency states, various epithelia were changed to stratified squamous keratinizing epithelium. ...
Coronavirus disease-19 (COVID-19) caused by SARS-CoV-2 is a novel viral infectious disease, which broke out in the end of winter season 2019 in China and soon became a pandemic. Characteristically there was severe local and systemic immune-inflammatory response to the virus, damaging the respiratory system and other organ systems. The morbidity and mortality caused by the disease are producing tremendous impact on health. The understanding about pathogenesis and manifestations of the disease was obscure. To date, no classic treatment or preventive measure was available for COVID-19 other than symptomatic and supportive care or few drugs under trial. A possibility exists that maintaining vitamin A adequate levels can protect the affected respiratory mucosa, increase antimicrobial activity, produce better antibody response, and have antiinflammatory effects, thereby promoting repair and healing as well. It has been discussed in the review that by various mechanisms, immune regulation through vitamin A supplementation is beneficial to boost immunity in the current outbreak situation when the population is susceptible to the disease. There is a high possibility that vitamin A supplementation to cases as well as population at risk of COVID-19 has a key role in prevention and control. Hence, it is believed that along with other therapeutic and preventive measures, maintaining vitamin A sufficiency during and prior to the development of active disease may act as an adjuvant in population at risk and cases to prevent and control COVID-19.
... RA is the ligand of several subfamilies of ligand-activated nuclear receptors that function as transcriptional regulators. It has been shown that in the absence of RA, the RAR/RXR heterodimers can bind RA-responsive elements (RAREs) in their target genes [146][147][148] and promote their repression via co-repressors like Ncor1 or Ncor2 [149][150][151][152][153]. RA promotes conformational changes of the repressive complex and promotes co-activator binding [154]. Thus, low RA levels would disrupt normal gene expression and the accompanying epigenetic changes that could result in cell death. ...
Multiple models were proposed to explain the mechanism(s) of alcohol (ethanol) teratogenesis inducing the wide range of developmental defects, neurobehavioral anomalies, and mental disabilities known collectively as Fetal Alcohol Spectrum Disorder (FASD). Competition between alcohol clearance and retinoic acid (RA) biosynthesis was proposed as both processes employ the same families of enzymes. Excess of ethanol or its clearance metabolite, acetaldehyde, will compete with vitamin A (retinol) or retinaldehyde and hamper the production of RA with teratogenic outcomes.Taking advantage of the ease of manipulation, external development, and large clutch sizes in Xenopus
, we have been studying and characterizing the alcohol/RA competition model. Xenopus embryos recapitulate many of the developmental malformations of Fetal Alcohol Syndrome (FAS), the more severe form of FASD. The effect of ethanol on development is most severe during gastrula stages and continues, but with milder outcomes throughout development. Ethanol targets the “embryonic organizer,” the earliest site of RA signaling. To support the connection between ethanol and RA, we show that all abnormal embryonic processes or molecular events induced by ethanol can be reproduced by reducing RA signaling levels. Importantly, the effects of ethanol can be rescued by increasing RA signaling, and RA reduction hypersensitizes the embryo to alcohol exposure. Biochemical studies demonstrated that RA biosynthetic enzymes can readily function in ethanol clearance. Additional syndromes linked to reduced RA signaling with partially overlapping phenotypes with FASD are discussed.Key wordsRetinoic acid signalingVertebrate embryogenesis
Xenopus
Retinaldehyde dehydrogenaseEmbryonic organizerAcetaldehydeMicrocephalyFetal Growth RestrictionCraniofacial malformations
... ATRA belongs to the retinoid family of molecules and regulates a wide variety of physiological functions through its nuclear receptors, known as the classical retinoic acid receptors (RARs) and nonclassical retinoid (36,37). To further verify and expand on the effect of ATRA on Cp secretion in WD-Hep and extrapolate the effect to clinically used retinoids, we employed four clinically approved retinoid derivatives. ...
Wilson’s disease (WD) is a copper metabolic disorder caused by a defective ATP7B function. Conventional therapies cause severe side effects and significant variation in efficacy, according to cohort studies. Thus, exploring new therapeutic approaches to prevent progression to liver failure is urgent. To study the physiology and pathology of WD, immortalized cell lines and rodent WD models have been used conventionally; however, a large gap remains among different species as well as in genetic backgrounds among individuals. We generated induced pluripotent stem cells (iPSCs) from 4 WD patients carrying compound heterozygous mutations in the ATP7B gene. ATP7B loss- and gain-of-functions were further manifested with ATP7B-deficient iPSCs and heterozygously corrected R778L WD patient-derived iPSCs using CRISPR-Cas9-based gene editing. Although the expression of ATP7B protein varied among WD-specific hepatocytes differentiated from these iPSCs, the expression and secretion of ceruloplasmin (Cp), a downstream copper carrier in plasma, was consistently decreased in WD-patient-derived and ATP7B-deficient hepatocytes. A transcriptome analysis detected abnormalities in the retinoid signaling pathway and lipid metabolism in WD-specific hepatocytes. Drug screening using WD-patient-derived hepatocytes identified retinoids as promising candidates for rescuing Cp secretion. All-trans retinoic acid (ATRA) also alleviates reactive oxygen species (ROS) production induced by lipid accumulation in WD-specific hepatocytes treated with oleic acid. These patient-derived iPSC-based hepatic models function as effective platforms for the development of potential therapeutics for hepatic steatosis in WD and other fatty liver diseases.
... Until now, most studies have focused on the effect of EDCs regulated by hormone receptors of the nuclear receptors (NRs) family, highlighting as one of the important mechanism of action of EDCs. It has now been more than 20 years since the identification of several receptors of the nuclear receptor superfamily such as estrogen and glucocorticoid receptors (Hollenberg et al. 1985), thyroid hormone receptor (Sap et al. 1986), retinoic acid receptor (Giguere et al. 1987), orphan receptors (Oro et al. 1988), ecdysone receptor (Koelle et al. 1991) and retinoid X-receptor (RXR) (Mangelsdorf et al. 1990). The discovery of these NRs together is considered as an important finding that led to our modern view of hormonal signalling. ...
The aim of this PhD thesis was to develop and assess the potential of novel zebrafish (zf)- based in vitro and in vivo reporter gene assays as bio-analytical tools to monitor estrogenic activity and their implementation in effect-directed analysis (EDA) approach to identify fishspecific
estrogenic compounds in complex mixtures. For this purpose, we first characterized the response of the assays towards a panel of (xeno)estrogens, revealing differences in the affinity of zebrafish estrogen receptor (zfER) subtypes to (xeno)-estrogens. Comparison with human cell-based (MELN-hERα) assay further highlighted inter-species differences showing different chemical ranking towards different classes of known ER ligands. Then, application of these tools to different environmental matrices demonstrated for the first time their functionality to detect and quantify estrogenic activity in complex mixtures, highlighting the zfERβ2 assay as the most sensitive among the different in vitro zfER assays. The above in vitro estrogenic activity was also confirmed in vivo at the most contaminated sites by using the EASZY assay, further adding eco-toxicological relevance. Interestingly, we also reported zebrafish-specific activities at several sites that were not active by the human MELN assay, suggesting the occurrence of fish-specific ER ligands. To address this hypothesis, we applied our zebrafish tools in specific higher tier-EDA studies that allowed isolating zebrafish-specific active fractions by multi-step sample fractionation procedures. Chemical analyses of these specific fractions so far identified several candidate compounds, of which few showed higher selectivity towards zfERβ2 than hERα, hence confirming inter-species differences. This work supports recommendations for the integration of these effect-based tools in future water monitoring strategies.
... RXR was the first adopted orphan receptor; its clone was first isolated in 1990 by Mangelsdorf et. al 15 and in 1992, Heyman R.A et. al identified the 9-cis-retinoic acid as a high affinity ligand for RXR 16 . ...
Nuclear receptors (NR) are a superfamily of structurally related proteins, which serve as the largest group of transcription factors in eukaryotes. They exert their transcriptional activity as a response to binding to their ligands; hence they are classified as ligand dependent transcription factors.
This family of receptors comprises 49 NRs in humans, and they sense a wide variety of ligands e.g. steroids, hormones, vitamins, metabolites, xenobiotics, etc. Many further molecules are being identified as ligands for NRs such as the signaling molecules; phospholipids and heme, which had extended the known types of ligands recognized by NRs.
Through binding to their ligands, NRs control a vast variety of biological process like development, cell differentiation, metabolism, and cell death. Consequently, dysfunctions in these pathways may end up with critical pathological issues ranging from simple metabolic diseases to cancers. Governing important signaling pathways spotlighted nuclear receptors as important druggable targets and a plethora of studies have been focusing on this field.
Typically, multiple heterodimeric partners of RXR are present in certain cell types. In cells where there is a limiting or sequestered pool of RXR combined with the expression of several RXR heterodimerization partners, the mechanism by which RXR partner selection is mediated remains unclear. We hypothesized that there is competition between RXR partners for binding to RXR and that binding of a specific agonist increases the affinity of a given NR to RXR and favors their heterodimerization. RXRα and three NR partners were included in this study: Peroxisome proliferator-activated receptor gamma (PPARγ), vitamin D receptor (VDR) and RARα.
Tracing the dynamic distribution pattern of EGFP-VDR, EGFP-PPAR/nlsm and EGFP-RARα/nlsm (homogeneous in the absence of RXRα and nuclear-enriched in response to RXRα binding) serves as a good model system for studying their competition for heterodimerization with RXRα. There is indeed dynamic competition between RXR partners, which is governed by two mechanisms. First, in the absence of agonist treatment, there is a hierarchy of affinities between RXRα and its partners in the following order: RARα > PPARγ > VDR. Second, in the presence of agonist treatment, RXRα partner selection is shifted towards the liganded partner.
Our results also show that RXR-NR heterodimerization and direct DNA binding are correlated events, and both are augmented by agonist treatment.
These results may explain certain side effects of drugs targeting NRs. Competition for RXR could be responsible for the symptoms of vitamin D deficiency developed in a child upon receiving systemic retinoid treatment for ichthyosis188, or the antagonistic effect of co-administered vitamin A on serum calcium response to vitamin D treatment189.
Our observations regarding these three RXR partners, consistently with metabolism regulation by two other RXR partners (LXR and PPARα) and similar previous findings on membrane-localized IL-2/15 receptors encourage us to generalize the concept that specific ligand binding may often govern competition between different partners of a promiscuous receptor.
Our findings are a proof-of-concept of a hierarchy of affinities between NRs and their common partner, RXRα. These studies can be extended to a larger number of receptors to uncover the network of hierarchies, and follow-up studies will also be focusing on testing this concept in a broader and physiological context.
... While the retinoic acid receptors (RARα, β, and γ) respond to ATRA, the retinoid X receptors (RXRα, β, and γ) are predominantly activated by the 9-cis-isomers of retinoic acid (Allenby et al., 1993), but due to isomerization of ATRA to 9-cis-isomers of retinoic acid (Urbach & Rando, 1994) a mixed RXRs activation by the micromolar concentration of ATRA has been observed (Mangelsdorf et al., 1990;Mic et al., 2003). ...
Using H9C2 cardiomyoblasts, we have shown that all-trans retinoic acid (ATRA), the biologically active metabolite of vitamin A, affects mitochondrial dynamics and functions. The low dose (10 nM) ATRA stimulates the expression of nuclear retinoid receptors and induces mechanisms that are protective against severe local damage caused by laser irradiation at the mitochondrial level. These changes include increased density of the mitochondrial network, higher number of mitochondrial junctions, and enhanced mitochondrial velocity. Moreover, the treated cells had lower basal level of reactive oxygen species (ROS) and could maintain mitochondrial potential (ΔΨm ) after photodamage. Cells treated with 10 nM ATRA had significantly better survival rate after photodamage in comparison to control cells. Cells treated with pharmacological concentration of ATRA (1 µM) expressed higher mitochondrial connectivity without increased motility, which did not lead to better survival or decreased ROS level as was in the case of low-dose ATRA. The proteomics analysis showed changes in proteins related to cellular metabolism (glycolysis) and respiration in ATRA-treated cells. The l-lactate assay confirmed the shift to anaerobic glycolysis in cells treated with 1 µm ATRA, whereas the 10 nM ATRA decreased the level of lactate in medium. The increased levels of cytochrome c or peroxiredoxins 5 level and also lower expression of retinoid and rexinoid receptors were observed in cells treated with 1 µM ATRA. The effect of ATRA is concentration-dependent; the increased mitochondrial dynamics and slower metabolism at 10 nM ATRA contributed significantly to the chance of survival of the cells after photodamage whereas the higher concentration of ATRA overrode the protective effect and led to the unfavorable ones.
... There are many findings that provide experimental evidences that RXR is mediating "imposex" in female gastropod mollusks (Nishikawa et al. 2004). As a member of nuclear receptors superfamily, retinoid X receptors (RXRs) were discovered in the early 1990s (Mangelsdorf et al. 1990), with the identification of 9-cis retinoic acid (9cRA) as the first endogenous ligand (Heyman et al. 1992;Levin et al. 1992). Then, following studies found that RXR exerted transcriptional effects on multiple biological processes such as embryonic development, cell proliferation and differentiation, and metabolism in vertebrates (Desvergne 2007;Evans and Mangelsdorf 2014;Lefebvre et al. 2010). ...
Retinoid X receptor (RXR) has been found to be a major target in various processes of endocrine disruption from the exposure to organotin compounds (OTCs), including imposex in gastropod mollusks. It was also reported in bivalves that OTCs caused intersex and skewed sex ratio. In order to evaluate the effect of these ligand-like OTCs, we constructed a reconstituted yeast system (CfRE system) based on GAL4 yeast two-hybrid principle using scallop Chlamys farreri retinoid X receptor (CfRXRa) and retinoid X response element (RXRE) to investigate the ligand-induced transactivation of CfRXRa. Responses of CfRXRa to 9-cis retinoic acid (9cRA) and tested four OTCs showed concentration-dependent response which is comparable with reported RXRa in vitro assay of human and gastropods. The detective limits of the CfRE system were found to be 100 nM for 9cRA and 10–1000 nM for the tested OTCs. While the tested non-Sn endocrine disrupting chemicals, including Benzo[a]pyrene, 2,4-Dichlorophenol, Nonylphenol, and Tetrabromobisphenol A, showed no effect on CfRXRa response. The present assay system may provide a valuable tool for screening assessments of unidentified environmental ligand chemicals on bivalve mollusks. It is also useful for comparison of sensitivity differences among species exposed to EDCs.
... RSV is also able to modulate vitamin D receptor (VDR)-signaling and it can induce dimerization of VDR with one of its partners, the nuclear retinoid X receptor (RXR) [9]. Three retinoic X receptors are known: RXRα [10], RXRβ [11], and RXRγ [12]. Although the distribution of RXR subtypes is different, their functions are similar: modulating gene expression they control numerous functions by dimerization with other nuclear hormone receptors, contributing thereby to activities of different cell fates [13]. ...
Objective
Ovarian cancer is the most lethal gynecologic cancer. Resveratrol (RSV) is known to alter metabolism in cancer. It affects the nuclear retinoid-X-receptor (RXR), which implies a modulating effect of RXR to gynaecologic cancers. Furthermore, RSV targets Sirtuin1 (Sirt1), a histone deacetylase.
Study design
123 tissue samples of patients with serous or mucinous ovarian cancer were examined for expression of Sirt1 and RXR. Ovarian cell lines were treated with RSV and consequences on viability and apoptosis were evaluated. The influence of RSV to Sirt1 and RXR expression was analyzed by western blotting
Results
A correlation of nuclear Sirt1 and RXRα expression could be detected ( p = 0.006). Co-expression of nuclear RXRα and cytoplasmic ( p = 0.026) or nuclear ( p = 0.041) Sirt1 was associated with significantly increased overall survival in advanced tumour stages. Viability was decreased in all cell lines after stimulation with resveratrol, while cell apoptosis was increased. RSV treatment led to significant lower Sirt1 expression in A2780 cells ( p = 0.025) and significant increased RXR expression in cisA2780 cells ( p = 0.012)
Conclusion
In order to use RSV as medical target, studies could be developed to improve the understanding of drug resistance mechanisms and consequently improve treatment outcome.
... The RXR belongs to the nuclear receptor superfamily and acts as a transcription factor following ligand binding [11,12]. RXR heterodimerizes with others in the nuclear receptors superfamily, including the retinoic acid receptor (RAR), vitamin D receptor (VDR), peroxisome proliferator-activated receptor (PPAR), or liver X receptor (LXR). ...
(1) Background: Notwithstanding numerous therapeutic advances, 176,000 deaths from breast and lung cancers will occur in the United States in 2021 alone. The tumor microenvironment and its modulation by drugs have gained increasing attention and relevance, especially with the introduction of immunotherapy as a standard of care in clinical practice. Retinoid X receptors (RXRs) are members of the nuclear receptor superfamily and upon ligand binding, function as transcription factors to modulate multiple cell functions. Bexarotene, the only FDA-approved RXR agonist, is still used to treat cutaneous T-cell lymphoma. (2) Methods: To test the immunomodulatory and anti-tumor effects of MSU42011, a new RXR agonist, we used two different immunocompetent murine models (MMTV-Neu mice, a HER2 positive model of breast cancer and the A/J mouse model, in which vinyl carbamate is used to initiate lung tumorigenesis) and an immunodeficient xenograft lung cancer model. (3) Results: Treatment of established tumors in immunocompetent models of HER2-positive breast cancer and Kras-driven lung cancer with MSU42011 significantly decreased the tumor burden and increased the ratio of CD8/CD4, CD25 T cells, which correlates with enhanced anti-tumor efficacy. Moreover, the combination of MSU42011 and immunotherapy (anti-PDL1 and anti-PD1 antibodies) significantly (p < 0.05) reduced tumor size vs. individual treatments. However, MSU42011 was ineffective in an athymic human A549 lung cancer xenograft model, supporting an immunomodulatory mechanism of action. (4) Conclusions: Collectively, these data suggest that the RXR agonist MSU42011 can be used to modulate the tumor microenvironment in breast and lung cancer.
... CVD, both congenital and acquired, constitutes a major global public health problem requiring the development of novel and more effective treatments and prevention strategies. Although CVD pathophysiology has been widely studied, there RXRs specifically respond to 9-cis retinoic acid (9cRA) and endogenous fatty acids (FA) [15,91,92]. The identification of natural RXR ligands is still a long matter of study. ...
The heart is the first organ to acquire its physiological function during development, enabling it to supply the organism with oxygen and nutrients. Given this early commitment, cardiomyocytes were traditionally considered transcriptionally stable cells fully committed to contractile function. However, growing evidence suggests that the maintenance of cardiac function in health and disease depends on transcriptional and epigenetic regulation. Several studies have revealed that the complex transcriptional alterations underlying cardiovascular disease (CVD) manifestations such as myocardial infarction and hypertrophy is mediated by cardiac retinoid X receptors (RXR) and their partners. RXRs are members of the nuclear receptor (NR) superfamily of ligand-activated transcription factors and drive essential biological processes such as ion handling, mitochondrial biogenesis, and glucose and lipid metabolism. RXRs are thus attractive molecular targets for the development of effective pharmacological strategies for CVD treatment and prevention. In this review, we summarize current knowledge of RXR partnership biology in cardiac homeostasis and disease, providing an up-to-date view of the molecular mechanisms and cellular pathways that sustain cardiomyocyte physiology.
... In contrast, we found that exposure to RAR agonists and a specific RAR antagonist results in increased penis length in C. atrasolea. While the natural ligand of RAR is thought to be ATRA (Germain et al., 2006a), ATRA can also bind to RXR (Mangelsdorf et al., 1990;Tsuji et al., 2015), including molluscan RXRs (Andr e et al., 2019). In addition, ATRA and 13-cis retinoic acid (but not 9-cis retinoic acid) are present in adult tissues of N. lapillus (Gutierrez-Mazariegos et al., 2014), and RAR-RXR dimers can bind to retinoic acid response element DNA motifs in R. clavigera, (Urushitani et al., 2013). ...
Sexual systems are surprisingly diverse, considering the ubiquity of sexual reproduction. Sequential hermaphroditism, the ability of an individual to change sex, has emerged multiple times independently across the animal kingdom. In molluscs, repeated shifts between ancestrally separate sexes and hermaphroditism are generally found at the level of family and above, suggesting recruitment of deeply conserved mechanisms. Despite this, molecular mechanisms of sexual development are poorly known. In molluscs with separate sexes, endocrine disrupting toxins bind the retinoid X receptor (RXR), activating ectopic male development in females, suggesting the retinoid pathway as a candidate controlling sexual transitions in sequential hermaphrodites. We therefore tested the role of retinoic acid signaling in sequentially hermaphroditic Crepidula snails, which develop first into males, then change sex, maturing into females. We show that retinoid agonists induce precocious penis growth in juveniles and superimposition of male development in females. Combining RXR antagonists with retinoid agonists significantly reduces penis length in induced juveniles, while similar treatments using retinoic acid receptor (RAR) antagonists increase penis length. Transcripts of both receptors are expressed in the induced penis. Our findings therefore show that retinoid signaling can initiate molluscan male genital development, and regulate penis length. Further, we show that retinoids induce ectopic male development in multiple Crepidula species. Species-specific influence of conspecific induction of sexual transitions correlates with responsiveness to retinoids. We propose that retinoid signaling plays a conserved role in molluscan male development, and that shifts in the timing of retinoid signaling may have been important for the origins of sequential hermaphroditism within molluscs.
The first evidence of the existence of vitamin A was the observation 1881 that a substance present in small amounts in milk was necessary for normal development and life. It was not until more than 100 years later that it was understood that vitamin A acts as a hormone through nuclear receptors. Unlike classical hormones, vitamin A cannot be synthesized by the body but needs to be supplied by the food as retinyl esters in animal products and ß-carotene in vegetables and fruits. Globally, vitamin A deficiency is a huge health problem, but in the industrialized world excess of vitamin A has been suggested to be a risk factor for secondary osteoporosis and enhanced susceptibility to fractures. Preclinical studies unequivocally have shown that increased amounts of vitamin A cause decreased cortical bone mass and weaker bones due to enhanced periosteal bone resorption. Initial clinical studies demonstrated a negative association between intake of vitamin A, as well as serum levels of vitamin A, and bone mass and fracture susceptibility. In some studies, these observations have been confirmed, but in other studies no such associations have been observed. One meta-analysis found that both low and high serum levels of vitamin A were associated with increased relative risk of hip fractures. Another meta-analysis also found that low levels of serum vitamin A increased the risk for hip fracture but could not find any association with high serum levels of vitamin A and hip fracture. It is apparent that more clinical studies, including large numbers of incident fractures, are needed to determine which levels of vitamin A that are harmful or beneficial for bone mass and fracture. It is the aim of the present review to describe how vitamin A was discovered and how vitamin A is absorbed, metabolized and is acting as a ligand for nuclear receptors. The effects by vitamin A in preclinical studies are summarized and the clinical investigations studying the effect by vitamin A on bone mass and fracture susceptibility are discussed in detail.
Recent advances have brought forth the complex interplay between tumor cell plasticity and its consequential impact on drug resistance and tumor recurrence, both of which are critical determinants of neoplastic progression and therapeutic efficacy. Various forms of tumor cell plasticity, instrumental in facilitating neoplastic cells to develop drug resistance, include epithelial-mesenchymal transition (EMT) alternatively termed epithelial-mesenchymal plasticity, the acquisition of cancer stem cell (CSC) attributes, and transdifferentiation into diverse cell lineages. Nuclear receptors (NRs) are a superfamily of transcription factors (TFs) that play an essential role in regulating a multitude of cellular processes, including cell proliferation, differentiation, and apoptosis. NRs have been implicated to play a critical role in modulating gene expression associated with tumor cell plasticity and drug resistance. This review aims to provide a comprehensive overview of the current understanding of how NRs regulate these key aspects of cancer biology. We discuss the diverse mechanisms through which NRs influence tumor cell plasticity, including EMT, stemness, and metastasis. Further, we explore the intricate relationship between NRs and drug resistance, highlighting the impact of NR signaling on chemotherapy, radiotherapy and targeted therapies. We also discuss the emerging therapeutic strategies targeting NRs to overcome tumor cell plasticity and drug resistance. This review also provides valuable insights into the current clinical trials that involve agonists or antagonists of NRs modulating various aspects of tumor cell plasticity, thereby delineating the potential of NRs as therapeutic targets for improved cancer treatment outcomes.
Graphical Abstract
Canonical retinoid signaling via nuclear receptors and gene regulation is critical for the initiation of developmental processes such as cellular differentiation, patterning and neurite outgrowth, but also mediates nerve regeneration and synaptic functions in adult nervous systems. In addition to canonical transcriptional regulation, retinoids also exert rapid effects, and there are now multiple lines of evidence supporting non-canonical retinoid actions outside of the nucleus, including in dendrites and axons. Together, canonical and non-canonical retinoid signaling provide the precise temporal and spatial control necessary to achieve the fine cellular coordination required for proper nervous system function. Here, we examine and discuss the evidence supporting non-canonical actions of retinoids in neural development and regeneration as well as synaptic function, including a review of the proposed molecular mechanisms involved.
Introduction:
Despite the recent full FDA approval of lecanemab, there is currently no disease modifying therapy (DMT) that can efficiently slow down the progression of Alzheimer's disease (AD) in the general population. This statement emphasizes the need to identify novel DMTs in the shortest time possible to prevent a global epidemic of AD cases as the world population experiences an increase in lifespan.
Areas covered:
Here, we review several classes of anti-cancer drugs that have been or are being investigated in Phase II/III clinical trials for AD, including immunomodulatory drugs, RXR agonists, sex hormone therapies, tyrosine kinase inhibitors, and monoclonal antibodies.
Expert opinion:
Given the overall course of brain pathologies during the progression of AD, we express a great enthusiasm for the repositioning of anti-cancer drugs as possible AD DMTs. We anticipate an increasing number of combinatorial therapy strategies to tackle AD symptoms and their underlying pathologies. However, we strongly encourage improvements in clinical trial study designs to better assess target engagement and possible efficacy over sufficient periods of drug exposure.
Glaucoma is a complex multifactorial eye disease manifesting in retinal ganglion cell (RGC) death and optic nerve degeneration, ultimately causing irreversible vision loss. Research in recent years has significantly enhanced our understanding of RGC degenerative mechanisms in glaucoma. It is evident that high intraocular pressure (IOP) is not the only contributing factor to glaucoma pathogenesis. The equilibrium of pro-survival and pro-death signalling pathways in the retina strongly influences the function and survival of RGCs and optic nerve axons in glaucoma. Molecular evidence from human retinal tissue analysis and a range of experimental models of glaucoma have significantly contributed to unravelling these mechanisms. Accumulating evidence reveals a wide range of molecular signalling pathways that can operate -either alone or via intricate networks - to induce neurodegeneration. The roles of several molecules, including neurotrophins, interplay of intracellular kinases and phosphates, caveolae and adapter proteins, serine proteases and their inhibitors, nuclear receptors, amyloid beta and tau, and how their dysfunction affects retinal neurons are discussed in this review. We further underscore how anatomical alterations in various animal models exhibiting RGC degeneration and susceptibility to glaucoma-related neuronal damage have helped to characterise molecular mechanisms in glaucoma. In addition, we also present different regulated cell death pathways that play a critical role in RGC degeneration in glaucoma.
The chick limb bud is a powerful experimental system in which to study pattern formation in vertebrate embryos. Exogenously applied retinoic acid, a vitamin A derivative, can bring about changes in pattern and, on several grounds, is a good candidate for an endogenous morphogen. As such, the local concentration of retinoic acid might provide cells with information about their position in relation to one axis of the limb. Alternatively, retinoic acid may be part of a more complex signalling system.
Homeobox genes are possible target genes for regulation by retinoic acid in the limb. In particular, one homeobox gene, XlHbox 1 is expressed locally in the mesenchyme of vertebrate forelimbs and might code for an anterior position. When the pattern of the chick wing is changed by retinoic acid or by grafts of signalling tissue such that anterior cells now form posterior structures, the domain of XlHbox 1 expression expands rather than contracts. The expansion of XlHbox 1 expression correlates with shoulder girdle abnormalities.
Retinoic acid application leads to visible changes in bud shape and this allows dissection of the way in which patterning is co-ordinated with morphogenesis. Results of recombination experiments and studies of changes in the apical ridge and proliferation in the mesenchyme suggest the following scheme: retinoic acid is involved in specification of position of mesenchyme cells; this specification determines their local interaction with the ridge that controls ridge morphology; the thickened apical ridge permits local proliferation in the underlying mesenchyme.
The recent advances in molecular biology that permit analysis of the expression of various interesting genes in developing limbs hold out the promise that further investigation may soon allow a complete account of the patterning process in one part of the vertebrate embryo.
Many epidemiological studies have emphasised the relation between carotenoid dietary intake and their circulating concentrations and beneficial health effect, such as lower risk of cardiometabolic diseases and cancer. However, there is dispute as to whether the attributed health benefits are due to native carotenoids or they are rather induced by their metabolites. Several categories of metabolites have been reported, most notably due to a) modifications at the cyclohexenyl-ring or the polyene chain, such as epoxides and geometric isomers, b) excentric cleavage metabolites with also alcohol-, aldehyde- or carboxylic acid-functional groups or c) centric cleaved metabolites with additional hydroxyl-, aldehyde- or carboxyl-functionalities, not counting their potential phase-II glucuronidated/sulphated derivatives. Of special interest are the apo-carotenoids, which originate in the intestine and other tissues from carotenoids cleavage by beta-carotene oxygenases 1/2 in a symmetrical/non-symmetrical fashion. These are more water soluble and more electrophilic, and therefore putative candidates for interactions with transcription factors such as NF-kB and Nrf2, as well as ligands for RAR-RXR nuclear receptor interactions. In this review, we discuss in vivo detected apo-carotenoids, their reported tissue concentrations, and potential associated health effects, focussing exclusively on the human situation and based on quantified/semi-quantified carotenoid-metabolites proven to be present in humans.
Rice crab co-culture is a new integrated farming model in China. The application of triazole plant growth regulators (PRGs) is often used as an advantageous option to combat rice lodging. However, there is still a gap regarding the toxicity of these PRGs on the growth and development of the Chinese mitten crab (Eriocheir sinensis, E. sinensis). Here the effect of triazoles (paclobutrazol and uniconazole) on the molting mechanism of E. sinensis was investigated. Monitoring of regulatory genes associated with molting showed that the two PRGs were found to inhibit the expression of ecdysteroid hormone (EH), ecdysteroid receptors gene (EcR), and retinoid X receptors gene (RXR) and induce secretion of molt-inhibiting hormone (MIH) gene. In addition, the activities of chitinase (CHIA) and N-acetyl-β-d-aminoglucosidase (β-NAGase) were also inhibited by exposure to PRGs. Exposure to PRGs also elevated the mRNA expression of the growth-related myostatin gene (MSTN). These results revealed that there is a long-term risk of exposure to triazoles PRGs that may inhibit molting and affect normal development and immune system of E. sinensis.
Tricyclazole is used as a common fungicide to control rice blast. However, studies on the toxicity of tricyclazole to crabs in the rice-crab co-culture system are still extremely rare. Here, the environmental dissipation of tricyclazole was monitored in this model, and the potential toxicity of tricyclazole to E. sinensis at environmental concentrations as well as the dietary risk was evaluated. The results showed that tricyclazole had no significant acute toxicity to E. sinensis (LC50 > 100 mg/L), while it promoted body weight gain. Tricyclazole in the hepatopancreas had a higher persistent bioaccumulation risk than in the muscle. Tricyclazole suppressed the immune response of E. sinensis under prolonged exposure and there should be gender differences, with females being more sensitive. Lipid metabolism enzymes were also significantly inhibited. While tricyclazole stimulated males molting but prolonged molting duration, both molting and duration of females were also disturbed. The dietary risk assessment indicated that tricyclazole intake from current crab consumption was low risk. This evidence demonstrated that tricyclazole may have potential risks to individual development, nutritional quality, and economic value on E. sinensis and should be used with caution in rice-crab co-culture system whenever possible.
Vit Α and its derivatives (retinoids) have been known since the beginning of the twentieth century to be essential for developing and maintaining various tissues, including the skin and hair [1, 2]. Retinoic acid, also known as Tretinoin, or all-trans-retinoic acid (ATRA) in its pharmaceutical form, is the carboxylic acid, an acidic, active metabolite of Vit A. Retinoic acid (RA), and retinoids, in general, are the most extensively studied class of drugs for acne treatment [3]. ATRA was patented in 1957, got approved for medical use in 1962, and notably, it is on the World Health Organization’s List of Essential Medicines along with the most effective and safe medicines needed in a health system [4].
β-catenin regulates HBV transcription in cell culture and viral biosynthesis in vivo in the transgenic mouse model of chronic HBV infection. Therefore, it is important to understand which transcription factor activities are coactivated by β-catenin to enhance HBV biosynthesis. The effect of β-catenin expression in the context of nuclear receptor-mediated HBV transcription was evaluated initially in the human embryonic kidney cell line, HEK293T. Reporter gene and viral replication assays revealed that β-catenin can coactivate HBV transcription through some, most predominantly liver receptor homolog 1 (LRH1), but not all nuclear receptors known to activate viral biosynthesis. Similarly, β-catenin activated nuclear receptor-mediated HBV transcription and replication in the human hepatoma cell line, Huh7, primarily through its effect on the farnesoid X receptor α (FXRα). These data indicate that β-catenin can enhance nuclear receptor-mediated HBV biosynthesis, but the relative importance of various transcription factors is dependent upon the precise cellular environment.
The functional aspects of fat soluble vitamins (A, D, E, and K) have been changed after the elucidation of their multidimensional effects. They are associated with systematic immune response exhibit immunomodulatory effects by regulating lymphocytes' functions, antibody secretion, and cytokines expression. Vitamins A and D help in the proliferation and maturation of lymphocytes; dendritic cells also increase the number of natural killer cells to potentiate innate immunity. Vitamin A specifically advances the gut-homing capacity of lymphocytes, differentiation of plasma cells, and antibody production. However, vitamin D decreases endoplasmic reticulum stress, foam cell formation, Th1 and Th17 cell production, and the activation of T helper cells. The antioxidant effect of vitamin E prevents lipid peroxidation maintains membrane integrity, signal transduction, and proliferation of lymphocytes. Vitamin K regulates complement activation decreases T cell proliferation. The antiinflammatory effect is precisely regulated by these vitamins. They potentially increase the numbers of Treg cells, Il -10 levels decrease the activity of NF-κB, cycloxygenase-2, levels of TNF-α, IL-6, Il-17, IFN-γ, and prostaglandin E2. This chapter deals with the mechanism of actions of fat-soluble vitamins on the modulation of the immune response.
Alterations in the nuclear retinoid X receptor (RXRs) signalling have been implicated in neurodegenerative disorders such as Alzheimer’s disease, Parkinson’s disease, stroke, multiple sclerosis and glaucoma. Single nucleotide polymorphisms (SNPs) are the main cause underlying single nucleic acid variations which in turn determine heterogeneity within various populations. These genetic polymorphisms have been suggested to associate with various degenerative disorders in population-wide analysis. This bioinformatics study was designed to investigate, search, retrieve and identify deleterious SNPs which may affect the structure and function of various RXR isoforms through a computational and molecular modelling approach. Amongst the 1,813 retrieved SNPs several were found to be deleterious with rs140464195_G139R, rs368400425_R358W and rs368586400_L383F RXRα mutant variants being the most detrimental ones causing changes in the interatomic interactions and decreasing the flexibility of the mutant proteins. Molecular genetics analysis identified seven missense mutations in RXRα/β/γ isoforms. Two novel mutations SNP IDs (rs1588299621 and rs1057519958) were identified in RXRα isoform. We used several in silico prediction tools such as SIFT, PolyPhen, I-Mutant, Protein Variation Effect Analyzer (PROVEAN), PANTHER, SNP&Go, PhD-SNP and SNPeffect to predict pathogenicity and protein stability associated with RXR mutations. The structural assessment by DynaMut tool revealed that hydrogen bonds were affected along with hydrophobic and carbonyl interactions resulting in reduced flexibility at the mutated residue positions but ultimately stabilizing the molecule as a whole. Summarizing, analysis of the missense mutations in RXR isoforms showed a mix of conclusive and inconclusive genotype–phenotype correlations suggesting the use of sophisticated computational analysis tools for studying RXR variants.
Communicated by Ramaswamy H. Sarma
Chronic disease states of aging should be viewed through the prism of metabolism and biophysical processes at all levels of physiological organization present in the human body. This book connects these insights to what causes them to go awry in the context of unhealthy human behaviors and aging, aiming to buttress scientific creativity. It also provides links between the art and science of medicine that strengthens problem-solving in patient care. New and important discoveries in the area of metabolic health and metabolic diseases are discussed in exquisite detail. Key Features: Broad and up-to-date overview of the field of metabolic aspects of health and chronic disease development, especially connecting the spectrum of topics that range from molecular clocks to stress response to nuclear hormone receptors and the role of microbiota in human health Provides a deeper basic science and interdisciplinary understanding of biological systems that broaden the perspectives and therapeutic problem solving by elaborating on the usefulness of the Physiological Fitness Landscape Describes the importance of insulin resistance in metabolic disease, especially diabetes but also includes links to cancer and Alzheimer's disease Examines the process of aging from the perspective of metabolic decline illustrating it with the Physiological Fitness Landscape This book, the second volume in a two-volume set, primarily targets an audience of clinical and science students, biomedical researchers and physicians who would benefit from understanding each other's language.
Apolipoprotein CIII (apoCIII), a lipid-binding protein involved in the transport of triglycerides and cholesterol in the plasma, is synthesized primarily in the liver and the intestine. A cis-acting regulatory element, C3P, located at -90 to -66 upstream from the apoCIII gene transcriptional start site (+1), is necessary for maximal expression of the apoCIII gene in human hepatoma (HepG2) and intestinal carcinoma (Caco2) cells. This report shows that three members of the steroid receptor superfamily of transcription factors, hepatocyte nuclear factor 4 (HNF-4), apolipoprotein AI regulatory protein 1 (ARP-1), and Ear3/COUP-TF, act at the C3P site. HNF-4 activates apoCIII gene expression in HepG2 and Caco2 cells, while ARP-1 and Ear3/COUP-TF repress its expression in the same cells. HNF-4 activation is abolished by increasing amounts of ARP-1 or Ear3/COUP-TF, and repression by ARP-1 or Ear3/COUP-TF is alleviated by increasing amounts of HNF-4. HNF-4 and ARP-1 bind with similar affinities to the C3P site, suggesting that their opposing transcriptional effects may be mediated by direct competition for DNA binding. HNF-4 and ARP-1 mRNAs are present within the same cells in the liver and intestine, and protein extracts from hepatic tissue, HepG2, and Caco2 cells contain significantly more HNF-4 than ARP-1 or Ear3/COUP-TF binding activities. These findings suggest that the transcription of the apoCIII gene in vivo is dependent, at least in part, upon the intracellular balance of these positive and negative regulatory factors.
At the level of transcription, all signals of the vitamin A derivative retinoic acid (RA) are mediated by the RA receptors (RARs) as well as the retinoid X receptors (RXRs). The control of expression of the various receptor subtypes and their specific isoforms appears to be strictly regulated and can be assumed to play a pivotal role during development and in the adult tissue. It has previously been shown that the RAR beta 2 isoform can regulate its own synthesis through an RA response element (RARE) in its promoter. Recent evidence suggests that the expression of other RAR isoforms, including that of RAR gamma 2, are also regulated by RA. We present evidence that expression of the RAR gamma 2 isoform can be regulated through the RARE in its own promoter region. Similar to the beta 2 RARE, the gamma 2 RARE consists of a 6-bp direct repeat with a 5-nucleotide spacer, but it has different functional features, including receptor specificity, basal-level activity, and affinity for RAR. In agreement with recent observations, this response element is bound most effectively by RAR/RXR heterodimers. Single-base-pair mutations had different effects on the activity of this RARE. The gamma 2 RARE is surrounded by several binding sites for the transcription factor Sp1. Cotransfected Sp1 enhanced strongly the activity of gamma 2 promoter reporter constructs in Drosophila cells. Our data suggest an important role for RAR-containing heterodimers and Sp1 in the regulation of RAR gamma 2 expression.
Retinoids regulate gene transcription by interacting with both retinoic acid (RA) receptors (RARs) and retinoid X receptors (RXRs). Since unliganded RXRs can act as heterodimerization partners for RARs and other nuclear hormone receptors, it is unclear whether ligand binding by RXRs actually regulates the expression of naturally occurring genes. To address this issue, we synthesized the RXR-selective retinoid SR11237 and confirmed its specificity in transient transfection and proteolytic susceptibility assays before using it to assess the contribution of ligand-activated RXRs to retinoid action. Unlike RAR ligands, SR11237 did not increase endogenous RAR beta mRNA levels in F9 embryonal carcinoma cells, even though it activated transcription of an RXR-responsive reporter gene in these cells. Thus, it is likely that RARs mediate the induction of RAR beta gene expression by RA. In contrast, the RXR-specific ligand induced rat growth hormone mRNA in GH3 pituitary cells, indicating that the effects of RA on growth hormone gene expression at least in part involve ligand binding to endogenous RXRs in vivo. Our results indicate that in addition to serving as cofactors for other nuclear hormone receptors, endogenous RXRs can function as ligand-dependent regulators of gene expression, i.e., classical nuclear hormone receptors.
A nonrandom chromosomal translocation breakpoint, t(15;17)(q22;q21), is found in almost all patients with acute promyelocytic leukemia (APL). Most of these breakpoints occur within the second intron of the retinoic acid receptor-alpha (RARA) gene. We screened a cDNA library of APL and have identified and sequenced a cDNA transcribed from the t(15;17) translocation breakpoint. The 5' end of cDNA p1715 consists of 503 bp of the RARA exon II sequence. A 1.76-kb cDNA without homology to any known gene available in GenBank was found truncated downstream. This cDNA sequence was assigned to chromosome 15 by dot blot hybridization of the flow cytometry-sorted chromosomes. We designate this fusion cDNA RARA/myl, which is different from myl/RARA reported by de The et al. (H. de The, C. Chomienne, M. Lanotte, L. Degos, and A. Dejean, Nature (London) 347:558-561, 1990). This result demonstrates that the two different types of hybrid mRNA can be transcribed from this breakpoint. We screened a non-APL cDNA library and identified a 2.8-kb myl cDNA. This cDNA is able to encode a polypeptide with a molecular weight of 78,450. Alternative splicing of the myl gene which resulted in myl proteins with different C terminals was found. Southern blot analysis of the genomic DNA isolated from 17 APL patients by using the myl DNA probe demonstrated that the myl gene in 12 samples was rearranged. Northern (RNA) blot analysis of RARA gene expression in two APL RNA samples showed abnormal mRNA species of 4.2 and 3.2 kb in one patient and of 4.8 and 3.8 kb in another patient; these were in addition to the normal mRNA species of 3.7 and 2.7-kb. The myl DNA probe detected a 2.6-kb abnormal mRNA in addition to the normal mRNA species of 3.2, 4.2, and 5.5 kb. Using the polymerase chain reaction, we demonstrated that both RARA/myl and myl/RARA were coexpressed in samples from three different APL patients. From this study, we conclude that the t(15;17) translocation breakpoint results in the transcription of two different fusion transcripts which are expected to be translated into fusion proteins.
The gene coding for apolipoprotein AI (apoAI), a lipid binding protein involved in the transport of cholesterol and other lipids in the plasma, is expressed in mammals predominantly in the liver and the intestine. Liver-specific expression is controlled by synergistic interactions between transcription factors bound to three separate sites, sites A (-214 to -192), B (-169 to -146), and C (-134 to -119), within a powerful liver-specific enhancer located between nucleotides -222 and -110 upstream of the apoAI gene transcription start site (+1). Previous studies in our laboratory have shown that ARP-1, a member of the nuclear receptor superfamily whose ligand is unknown (orphan receptor), binds to site A and represses transcription of the apoAI gene in liver cells. In a more recent series of experiments, we found that site A is a retinoic acid (RA) response element that responds preferentially to the recently identified RA-responsive receptor RXR alpha over the previously characterized RA receptors RAR alpha and RAR beta. In this study we investigated the combined effects of ARP-1 and RXR alpha on apoAI gene expression in liver cells. Transient transfection assays showed that site A is necessary and sufficient for RXR alpha-mediated transactivation of the apoAI gene basal promoter in human hepatoma HepG2 cells in the presence of RA and that this transactivation is abolished by increasing amounts of cotransfected ARP-1. Electrophoretic mobility shift assays and subsequent Scatchard analysis of the data revealed that ARP-1 and RXR alpha bind to site A with similar affinities. These assays also revealed that ARP-1 and RXR alpha bind to site A as heterodimers with an affinity approximately 10 times greater than that of either ARP-1 or RXR alpha alone. Further transfection assays in HepG2 cells, using as a reporter a construct containing the apoAI gene basal promoter and its upstream regulatory elements (including site A) in their natural context, revealed that RXR alpha has very little effect on the levels of expression regardless of the presence or absence of RA. However, while ARP-1 alone or ARP-1 and RXR alpha together dramatically repress expression in the absence of RA, the repression by ARP-1 and RXR alpha together, but not ARP-1 alone, is almost completely alleviated in the presence of RA. These results indicate that transcriptional repression by ARP-1 sensitizes apoAI gene responsiveness to RXR alpha and RA and suggest that the magnitude of this responsiveness is regulated by the intracellular ratio of ARP-1 to RXR alpha. These observations raise the possibility that transcriptional repression is a general mechanism for switching gene transcription between alternative transcription activation pathways.
The complex and diverse biological effects of retinoic acid (RA) are mediated through specific receptors that are members of the steroid hormone family of nuclear transcription factors. The RA receptor family consists of multiple structurally distinct RA receptors, which diverge primarily at the NH2-terminal domain. The evolutionary conservation of this divergent region in individual RA receptors among different species together with their tissue-specific patterns of expression suggest that the biological function and activity of the individual RA receptors may be confined to specific tissues. To test this hypothesis in hematopoietic cells, we used retrovirus-mediated gene transduction to introduce the RA receptors RAR-alpha, RAR-beta, and RAR-gamma as well as RXR-alpha into a mutant subclone of the HL-60 promyelocytic leukemia cell line (designated HL-60R) that is relatively resistant to RA-induced granulocytic differentiation. We found that each of these structurally distinct RA receptors could restore sensitivity of the HL-60R cells to RA. A critical threshold number of transduced receptors per cell appears to be necessary to restore this functional activity. Thus, the capability to mediate granulocytic differentiation of HL-60 cells is shared among distinctly different RA receptors.
Thyroid hormone (T3) receptor (TR) is a ligand-dependent transcription factor that acts through specific binding sites in the promoter region of target genes. In order to identify new genes that are regulated by T3, we used anti-TR antiserum to immunoprecipitate TR-DNA complexes from GH4 cell nuclei that had previously been treated with a restriction enzyme. Screening of the immunopurified, cloned DNA for TR binding sites by electrophoretic mobility shift assay yielded 53 positive clones. A subset of these clones was specifically immunoprecipitated with anti-TR antiserum and may therefore represent biologically significant binding sites. One of these clones, clone 122, was characterized in detail. It includes sequences highly related to the NICER long terminal repeat-like element and contains three TR binding sites as determined by DNase I footprinting. Two of the clone 122 TR binding sites are located upstream of the TATA box, and one is located downstream. The TR binding site downstream from the promoter was necessary and sufficient to confer T3-dependent regulation in transient transfection experiments. Expression of a reporter construct under the control of the clone 122 promoter region was activated by TR in the absence of ligand and returned to basal levels after T3 addition. Clone 122 sequences hybridize to at least two different mRNAs of approximately 6 and 10 kb from GH4 cells. The levels of both of these mRNAs increased upon removal of T3. Our studies suggest that specific immunoprecipitation of chromatin allows identification of binding sites and target genes for transcription factors.
The vitamin hormone retinoic acid (RA) regulates many complex biological programs. The hormonal signals are mediated at the level of transcription by multiple nuclear receptors. These receptors belong to the steroid/thyroid hormone receptor superfamily that also includes a large number of orphan receptors whose biological roles have not yet been determined. Although much has been learned in recent years about RA receptor (RAR) functions, little is known about how specific RA response programs are restricted to certain tissues and cell types during development and in the adult. It has been recently shown that RAR activities are regulated by retinoid X receptors (RXR) through heterodimer formation. In an effort to isolate and further characterize nuclear receptors that modulate RAR and/or RXR activities, we have screened cDNA libraries by using a RXR alpha cDNA probe. Two clones, COUP alpha and COUP beta, identical and closely related to the orphan receptor COUP-TF, were obtained. We show that COUP proteins dramatically inhibit retinoid receptor activities on certain response elements that are activated by RAR/RXR heterodimers or RXR homodimers. COUP alpha and -beta bind strongly to these response elements, including a palindromic thyroid hormone response element and a direct repeat RA response element as well as an RXR-specific response element. In addition, we found that the previously identified COUP-TF binding site in the ovalbumin gene functions in vitro as an RA response element that is repressed in the presence of COUP. Our data suggest that the COUP receptors are a novel class of RAR and RXR regulators that can restrict RA signaling to certain elements. The COUP orphan receptors may thus play an important role in cell- or tissue-specific repression of subsets of RA-sensitive programs during development and in the adult.
Terminal differentiation of epidermal keratinocytes is inhibited by 1 microM retinoic acid, a concentration which induces differentiation in a number of cell types, including F9 teratocarcinoma cells. The molecular basis for these opposing retinoid responses is unknown, although retinoic acid receptors (RARs) and retinoid X receptors (RXRs) have been detected in both cell types. When F9 cells are stably transfected with a truncated RAR alpha lacking the E/F domain necessary for ligand binding and RAR/RXR dimerization, action at retinoid response elements is suppressed and cells produce a retinoic acid-resistant phenotype; i.e., they are blocked in differentiation (A. S. Espeseth, S. P. Murphy, and E. Linney, Genes Dev. 3:1647-1656, 1989). If retinoid receptors influence epidermal differentiation only in a negative fashion, then suppression of transactivation at retinoid response elements would be expected to enhance, rather than block, keratinocyte differentiation. In this study, we show that surprisingly, even though constitutive expression of an analogous truncated RAR gamma in keratinocytes specifically suppressed transactivation at retinoid response elements, keratinocytes were blocked, rather than enhanced, in their ability to undergo morphological and biochemical features of differentiation. These findings demonstrate a direct and hitherto unrecognized role for RARs and RXRs in positively as well as negatively regulating epidermal differentiation. Additionally, our studies extend those of Espeseth et al. (Genes Dev. 3:1647-1656, 1989), indicating a novel RAR function independent of the E/F domain.
The ability of a retinoic acid (RA) response element (RARE) in the phosphoenolpyruvate carboxykinase (PEPCK) gene promoter to mediate effects of either RA or thyroid hormone (T3) on gene expression was studied. Fusion gene constructs consisting of PEPCK promoter sequences ligated to the chloramphenicol acetyltransferase (CAT) reporter gene were used for this analysis. While T3 induced CAT expression to a small degree (about twofold) when such constructs were transiently transfected into H4IIE rat hepatoma cells, along with an expression vector encoding the alpha subtype of the T3 receptor (TR), this effect was mediated by promoter sequences distinct from the PEPCK RARE. Although TRs were capable of binding the PEPCK RARE in the form of putative monomers, dimers, and heterodimers with RA receptors (RARs), this element failed to mediate any positive effect of T3 on gene expression. In contrast, the PEPCK RARE mediated six- to eightfold induction of CAT expression by RA. When TRs were coexpressed along with RARs in transfected H4IIE cells, this RA induction was substantially blunted in a T3-independent manner. This inhibitory effect may be due to the binding of nonfunctional TRs or TR-RAR heterodimers to the PEPCK RARE. A model is proposed to explain the previously observed in vivo effects of T3 on PEPCK gene expression.
We report that thyroid hormone (T3) receptor (T3R) can activate the human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR). Purified chick T3R-alpha 1 (cT3R-alpha 1) binds as monomers and homodimers to a region in the LTR (nucleotides -104 to -75 [-104/-75]) which contains two tandem NF-kappa B binding sites and to a region (-80/-45) which contains three Sp1 binding sites. In contrast, human retinoic acid receptor alpha (RAR-alpha) and mouse retinoid X receptor beta (RXR-beta) do not bind to these elements. However, RXR-beta binds to these elements as heterodimers with cT3R-alpha 1 and to a lesser extent with RAR-alpha. Gel mobility shift assays also revealed that purified NF-kappa B p50/65 or p50/50 can bind to one but not both NF-kappa B sites simultaneously. Although the binding sites for p50/65, p50/50, and T3R, or Sp1 and T3R, overlap, their binding is mutually exclusive, and with the inclusion of RXR-beta, the major complex is the RXR-beta-cT3R-alpha 1 heterodimer. The NF-kappa B region of the LTR and the NF-kappa B elements from the kappa light chain enhancer both function as T3 response elements (TREs) when linked to a heterologous promoter. The TREs in the HIV-1 NF-kappa B sites appear to be organized as a direct repeat with an 8- or 10-bp gap between the half-sites. Mutations within the NF-kappa B motifs which eliminate binding of cT3R-alpha 1 also abolish stimulation by T3, indicating that cT3R-alpha 1 binding to the Sp1 region does not independently mediate activation by T3. The Sp1 region, however, is converted to a functionally strong TRE by the viral tat factor. These studies indicate that the HIV-1 LTR contains both tat-dependent and tat-independent TREs and reveal the potential for T3R to modulate other genes containing NF-kappa B- and Sp1-like elements. Furthermore, they indicate the importance of other transcription factors in determining whether certain T3R DNA binding sequences can function as an active TRE.
Rev-ErbA alpha (Rev-Erb) is a nuclear hormone receptor-related protein encoded on the opposite strand of the alpha-thyroid hormone receptor (TR) gene. This unusual genomic arrangement may have a regulatory role, but the conservation of human and rodent Rev-Erb amino acid sequences suggests that the protein itself has an important function, potentially as a sequence-specific transcriptional regulator. However, despite its relationship to the TR, Rev-Erb bound poorly to TR binding sites. To determine its DNA-binding specificity in an unbiased manner, Rev-Erb was synthesized in Escherichia coli, purified, and used to select specific binding-sites from libraries of random double-stranded DNA sequences. We found that Rev-Erb binds to a unique site consisting of a specific 5-bp A/T-rich sequence adjacent to a TR half-site. Rev-Erb contacts this entire asymmetric 11-bp sequence, which is the longest nonrepetitive element specifically recognized by a member of the thyroid/steroid hormone receptor superfamily, and mutations in either the A/T-rich or TR half-site regions abolished specific binding. The binding specificity of wild-type Rev-Erb was nearly identical to that of C- and N-terminally truncated forms. This binding was not enhanced by retinoid X receptor, TR, or other nuclear proteins, none of which formed heterodimers with Rev-Erb. Rev-Erb also appeared to bind to the selected site as a monomer. Furthermore, Rev-Erb activates transcription through this binding site even in the absence of exogenous ligand. Thus, Rev-Erb is a transcriptional activator whose properties differ dramatically from those of classical nuclear hormone receptors, including the TR encoded on the opposite strand of the same genomic locus.
A new method of total RNA isolation by a single extraction with an acid guanidinium thiocyanate-phenol-chloroform mixture is described. The method provides a pure preparation of undegraded RNA in high yield and can be completed within 4 h. It is particularly useful for processing large numbers of samples and for isolation of RNA from minute quantities of cells or tissue samples.
A new method of total RNA isolation by a single extraction with an acid guanidinium thiocyanate-phenol-chloroform mixture is described. The method provides a pure preparation of undegraded RNA in high yield and can be completed within 4 h. It is particularly useful for processing large numbers of samples and for isolation of RNA from minute quantities of cells or tissue samples.
Retinoic acid receptors (RARs) are retinoic acid (RA)-inducible enhancer factors belonging to the superfamily of steroid/thyroid nuclear receptors. We have previously characterized two human RAR (hRAR-alpha and hRAR-beta) cDNAs and have recently cloned their murine cognates (mRAR-alpha and mRAR-beta) together with a third RAR (mRAR-gamma) whose RNA was detected predominantly in skin, a well-known target for RA. mRAR-gamma cDNA was used here to clone its human counterpart (hRAR-gamma) from a T47D breast cancer cell cDNA library. Using a transient transfection assay in HeLa cells and a reporter gene harboring a synthetic RA responsive element, we demonstrate that hRAR-gamma cDNA indeed encodes a RA-inducible transcriptional trans-activator. Interestingly, comparisons of the amino acid sequences of all six human and mouse RARs indicate that the interspecies conservation of a given member of the RAR subfamily (either alpha, beta, or gamma) is much higher than the conservation of all three receptors within a given species. These observations indicate that RAR-alpha, -beta, and -gamma may perform specific functions. We show also that hRAR-gamma RNA is the predominant RAR RNA species in human skin, which suggests that hRAR-gamma mediates some of the retinoid effects in this tissue.
Transcription of major histocompatibility complex (MHC) class I genes is regulated by the conserved MHC class I regulatory element (CRE). The CRE has two factor-binding sites, region I and region II, both of which elicit enhancer function. By screening a mouse lambda gt 11 library with the CRE as a probe, we isolated a cDNA clone that encodes a protein capable of binding to region II of the CRE. This protein, H-2RIIBP (H-2 region II binding protein), bound to the native region II sequence, but not to other MHC cis-acting sequences or to mutant region II sequences, similar to the naturally occurring region II factor in mouse cells. The deduced amino acid sequence of H-2RIIBP revealed two putative zinc fingers homologous to the DNA-binding domain of steroid/thyroid hormone receptors. Although sequence similarity in other regions was minimal, H-2RIIBP has apparent modular domains characteristic of the nuclear hormone receptors. Further analyses showed that both H-2RIIBP and the natural region II factor bind to the estrogen response element (ERE) of the vitellogenin A2 gene. The ERE is composed of a palindrome, and half of this palindrome resembles the region II binding site of the MHC CRE. These results indicate that H-2RIIBP (i) is a member of the superfamily of nuclear hormone receptors and (ii) may regulate not only MHC class I genes but also genes containing the ERE and related sequences. Sequences homologous to the H-2RIIBP gene are widely conserved in the animal kingdom. H-2RIIBP mRNA is expressed in many mouse tissues, in agreement with the distribution of the natural region II factor.
Identification of complementary DNAs encoding the human glucocorticoid receptor predicts two protein forms, of 777 (alpha) and 742 (beta) amino acids, which differ at their carboxy termini. The proteins contain a cysteine/lysine/arginine-rich region which may define the DNA-binding domain. Pure radiolabelled glucocorticoid receptor, synthesized in vitro, is immunoreactive and possesses intrinsic steroid-binding activity characteristic of the native glucocorticoid receptor.
Steroid hormone receptors activate specific gene transcription by binding as hormone-receptor complexes to DNA enhancer elements termed hormone responsive elements. A highly conserved 66-amino-acid region of the oestrogen and glucocorticoid receptors which corresponds to part of the receptor DNA-binding domain (region C) determines the specificity of target gene recognition. This region contains two subregions (CI and CII), encoded in two separate exons, that are analogous to the 'zinc fingers' of the transcription factor TFIIIA. The N-terminal CI finger determines the recognition specificity of the hormone responsive element. A chimaeric oestrogen receptor, in which the CI finger is replaced with the corresponding glucocorticoid receptor CI finger region, activates transcription from a reporter gene containing a glucocorticoid-responsive element, but not from a reporter gene containing an oestrogen-responsive element. We report here that three amino acids located at the C-terminal side of the oestrogen receptor CI finger play a key part in this specificity.
Androgenic hormones mediate their effects on male sex differentiation and development through a high affinity receptor protein. We report here cloning of the complete coding sequence of the human androgen receptor (hAR). By sequence homology hAR is a member of the nuclear receptor family, with closest sequence identity to the progesterone, mineralocorticoid, and glucocorticoid receptors. Regions of highest homology include the DNA-binding domain and a small region within the hydrophobic ligand-binding domain. Comparison of the deduced 919 amino acid sequence of hAR (98,999 mol wt) to the 902 amino acid sequence of rat AR (98,227 mol wt) reveals identical sequences in the DNA- and hormone-binding domains, with an overall homology of 85%. In human prostate, the major androgen receptor mRNA species is 10 kilobases while a less abundant mRNA is approximately 7 kilobases. Rabbit polyclonal antibodies were raised against a synthetic peptide from the N-terminal region of hAR. Immunocytochemical analysis of human prostate tissue demonstrated that AR is localized predominantly in nuclei of glandular epithelial cells.
5'-Noncoding sequences have been compiled from 699 vertebrate mRNAs. (GCC)GCCAGCCATGG emerges as the consensus sequence for initiation of translation in vertebrates. The most highly conserved position in that
motif is the purine in position -3 (three nucleotides upstream from the ATG codon); 97% of vertebrate mRNAs have a purine,
most often A, in that position- The periodical occurrence of G (in positions −3, −6, −9) is discussed. Upstream ATG codons
occur in fewer than 10% of vertebrate mRNAs-at-large; a notable exception are oncogene transcripts, two-thirds of which have
ATG codons preceding the start of the major open reading frame. The leader sequences of most vertebrate mRNAs fall in the
size range of 20 to 100 nucleotides. The significance of shorter and longer 5' -noncoding sequences is discussed.
Several embryonal carcinoma (EC) cell lines were tested in culture for their ability to metabolize all-trans-[3H]retinol, all-trans-[3H]retinyl acetate, and all-trans-[3H]retinoic acid. There was little, if any, metabolism of all-trans-retinol to more polar compounds; we failed to detect conversion to acidic retinoids by reverse-phase high performance liquid chromatography and derivatization. We also did not observe [3H]retinoic acid when EC cells were incubated with [3H]retinyl acetate. Unlike the other retinoids, all-trans-[3H]retinoic acid, even at micromolar levels, was almost totally modified by cells from several EC lines within 24 h. Most of the labeled products were secreted into the medium. Some EC lines metabolized retinoic acid constitutively, whereas others had an inducible enzyme system. A differentiation-defective line, which contains little or no cellular retinoic acid-binding protein activity, metabolized retinoic acid poorly, even after exposure to inducers. At least eight retinoic acid metabolites were generated; many contain hydroxyl residues. Our data lead us to propose that retinol does not induce differentiation of EC cells in vitro via conversion to retinoic acid. Also, the relatively rapid metabolism of retinoic acid by EC cells suggests either that the induction of differentiation need involve only a transient exposure to this retinoid or that one or more of the retinoic acid metabolites can also promote differentiation.
Administration of a physiological dose of radioactive vitamin D3 to vitamin D-deficient chicks results in a localization of the radioactivity isolated from intestinal mucosa within the nuclear chromatin fraction. Extraction and chromatography of this chromatin-bound radioactivity in several systems indicates that 87% of it exists as a polar metabolite of vitamin D3. This polar metabolite has biological activity equivalent to the parent vitamin.
The association of this metabolite with the chromatin fraction occurs only in the target intestinal mucosa and is specifically inhibited by pretreatment of the rachitic chick with nonradioactive vitamin D3 or vitamin D3 analogues such as vitamin D2 and dihydrotachysterol3. The time course of appearance of the polar metabolite in the entire intestine parallels the location of radioactivity in the chromatin fraction and is consistent with the lag in the physiological response to vitamin D.
Retinoic acid is known to have dramatic effects on vertebrate limb pattern in development and regeneration, supporting a model in which a gradient of retinoic acid serves as a morphogen to differentially supply positional information to a developing limb. The discovery of a retinoic acid receptor (RAR) and its homology to the steroid and thyroid hormone receptors provided a potential molecular mechanism for limb morphogenesis. One prediction of this model is that the receptor must be expressed in the developing and regenerating limb anlage. We investigated the expression of the RAR in the adult newt, Notophthalmus viridescens, whose amputated limbs are capable of regenerating and upon which retinoic acid can act to alter pattern. We report the cloning of cDNAs encoding a functional newt RAR and the localization of high and uniform levels of RAR mRNA specifically in the regenerating cells that control limb pattern. These results indicate that the morphogenic field is established through differential activation of pre-existing retinoic acid receptors rather than differential expression of the RAR gene.
Embryonal carcinoma cells, the stem cells of teratocarcinomas, usually undergo extensive differentiation in vivo and in vitro to a wide variety of cell types. There exist, however, several embryonal carcinoma cell lines that have almost completely lost the capacity to differentiate, so that the cells are propagated primarily as the stem cells. Using one such cell line, F9, we have found that retinoic acid at concentrations as low as 10(-9) M induces multiple phenotypic changes in the cultures in vitro. These changes include morphological alteration at the resolution of the light microscope, elevated levels of plasminogen activator production, sensitivity to cyclic AMP compounds and increased synthesis of collagen-like proteins. The nature of these changes, as well as their independence of the continued presence of retinoic acid, are consistent with the proposition that retinoic acid induces differentiation of embryonal carcinoma cells into endoderm.
Retinoic acid stimulates several murine embryonal carcinoma (EC) cell lines, even those previously considered to be incapable of differentiating, to give rise to cell types distinguishable from the parental phenotype in morphology, production of plasminogen activator and surface protein properties. Retinoic acid promotes these changes over a range of low concentrations (10−9–10−5 M) which are generally non-toxic to the cells. The effects are clearly demonstrated when EC cells are aggregated prior to exposure to retinoic acid. It is concluded that the observed phenotypic alterations induced by retinoic acid reflect differentiation of the EC cells since non-EC cell characteristics are maintained by cloned cells several generations after retinoic acid is removed from the cultures. Our studies suggest that although retinoic acid stimulates the conversion of EC cells to differentiated derivatives, it does not influence the direction of differentiation. Furthermore, the effectiveness of retinoic acid in stimulating differentiation of EC cells from lines such as Nulli-SCC1 raises the question of whether true ‘nullipotent’ EC lines really exist.
This paper describes a method of transferring fragments of DNA from agarose gels to cellulose nitrate filters. The fragments can then be hybridized to radioactive RNA and hybrids detected by radioautography or fluorography. The method is illustrated by analyses of restriction fragments complementary to ribosomal RNAs from Escherichia coli and Xenopus laevis, and from several mammals.
The molecular specificity of the receptors for steroid and thyroid hormones is achieved by their selective interaction with DNA binding sites referred to as hormone response elements (HREs). HREs can differ in primary nucleotide sequence as well as in the spacing of their dyadic half-sites. The target gene specificity of the glucocorticoid receptor can be converted to that of the estrogen receptor by changing three amino acids clustered in the first zinc finger. Remarkably, a single Gly to Glu change in this region produces a receptor that recognizes both glucocorticoid and estrogen response elements. Further replacement of five amino acids in the stem of the second zinc finger transforms the specificity to that of the thyroid hormone receptor. These findings localize structural determinants required for discrimination of HRE sequence and half-site spacing, respectively, and suggest a simple pathway for the coevolution of receptor DNA binding domains and hormone-responsive gene networks.
The specificity of target gene activation by steroid receptors is encoded within a small, cysteine-rich domain that is believed to form two zinc-coordinated fingers. Here we show that the ability of glucocorticoid and estrogen receptors to discriminate between their closely related response elements resides in the two amino acids located between the two cysteines in the C-terminal half of the first finger. Unexpectedly, chimeric glucocorticoid receptors harboring portions of the interfinger and/or second finger of the estrogen receptor have the ability to activate transcription from either a GRE- or ERE-containing promoter. We surmise that whereas the "knuckle" region of the first finger may be the primary determinant of sequence recognition, the remainder of the DNA binding domain normally confers structural information required for preventing promiscuous HRE recognition.
To further our understanding of the action of retinoids on the respecification of pattern in the regenerating axolotl limb we have studied the relative potencies of a range of synthetic and natural retinoids administered locally to the blastema. Alterations in the polar end group of the retinoic acid (RA) molecule to produce esters, the alcohol, or the aldehyde abolish the ability of the molecule to respecify pattern. On the other hand, alterations of the ring or side chain to produce the synthetic retinoids arotinoid and TTNPB considerably increases the potency of the molecule to respecify pattern--TTNPB is at least 100X more potent than retinoic acid. To examine the role of cellular retinoic acid-binding protein (CRABP) in the respecification process we determined the relative binding affinities of these retinoids for CRABP. These data correlated well with the respecification series: retinoids which showed no affinity for CRABP did not respecify pattern and those which did show affinity for CRABP did respecify pattern. Furthermore the most potent retinoid, TTNPB, has a higher affinity for CRABP than RA itself. This suggests that CRABP may be playing an important role in the action of RA on pattern formation in the regenerating limb.
In addition to having profound effects on embryonic pattern formation, retinoic acid (RA) has striking effects on differentiation and maintenance of epithelial cells in vivo and in vitro Skin is a major target organ for retinoids both in its normal and pathological states. The discovery of two human nuclear receptors for RA (hRAR alpha and hRAR beta) acting as transcriptional RA-inducible enhancer factors has provided a basis for understanding how RA controls gene expression. To investigate the specific role that RARs might play during development and in adult tissues, we have cloned the mouse RAR alpha and RAR beta (mRAR alpha and mRAR beta). Their amino-acid sequences are much more homologous to those of hRAR alpha and hRAR beta, respectively, than to each other, which suggests strongly that RAR alpha- and beta-subtypes have different functions. Most interestingly we have discovered a novel RAR subtype (mRAR gamma) whose expression in adult mouse seems to be highly restricted to skin, whereas RAR alpha and RAR beta are expressed in a variety of adult tissues. Furthermore, both mRAR alpha and mRAR gamma RNAs are readily detected in undifferentiated F9 embryocarcinoma (EC) cells, whereas mRAR beta messenger RNA is induced at least 30-fold in RA-differentiated F9 cells.
In urodele amphibians, the progenitor cells that regenerate amputated limbs (known as the blastema) normally replace only the missing structures. After systemic delivery of retinoic acid (RA), more proximal structures are also formed, indicating that RA can control position specification in the proximal-distal axis of the regenerating limb. According to dose and experimental context, retinoids can also re-specify the anteroposterior axis of the limb, induce deletions of skeletal elements, or block re-growth completely. To study the molecular basis of these morphogenetic effects, we screened complementary DNA libraries of newt regenerative tissues (limbs and tails) for hormone nuclear receptors activated by RA. Two functional retinoic acid receptors (RARs) were identified, one of which is the newt homologue of the human alpha-receptor (RAR alpha). The second receptor, called RAR delta, is novel. Sequence analysis suggests that the composite newt RAR previously reported is chimaeric, consisting of 5'RAR-beta-like and 3' RAR delta clones. We conclude that multiple RARs are expressed during limb regeneration in amphibians and suggest that receptor heterogeneity may underlie the different effects of retinoids on limb morphogenesis.
Retinoic acid has profound effects on vertebrate limb morphogenesis (refs 1-6, reviewed in refs 7-9), including in the mouse, where it can act as a teratogen generating phocomelia and bone defects. A retinoic acid gradient, possibly amplified by a graded distribution of cellular retinoic acid-binding protein (CRABP), could provide positional information across the antero-posterior axis of the chick limb bud. The discovery of nuclear retinoic acid receptors (RARs) acting as retinoic acid-inducible enhancer factors provided a basis for understanding how retinoic acid signals could be transduced at the level of gene expression. We have now used in situ hybridization to study the distribution of messenger RNA transcripts of the three murine receptors (mRARs) and CRABP during mouse limb development. Both mRAR alpha and mRAR gamma transcripts, but not those for mRAR beta, are present and uniformly distributed in the limb bud at day 10 post-coitum, whereas CRABP transcripts have a graded proximo-distal distribution, indicating that differential expression of CRABP, but not of mRAR alpha or mRAR gamma, could participate in the establishment of the morphogenetic field. At later stages, mRAR gamma transcripts become specific to the cartilage cell lineage and to the differentiating skin and mRAR beta transcripts are mostly restricted to the interdigital mesenchyme. CRABP transcripts, however, are excluded from regions expressing mRAR gamma and mRAR beta. These results indicate that all three RARs and CRABP have specific functions during morphogenesis and differentiation of the mouse limb.
Homeotic genes of Drosophila melanogaster such as Ultrabithorax (Ubx) and Antennapedia (Antp) have long been thought to select metameric identity during development by controlling the expression of various target genes. Here we describe a cotransfection assay in cultured D. melanogaster cells that is used to demonstrate that Ubx proteins (UBX) can repress an Antp promoter fusion and activate a Ubx promoter fusion, activities predicted from genetic studies. We show (a) that UBX proteins regulated the level of accurately initiated Antp P1 and Ubx transcripts, (b) that activation of the Ubx promoter required a downstream cluster of UBX binding sites, and (c) that binding site sequences were sufficient to confer regulation on a heterologous promoter, regardless of their orientation or precise position. We conclude that UBX proteins are transcriptional repressors and activators, and that their actions are mediated by binding to promoter region sequences. Each member of the UBX protein family has similar regulatory abilities, but the properties of synthetic mutant forms suggest that UBX proteins may have a modular design similar to other transcriptional regulators.
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The effects of thyroid hormones are mediated through nuclear receptor proteins that modulate the transcription of specific genes in target cells. We previously isolated cDNAs encoding two different mammalian thyroid hormone receptors, one from human placenta (hTR beta) and the other from rat brain (rTR alpha), and showed that their in vitro translation products bind thyroid hormones with the characteritistic affinities of the native thyroid hormone receptor. We now demonstrate that both of the cloned receptors activate transcription from a thyroid hormone-responsive promoter in a hormone-dependent manner, with rTR alpha eliciting a greater response than hTR beta. The putative functional domains of the thyroid hormone receptors were examined by creating chimeric thyroid hormone/glucocorticoid receptors, producing receptors with hybrid functional properties. These experiments support the proposal that the thyroid hormone receptors are composed of interchangeable functional domains, and indicate that the mechanism of hormone-inducible gene regulation has been conserved in steroid and thyroid hormone receptors.
A series of conformationally restricted analogues of (E)-4-[2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)propenyl ] benzoic acid--(E)-4-[1-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-2 - propenyl]benzoic acid, (E)-4-[3-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-2-bu ten- 2-yl]benzoic acid, trans-4-[2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl) cyclopropyl]benzoic acid, 4-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-anthracenyl)benzoic acid, 6-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-2- naphthalenecarboxylic acid, 6-(5,6,7,8-tetrahydro-3,5,5,8,8-pentamethyl-2-naphthalenyl)-2- naphthalenecarboxylic acid and 6-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-5-methyl-2- naphthalenecarboxylic acid--were synthesized and screened for retinoid biological activity. Comparison of the conformers of these analogues generated by molecular mechanics calculations with the biological activity profiles of these compounds indicates that geometric constraints required for high biological activity are imposed on the bridge joining the two aromatic ring systems by the retinoid receptor.
Analysis of complementary DNA encoding a novel gene product reveals striking similarity to the steroid and thyroid hormone receptors. Binding and transcription activational studies show it to be a receptor for the vitamin A-related morphogen retinoic acid.
A cDNA encoding a protein that binds retinoic acid with high affinity has been cloned. The protein is homologous to the receptors for steroid hormones, thyroid hormones and vitamin D3, and appears to be a retinoic acid-inducible trans-acting enhancer factor, suggesting that the molecular mechanisms of the effect of retinoids (vitamin A) on embryonic development, differentiation and tumour cell growth are similar to those described for other members of this nuclear receptor family.
We have previously described a human complementary DNA that encodes a novel protein which is homologous to members of the steroid/thyroid nuclear receptor multigene family. This novel protein (hap for hepatoma) exhibits strong homology with the human retinoic acid receptor (RAR) which has been recently characterized. To test the possibility that the hap protein might also be a retinoid receptor, a chimaeric receptor was created by replacing the putative DNA binding domain of hap with that of the human oestrogen receptor (ER). The resulting hap-ER chimaera was then tested for its ability to trans-activate an oestrogen-responsive reporter gene (vit-tk-CAT) in the presence of possible receptor ligands. Here we show that retinoic acid (RA) at physiological concentrations is effective in inducing the expression of this reporter gene by the hap-ER chimaeric receptor. This demonstrates the existence of two human retinoic acid receptors designated RAR-alpha and RAR-beta.
Processes as diverse as growth, vision and reproduction depend on the presence of vitamin A and its metabolites (retinoids), but the molecular mechanisms which govern these diverse actions remain unclear (for reviews see refs 1,2). A crucial advance recently was the isolation of a specific nuclear receptor for retinoic acid, one of the physiologically active vitamin A derivatives. This nuclear receptor is a member of the steroid/thyroid hormone receptor family. Our analysis of an uncharacterized member of this class of intracellular receptors, encoded by a complementary DNA clone from a human placental library, has led us to discover a second retinoic acid receptor. This new receptor is expressed at high levels in a number of epithelial-type tissues. The gene for the receptor was first identified in a hepatocellular carcinoma where it surrounds a site of integration of hepatitis B virus. Activation by this virus may play a role in tumour development in liver cells, where it is normally not expressed.
Studies of steroid receptors have led to the identification of a superfamily of ligand-inducible regulatory proteins that includes receptors for thyroid hormones and retinoic acid. This family of receptors regulates gene expression through binding to short cis-acting sequences referred to as hormone-response elements. Identification of a functional retinoic acid responsive element is crucial to our understanding of the mechanisms by which retinoic acid receptors activate gene expression and regulate cell differentiation. One impediment to such a study is the absence of any identified gene whose transcription is directly dependent on the receptor-hormone complex. Because the DNA-binding domains of the retinoic acid and thyroid hormone receptors are highly related (62% identical in their amino acid sequences), we have investigated the possibility that the retinoic acid receptor could activate gene expression through a thyroid hormone response element. We now report that a human retinoic acid receptor expressed from cloned complementary DNA or the endogenous retinoic acid receptor present in F9 teratocarcinoma cells can activate gene expression from promoters fused to a natural or synthetic thyroid hormone response element. The product translated in vitro from the human retinoic acid receptor cDNA can bind to a thyroid hormone response element with high affinity. The unexpected implication of these findings is that retinoic acid and thyroid hormones, acting through their respective receptors, could control overlapping gene networks involved in the regulation of vertebrate morphogenesis and homeostasis.
Receptors for retinoic acid, vitamin D3 and the steroid and thyroid hormones belong to a family of ligand-activated enhancer-binding factors which are composed of a number of functional domains required for ligand and DNA binding, nuclear translocation, dimerization and trans-activation of transcription. Ligand binding, which may promote dissociation of the receptor from a heat shock protein, results in the binding of the ligand-receptor complex as a dimer to its cognate palindromic responsive element. The mechanism by which the DNA-bound receptor activates transcription is unknown but appears to involve more than one trans-activating domain.
The cDNA sequence of human c-erb-A, the cellular counterpart of the viral oncogene v-erb-A, indicates that the protein encoded by the gene is related to the steroid hormone receptors. Binding studies with the protein show it to be a receptor for thyroid hormones.
A full-length human phenylalanine hydroxylase complementary DNA (cDNA) clone was isolated from a human liver cDNA library, and the nucleotide sequence encoding the entire enzyme was determined. The cDNA clone contains an inserted DNA fragment of 2448 base pairs, including 19 base pairs of poly(A) at the 3' end. The first methionine codon occurs at nucleotide position 223, followed by an open reading frame of 1353 base pairs, encoding 451 amino acids. Translation of the nucleotide sequence in the open reading frame predicts the amino acid sequence of human phenylalanine hydroxylase. The human protein shows a 96% amino acid sequence homology with the corresponding rat enzyme. The determination of the complete primary structure for phenylalanine hydroxylase represents the first among mixed-function oxidases.
Vitamin D3 receptors are intracellular proteins that mediate the nuclear action of the active metabolite 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]. Two receptor-specific monoclonal antibodies were used to recover the complementary DNA (cDNA) of this regulatory protein from a chicken intestinal lambda gt11 cDNA expression library. The amino acid sequences that were deduced from this cDNA revealed a highly conserved cysteine-rich region that displayed homology with a domain characteristic of other steroid receptors and with the gag-erbA oncogene product of avian erythroblastosis virus. RNA selected via hybridization with this DNA sequence directed the cell-free synthesis of immunoprecipitable vitamin D3 receptor. Northern blot analysis of polyadenylated RNA with these cDNA probes revealed two vitamin D receptor messenger RNAs (mRNAs) of 2.6 and 3.2 kilobases in receptor-containing chicken tissues and a major cross-hybridizing receptor mRNA species of 4.2 kilobases in mouse 3T6 fibroblasts. The 4.2-kilobase species was substantially increased by prior exposure of 3T6 cells to 1,25(OH)2D3. This cDNA represents perhaps the rarest mRNA cloned to date in eukaryotes, as well as the first receptor sequence described for an authentic vitamin.
Low-stringency hybridization with human glucocorticoid receptor (hGR) complementary DNA was used to isolate a new gene encoding
a predicted 107-kilodalton polypeptide. Expression studies demonstrate its ability to bind aldosterone with high affinity
and to activate gene transcription in response to aldosterone, thus establishing its identity as the human mineralocorticoid
receptor (hMR). This molecule also shows high affinity for glucocorticoids and stimulates a glucocorticoid-responsive promoter.
Together the hMR and hGR provide unexpected functional diversity in which hormone-binding properties, target gene interactions,
and patterns of tissue-specific expression may be used in a combinatorial fashion to achieve complex physiologic control.
We describe nine P-element vectors that can be used to study gene regulation and function in Drosophila. These vectors were designed for use in germline transformation and cell culture transfection assays. One set consists of five P elements that can be used to study transcriptional regulatory sequences. These vectors contain several unique restriction sites for insertion of a foreign promoter upstream from either a cat or lacZ reporter gene. Two of the beta-galactosidase-coding vectors also require the insertion of a start codon for translation of the reporter enzyme and thus can be used to study translational regulatory sequences. The second set of P elements consists of four vectors that contain the Drosophila cytoplasmic actin 5C promoter and polyadenylation signals. Upon insertion of a foreign DNA segment, these vectors direct constitutive expression of the encoded RNA and protein.
Analyses of steroid receptors are important for understanding molecular details of transcriptional control, as well as providing
insight as to how an individual transacting factor contributes to cell identity and function. These studies have led to the
identification of a superfamily of regulatory proteins that include receptors for thyroid hormone and the vertebrate morphogen
retinoic acid. Although animals employ complex and often distinct ways to control their physiology and development, the discovery
of receptor-related molecules in a wide range of species suggests that mechanisms underlying morphogenesis and homeostasis
may be more ubiquitous than previously expected.
We report that the thyroid hormone (T3) receptor binds to DNA recognition sequences that are variations of the palindromic motif 5'-TCAGGTCA.TGACCTGA-3', resulting in transcriptional activation in vivo. This sequence is identical to the vitellogenin A2 estrogen response element except for the absence of 3 bp at the center of dyad symmetry, which we have termed a "gap." The T3 receptor binds to estrogen response elements with high affinity in vitro, as well as to variations of the same sequence containing gaps of 1-6 bp. The T3 receptor not only fails to activate transcription from estrogen response elements in vivo, but inhibits estrogen-dependent transactivation. We propose that the T3 receptor binds to estrogen response elements in a transcriptionally inactive form and competes for estrogen receptor binding, resulting in a net decrease in gene expression. These data reveal that only a subset of T3 receptor DNA-binding elements function as T3 response elements.
Vitamin A and its derivatives (retinoids) exhibit profound effects on the proliferation and differentiation of many cell types. However, the molecular mechanism by which retinoids exert these effects is unknown. Cultured murine F9 teratocarcinoma stem cells, which differentiate into nontumorigenic endoderm cells in response to retinoic acid (RA), have been used to identify genes regulated by RA. A cDNA library synthesized from F9 cells treated with RA for 8 hr has been screened with a cDNA probe enriched for sequences rapidly induced by RA, and a gene that exhibits the characteristics of a primary target for RA has been identified. This gene, early retinoic acid-1 (Era-1), encodes a 2.2- to 2.4-kilobase polyadenylylated RNA; the level of Era-1 mRNA rapidly and transiently increases up to 35-fold, depending on the concentration of exogenous RA. The increase in Era-1 mRNA is dependent on the continuous presence of exogenous RA. The RA-associated increase in Era-1 mRNA is seen even in the presence of protein synthesis inhibitors, but the increase is prevented by inhibitors of RNA synthesis such as actinomycin D. This increase in the steady-state level of Era-1 mRNA in F9 cells is a very early effect of retinoic acid on gene expression in this differentiation system.
All-trans-retinoic acid (RA) induces striking digit pattern duplications when locally applied to the developing chick limb bud. Instead of the normal digit pattern (234) a mirror-symmetrical 432234 pattern can be specified. Hence, RA closely mimics posterior limb bud tissue (the zone of polarizing activity, ZPA) that causes very similar duplications when grafted to an anterior site of a host limb bud. This resemblance raises an intriguing possibility: that RA is related to the as yet unidentified inducer substance thought to be released by the ZPA. Here we report that chick limb buds contain endogenous RA and we show that RA, but not its biosynthetic precursor retinol, forms a concentration gradient across the limb anlage with a high-point in the posterior domain of the limb bud, the part that also contains the ZPA.
Using a combination of a transient expression assay and in vitro mutagenesis, we showed previously that the human glucocorticoid receptor (hGR) is composed of a series of discrete functional domains. Here we report the effects of selective deletion of each of these domains on hGR ability to activate transcription of the MTV-CAT fusion gene. Deletion of the immunogenic domain or the entire amino-terminal half of the protein reduces but does not abolish the ability of the hGR to induce transcriptional activation. Somewhat surprisingly, deletion of the steroid-binding domain engenders a constitutively active receptor, revealing that this domain normally represses receptor function. However, the central, cysteine-rich region contains all the information required for both DNA binding and trans-activation. Taken together, these data delineate a core domain in the hGR spanning 88 amino acids that determines both DNA-binding and transcriptional activation functions. This physical linkage distinguishes the glucocorticoid receptor from other described eukaryotic regulatory proteins, where these two functions have been shown to be separable.
Retinol and retinoic acid dose-response curves were obtained for promotion of the differentiation of F9 murine embryonal carcinoma cells with an enzyme-linked immunoadsorbent assay for laminin, a product of differentiated F9 cells. Retinoic acid produced a half-maximum response at 1.3 nM and a maximum response at about 30 nM; retinol was 1/175th as potent. Maximum differentiation required 48 hr of continuous exposure to retinoic acid, whereas retinol required 72 hr of exposure. The half-time of retinoic acid conversion into polar metabolites was 3.5 hr; metabolism was accelerated by pretreating F9 cells with retinoic acid. An inhibitor of oxidative metabolism, ketoconazole, decreased the rate of retinoic acid metabolism and decreased the concentration of retinoic acid required to produce a half-maximum response. Unchanged retinoic acid was the sole compound isolated from nuclei of F9 cells incubated with retinoic acid. Retinol had a half-life approximately 5-fold longer than retinoic acid, attained greater cell concentrations, and was converted into retinoic acid by F9 cells. These data indicate that retinoic acid itself directs the differentiation of F9 cells and may mediate differentiation induced by retinol.
Two distinct nonoverlapping enhancer elements can be defined within the polyoma enhancer region. In mouse fibroblasts, element A provides a 3-fold higher enhancement of the alpha 2-collagen promoter than element B. In mouse embryonal carcinoma cells, element B shows the same efficiency as in fibroblasts, whereas that of element A decreases by a factor of 3.5. Moreover, a single point mutation (PyEC F9.1) increases the efficiency of element B in both cell types, making it superior to element A in embryonal carcinoma cells. The core of element A is located within a 35 bp region tandem duplicated in several wild-type strains, mostly homologous to a crucial repeated sequence of adenovirus E1a enhancer (Hearing and Shenk, 1983). Element B contains the consensus sequence of Weiher et al. (1982). These two homologies are precisely adjacent to the two DNAase l-hypersensitive sites present in viral chromatin.
The University of Wisconsin Genetics Computer Group (UWGCG) has been organized to develop computational tools for the analysis
and publication of biological sequence data. A group of programs that will interact with each research-article has been developed
for the Digital Equipment Corporation VAX computer using the VMS operating system. The programs available and the conditions
for transfer are described.
Previous experiments in which vitamin A has been administered to developing or regenerating limbs have shown that different limb axes are affected. In regenerating axolotl limbs, serial reduplications in the proximodistal axis are produced. In the developing chick limb bud, mirror-imaged reduplications in the anteroposterior axis are produced. Results reported here on Rana temporaria limb buds reveal that vitamin A causes both effects to occur. That is, limbs are both serially reduplicated in the proximodistal axis and mirror imaged in the anteroposterior axis. Time and concentration effects are explored and the significance of these results for our current understanding of axial organisation in limbs is discussed.
The HL-60 cell line, derived from a patient with acute promyelocytic leukemia, proliferates continuously in suspension culture and consists predominantly (greater than 90%) of promyelocytes. These cells can be induced to differentiate to morphologically and functionally mature granulocytes by incubation with a wide variety of compounds, including butyrate and hypoxanthine and polar planar compounds such as dimethyl sulfoxide and hexamethylene bisacetamide. We have now found that retinoic acid (all-trans-retinoic acid) induces differentiation (as measured morphologically and by the ability to reduce nitroblue tetrazolium) of HL-60 at concentrations as low as 1 nM. Maximal differentiation (approximately 90%) occurs at 1 micro M, a concentration 1/500th to 1/160,000th the concentrations of butyrate (0.5 mM) and dimethyl sulfoxide (160 mM) that promote a similar increase in differentiation. Continuous exposure to retinoic acid is necessary for optimal differentiation, with the percentage of mature cells in the culture directly related to the length of time of exposure to retinoic acid. Retinoic acid and 13-cis-retinoic acid are equally effective in inducing differentiation of HL-60. Retinol (vitamin A), retinal, and retinyl acetate are approximately 1/1000th less potent. This study suggests that retinoids could provide a therapeutic tool in the treatment of acute myeloid leukemia, a disease that has been looked upon as primarily involving a block in myeloid differentiation, and indicates that retinoids, in addition to their well-characterized involvement in epithelial cell differentiation, may also be involved in the differentiation of certain hematopoietic cells.
We constructed a series of recombinant genomes which directed expression of the enzyme chloramphenicol acetyltransferase (CAT) in mammalian cells. The prototype recombinant in this series, pSV2-cat, consisted of the beta-lactamase gene and origin of replication from pBR322 coupled to a simian virus 40 (SV40) early transcription region into which CAT coding sequences were inserted. Readily measured levels of CAT accumulated within 48 h after the introduction of pSV2-cat DNA into African green monkey kidney CV-1 cells. Because endogenous CAT activity is not present in CV-1 or other mammalian cells, and because rapid, sensitive assays for CAT activity are available, these recombinants provided a uniquely convenient system for monitoring the expression of foreign DNAs in tissue culture cells. To demonstrate the usefulness of this system, we constructed derivatives of pSV2-cat from which part or all of the SV40 promoter region was removed. Deletion of one copy of the 72-base-pair repeat sequence in the SV40 promoter caused no significant decrease in CAT synthesis in monkey kidney CV-1 cells; however, an additional deletion of 50 base pairs from the second copy of the repeats reduced CAT synthesis to 11% of its level in the wild type. We also constructed a recombinant, pSV0-cat, in which the entire SV40 promoter region was removed and a unique HindIII site was substituted for the insertion of other promoter sequences.