Mammalian RXR - interactions during transcriptional activation

An isoform of hRXR beta, termed hRXR beta3, contains an in-frame insertion of four amino acids (SLSR) in the ligand binding domain at codon 419. The isoform is generated by alternate use of a 3' splice acceptor site and is detectable by reverse transcription polymerase chain reaction (RT-PCR) in all human tumor cell lines and mouse tissues examined. Chimeric receptors, in which the ligand-binding domain of hRXR alpha was substituted by the corresponding domain from hRXR beta3, were used to investigate the consequences of the SLSR insertion on the transactivation and DNA-binding functions of the chimeric receptor. Co-transfection assays reveal that a chimera RXR alpha/beta3 receptor fails to transactivate the RXR-specific CRBPII promoter, whereas the identical chimera lacking the SLSR insertion is active. The RXR alpha/beta3 receptor exhibits dominant negative activity against active retinoid X and retinoic acid receptors on retinoid-responsive promoters. Moreover, the RXR alpha/beta3 protein fails to interact physically with the retinoic acid receptor (RAR) to form heterodimers as detected by physical association assays, and fails to bind DNA containing an RAR-responsive element. Therefore, this suggests that the SLSR insertion in the ligand-binding domain of the RXR alpha/beta3 receptor is responsible for the altered behavior of the chimera. These findings raise the possibility that RXR alpha/beta3, and perhaps hRXR beta3 isoform, function by titrating a limiting adaptor molecule that is involved in mediating retinoid function (Mahajna, 1997).

The retinoid X receptor (RXR) participates in a wide array of hormonal signaling pathways, either as a homodimer or as a heterodimer, with other members of the steroid and thyroid hormone receptor superfamily. In this report the ligand-dependent transactivation function of RXR has been characterized, and the ability of RXR to interact with components of the basal transcription machinery has been examined. In vivo and in vitro experiments indicate the RXR ligand-binding domain makes a direct, specific, and ligand-dependent contact with a highly conserved region of the TATA-binding protein. The ability of mutations that reduce ligand-dependent transcription by RXR to disrupt the RXR-TATA-binding protein interaction in vivo and in vitro suggests that RXR makes direct contact with the basal transcription machinery to achieve activation (Schulman, 1995).

RXR interactions with co-activators

PNRC (proline-rich nuclear receptor coregulatory protein) was identified using bovine SF1 (steroidogenic factor 1) as the bait in a yeast two-hybrid screening of a human mammary gland cDNA expression library. PNRC is unique in that it has a molecular mass of 35 kDa, significantly smaller than most of the coregulatory proteins reported so far, and it is proline-rich. PNRC's nuclear localization has been demonstrated. In the yeast two-hybrid assays, PNRC interacted with the orphan receptors SF1 and ERRalpha1 in a ligand-independent manner. PNRC was also found to interact with the ligand-binding domains of all the nuclear receptors tested in a ligand-dependent manner, including estrogen receptor (ER), androgen receptor (AR), glucocorticoid receptor (GR), progesterone receptor (PR), thyroid hormone receptor (TR), retinoic acid receptor (RAR), and retinoid X receptor (RXR). Functional AF2 domain is required for nuclear receptors to bind to PNRC. Furthermore, in vitro glutathione-S-transferase pull-down assay was performed to demonstrate a direct contact between PNRC and nuclear receptors such as SF1. A coimmunoprecipitation experiment using Hela cells that express PNRC and ER was performed to confirm the interaction of PNRC and nuclear receptors in vivo in a ligand-dependent manner. PNRC functions as a coactivator to enhance the transcriptional activation mediated by SF1, ERR1 (estrogen related receptor alpha-1), PR, and TR. A 23-amino acid sequence in the carboxy-terminal region, amino acids 278-300, is critical and sufficient for the interaction with nuclear receptors. This region is proline rich and contains a SH3-binding motif, S-D-P-P-S-P-S. The two conserved proline (P) residues in this motif are crucial for PNRC to interact with the nuclear receptors. The exact 23-amino acid sequence was also found in another protein isolated from the same yeast two-hybrid screening study. These two proteins belong to a new family of nuclear receptor coregulatory proteins (Zhou, 2000).

PNRC2 (proline-rich nuclear receptor co-regulatory protein 2) was identified using mouse steroidogenic factor 1 (SF1) as bait in a yeast two-hybrid screening of a human mammary gland cDNA expression library. PNRC2 is an unusual coactivator in that it is the smallest coactivator identified so far, with a molecular weight of 16 kDa, and interacts with nuclear receptors using a proline-rich sequence. In yeast two-hybrid assays PNRC2 interacted with orphan receptors SF1 and estrogen receptor-related receptor alpha1 in a ligand-independent manner. PNRC2 was also found to interact in a ligand-dependent manner with the ligand-binding domains of estrogen receptor, glucocorticoid receptor, progesterone receptor, thyroid receptor, retinoic acid receptor and retinoid X receptor. A functional activation function 2 domain is required for nuclear receptors to interact with PNRC2. Using the yeast two-hybrid assay, the region amino acids 85-139 were found to be responsible for the interaction with nuclear receptors. This region contains an SH3 domain-binding motif (SEPPSPS) and an NR box-like sequence (LKTLL). A mutagenesis study has shown that the SH3 domain-binding motif is important for PNRC2 to interact with all the nuclear receptors tested. These results reveal that PNRC2 has a structure and function similar to PNRC, a previously characterized coactivator. These two proteins represent a new type of nuclear receptor co-regulatory proteins (Zhou, 2001).

Mammalian RXR - differential expression

Retinoic acid (RA) is known to exert profound effects on growth and differentiation in human dermal fibroblasts. The regulation of expression of members of the RXR multigene family was examined in human dermal fibroblasts. The messenger RNAs for both RXRalpha and RXRbeta are expressed in human fibroblasts, but the messenger RNA for RXRgamma is not detectable in these cells. Electrophoretic mobility shift studies of binding to the beta2RARE in human dermal fibroblasts demonstrate that a single complex binds to beta2RARE in the absence of RA. Stimulating cells with all-trans RA induces a second complex. An antibody to the RXRbeta protein supershifts both complexes, while an antibody to the RXRalpha S/B protein has no effect on the binding. These data demonstrate that (1) RXRbeta plays an important role in the retinoid-regulated signal transduction pathways in human dermal fibroblasts and (2) that the regulation of expression of the RXR gene family is different from that of the RAR gene family (Tsou, 1997).

Mammalian RXR - roles in differentiation

Embryos carrying null mutations of both retinoid X receptors alpha and beta were generated. These mutant embryos die between 9.5 and 10.5 days of gestation and display a wide range of abnormalities. The cause of the lethality appears to be the lack of formation of the labyrinthine zone of the chorioallantoic placenta. In a thorough analysis of mutant conceptuses, it was established that RXRs, through heterodimerization with retinoic acid receptors, are essential for postimplantation embryonic development before placentogenesis. RXRs are also essential for the formation of the chorioallantoic placenta, most probably through RXR/peroxisomal proliferator-activated receptor-gamma heterodimers. Interestingly, as a RXR ligand appears dispensable, placentogenesis must be controlled by a yet unknown hormonal ligand(s) activating the heterodimerization partner(s) of RXRs (Wendling, 1999).

The active derivatives of vitamin A (the retinoids) play important and multiple roles in mammalian development and homeostasis. Specific retinoic acid receptors are expressed in the chorioallantoic placenta of the mouse: among these, RXRalpha is strongly expressed in the developing labyrinthine zone. Mouse fetuses with a targeted disruption of the RXRalpha gene develop defects of the chorioallantoic placenta. Both morphological abnormalities and alterations in the expression of molecular markers are found, mostly confined to the labyrinthine zone of placentas from mid-late gestation mutants. This region exhibits edema, abnormal stasis of maternal blood, and signs of disruption of the endothelial layer of fetal vessels. A reduction in the number of lipid droplets was detected in the trophoblastic layer and abnormal fibrin deposits in the junctional zone of the mutant placentas. These abnormalities most probably result in an impairment of the functional capacities of exchange between the maternal and fetal circulations in the mutant placentas. Thus, placental defects could represent an extraembryonic cause of lethality for RXRalpha null mutant fetuses, in addition to the previously described embryonic cardiac defects (Sapin, 1997).

Even though previous studies have shown that transcripts encoding the murine retinoid X receptor gamma (RXRgamma) are present in the skeletal muscle of mouse embryos and that cultured myoblasts are induced to differentiate upon retinoid treatment, a function for RXRgamma and retinoids in mammalian myogenesis has not yet been identified. To begin to understand the possible role of RXRgamma during mammalian myogenesis novel rat RXRgamma cDNA sequences were isolated and the spatio-temporal expression pattern of RXRgamma transcripts was examined in detail in relation to skeletal muscle differentiation in rat embryos and cultured myoblasts. The onset of RXRgamma expression coincides with the differentiation of limb myoblasts in vivo. In vitro, RXRgamma is expressed in differentiating myoblasts, but not in proliferating myoblasts. In the myotome, however, RXRgamma is first expressed after myoblast differentiation, with RXRgamma transcripts being confined initially to its ventral region. Subsequently, RXRgamma becomes expressed throughout limb and myotome-derived muscle masses, and by the end of the primary myogenic wave, RXRgamma transcripts are mainly confined to muscle mass periphery. This dynamic expression pattern of RXRgamma during myogenesis suggests its possible involvement in the differentiation of limb myoblasts but excludes a role in the differentiation of early myotomal myoblasts (Georgiades, 1997).

Mouse embryos lacking the retinoic acid receptor gene RXRalpha die in midgestation from hypoplastic development of the myocardium of the ventricular chambers and consequent cardiac failure. The issue of whether the RXRalpha gene is required in the cardiomyocyte lineage has been addressed by generating mice that harbor a ventricular restricted deficiency in RXRalpha at the earliest stages of ventricular chamber specification. A conditional ('floxed') allele of RXRalpha was created by flanking a required exon of the gene with loxP recombination sequences. To achieve ventricular myocardium-specific gene targeting, and to avoid potential transgenic artifacts, a knock-in strategy was employed to place cre recombinase coding sequences into the myosin light chain 2v (MLC2v) genomic locus, a gene that, in the heart, is expressed exclusively in ventricular cardiomyocytes at the earliest stages of ventricular specification. Crossing the MLC2v-cre allele with the floxed RXRalpha gene results in embryos in which approximately 80% of the ventricular cardiomyocytes lack RXRalpha function, but which yet display a completely normal phenotype, without evidence of the wide spectrum of congenital heart disease phenotype seen in RXRa-/- embryos, and normal adult viability. It is concluded that the RXRalpha mutant phenotype is not cell autonomous for the cardiomyocyte lineage, and it is suggested that RXRalpha functions in a neighboring compartment of the developing heart to generate a signal that is required for ventricular cardiomyocyte development and chamber maturation (Chen, 1998).

Germline mutation in mice of the retinoic acid receptor gene RXR(alpha) results in a proliferative failure of cardiomyocytes, which leads to an underdeveloped ventricular chamber and midgestation lethality. Mutation of the cell cycle regulator N-myc gene also leads to an apparently identical phenotype. Chimera analysis demonstrates that the cardiomyocyte phenotype in RXRalpha-/- embryos is a non-cell-autonomous phenotype. In chimeric embryos made with embryonic stem cells lacking RXRalpha, cardiomyocytes deficient in RXRalpha develop normally and contribute to the ventricular chamber wall in a normal manner. Because the ventricular hypoplastic phenotype reemerges in highly chimeric embryos, it is concluded that RXRalpha functions in a non-myocyte lineage of the heart to induce cardiomyocyte proliferation and accumulation, in a manner that is quantitatively sensitive. It is further shown that RXRalpha is not epistatic to N-myc, and that RXRalpha and N-myc regulate convergent obligate pathways of cardiomyocyte maturation (Tran, 1998).

Vitamin A requirement for early embryonic development is clearly evident in the gross cardiovascular and central nervous system abnormalities that lead to an early death for the vitamin A-deficient quail embryo. This retinoid knockout model system was used to examine the biological activity of various natural retinoids in early cardiovascular development. All-trans-, 9-cis-, 4-oxo-, and didehydroretinoic acids, and didehydroretinol and all-trans-retinol induce and maintain normal cardiovascular development as well as induce expression of the retinoic acid receptor beta2 in the vitamin A-deficient quail embryo. The expression of RARbeta2 is at the same level and at the same sites where it is expressed in the normal embryo. Until the 5-somite stage of development, but not later, retinoids provided to the vitamin A-deficient embryo completely rescue embryonic development, suggesting the 5-somite stage as a critical retinoid-sensitive time point during early avian embryogenesis. Retinoid receptors RARalpha, RARgamma, and RXRalpha are expressed in both the precardiac endoderm and mesoderm in the normal and the vitamin A-deficient quail embryo, while the expression of RXRgamma is restricted to precardiac endoderm. Vitamin A deficiency downregulates the expression of RARalpha and RARbeta. These studies provide strong evidence for a narrow, retinoid-requiring, developmental window during early embryogenesis, in which the presence of bioactive retinoids and their receptors are essential for a subsequent normal embryonic development (Kostetskii, 1998).

Retinoids regulate gene expression via nuclear retinoic acid receptors, the RARs and RXRs. To investigate the functions of retinoid receptors during early neural development, a dominant negative RARbeta was expressed in early Xenopus embryos. Dominant negative RARbeta specifically inhibits RAR/RXR heterodimer-mediated, but not RXR homodimer-mediated, transactivation. Both all-trans- and 9-cis-RA-induced teratogenesis are, however, efficiently opposed by ectopic expression of dominant negative RARbeta, indicating that only RAR/RXR transactivation is required for retinoid teratogenesis by each of these ligands. Experiments with two RXR-selective ligands confirms that activation of RXR homodimers does not cause retinoid teratogenesis. Dominant negative RARbeta thus specifically interferes with the retinoid signalling pathway that is responsible for retinoid teratogenesis. Dominant negative RARbeta-expressing embryos have a specific developmental phenotype leading to disorganization of the hindbrain. Mauthner cell multiplications in the posterior hindbrain, and (both anteriorly and posteriorly) expanded Krox-20 expression domains indicate (partial) transformation of a large part of the hindbrain into (at least partial) rhombomere 3, 4 and/or 5 identity. In contrast, the fore- and mid-brain and spinal cord appeared to be less affected. These data indicate that RARs play a role in patterning the hindbrain (van der Wees, 1998).

Classes of both retinoic acid receptors (RARs) and the retinoic X receptors (RXRs) have (respectively) three different subtypes (RARalpha, RARbeta, and RARgamma, and RXRalpha, RXRbeta, and RXRgamma) that act as ligand-dependent transcription factors. To examine the involvement of the different receptor classes and their subtypes in the biological responses of neuroblastoma cells to retinoids, the effects of a panel of receptor-selective retinoids were analyzed for cell growth, differentiation, and gene expression, using in vitro cultured KCNR cells. Activation of three distinct RXR/RAR heterodimers induces growth arrest and differentiation of neuroblastoma cells. Any association of per se inactive RXR-selective with RAR-selective ligands efficiently regulates growth inhibition, differentiation (neurite extension), and expression of RARbeta, TrkB, and N-myc. SR11383 alone, a very potent retinoid, entirely reproduces the pattern of biological responses induced by naturally occurring retinoids. In contrast to other tumor cell lines, the growth of neuroblastoma cell lines is not altered using AP1-antagonistic retinoids. These studies raise the possibility that three distinct RXR/RAR heterodimers mediate the effects of retinoids on neuroblastoma cells through an AP-1 antagonism-independent mechanism (Giannini, 1997).

Retinaldehyde dehydrogenase type 2 (RALDH-2) was identified as a major retinoic acid generating enzyme in the early mouse embryo. The expression domains of the RALDH-2 are likely to indicate regions of endogenous retinoic acid (RA) synthesis. During early gastrulation, RALDH-2 is expressed in the mesoderm adjacent to the node and primitive streak. This suggests that RALDH-2 gene expression may be switched on during the process of cell ingression through the primitive streak. Expression extends rostrally along each side of the node, whereas the node itself is unlabelled. At the headfold stage, mesodermal expression is restricted to posterior regions up to the base of the headfolds. No expression is detected in mesoderm underneath the rhomboencephalon. Later, RALDH-2 is transiently expressed in the undifferentiated somites and the optic vesicles, and more persistently along the lateral walls of the intraembryonic coelom and around the hindgut diverticulum. The RALDH-2 expression domains in differentiating limbs, which include presumptive interdigital regions, coincide with, but slightly precede, those of the RA-inducible RAR beta gene. The RALDH-2 gene is also expressed in specific regions of the developing head, including the tooth buds, inner ear, meninges and pituitary gland, and in several viscera. Administration of a teratogenic dose of RA at embryonic day 8.5 results in downregulation of RALDH-2 transcript levels in caudal regions of the embryo, and may reflect a mechanism of negative feedback regulation of RA synthesis (Niederreither, 1997).

NB4, a human acute promyelocytic leukemia cell line expressing the promyelocyte-retinoic acid receptor alpha (PML-RAR alpha) hybrid protein was treated with RAR- and retinoid X receptor (RXR)-selective analogs to determine their effects on cell proliferation, retinoblastoma (RB) tumor-suppressor protein phosphorylation, and differentiation. An RAR- or just RAR alpha-selective analog alone induces similar cell population growth arrest, cell cycle arrest without restriction to G1, hypophosphorylation of RB, and myelomonocytic cell surface differentiation marker expression (CD11b). An RAR alpha antagonist can inhibit the effects of the RAR alpha agonist completely. The RAR alpha-selective analog-elicited response is attenuated by simultaneous addition of various RXR-selective analogs. In contrast, each of the RXR-selective analogs is unable to induce any of the cellular responses analyzed. The growth arrest of NB4 cells is not G1-restricted and occurs at all points in the cell cycle. Cells growth arrested by treatment with an RAR alpha-selective analog shows primarily hypophosphorylated RB. When these cells are sorted into G1 or S + G2/M subpopulations by flow cytometry, hypophosphorylated RB protein is found in G1 as well as S + G2/M cells. This suggests that the hypophosphorylated RB protein may be mediating the growth arrest of NB4 cells at all points in the cell cycle. These results are consistent with an involvement of PML-RAR alpha and/or RAR alpha in the transduction of the retinoid signal in NB4 cells (Brooks, 1997).

Mammalian RXR - Roles in development

The cytokine erythropoietin (Epo) promotes erythropoietic progenitor cell proliferation and is required for erythropoietic differentiation. The primary physiological regulator of Epo expression in late embryos and in postnatal stages is oxygen tension. A mostly unknown hypoxia sensing mechanism results in the activity of the transcription factor HIF1 (hypoxia-inducible factor 1), which binds to a defined sequence in the 3' enhancer of the Epo gene and initiates Epo expression. In the fetal liver, Epo is expressed primarily by hepatocytes, a property which is conserved in hepatocellular carcinoma cell lines such as Hep3B and HepG2, in which Epo expression is induced in response to hypoxia. Adjacent to the HIF1-binding site in the mouse Epo 3' enhancer is the sequence TGACCTCTTGACCC, which is known as a DR2 element because of the direct repeat of the hexameric sequence TGACC(C/T) spaced by two nucleotides. The Epo enhancer DR2 element substantially augments hypoxic induction of Epo gene reporter constructs in transfected Hep3B cells, but is not itself responsible for responding to hypoxia. HNF4 is currently believed to be the primary factor that is responsible for Epo gene regulation through the DR2 element. HNF4 is expressed in the fetal liver and postnatal kidney, the two major sites of Epo expression, and introduction of an HNF4 expression construct in transfected HeLa cells (which do not normally express HNF4) confers hypoxic inducibility to an Epo reporter gene. HNF4 appears to function synergistically with HIF1 on the Epo enhancer by direct protein-protein interaction and through the recruitment of transcriptional coactivators (Makita, 2001 and references therein).

The Epo gene is a direct transcriptional target gene of retinoic acid signaling during early erythropoiesis (prior to embryonic day E12.5) in the fetal liver. Mouse embryos lacking the retinoic acid receptor gene RXRalpha have a morphological and histological phenotype that is comparable with embryos in which the Epo gene itself has been mutated, and flow cytometric analysis indicates that RXRalpha-deficient embryos are deficient in erythroid differentiation. Epo mRNA levels are reduced substantially in the fetal livers of RXRalpha^-/- embryos at E10.25 and E11.25, and genetic analysis shows that the RXRalpha and Epo genes are coupled in the same pathway. The Epo gene is shown to be retinoic acid inducible in embryos, and the Epo gene enhancer contains a DR2 sequence that represents a retinoic acid receptor-binding site and a retinoic acid receptor transcriptional response element. However, unlike Epo-deficient embryos that die from anemia, the erythropoietic deficiency in RXRalpha^-/- embryos is transient; Epo mRNA is expressed at normal levels by E12.5, and erythropoiesis and liver morphology are normal by E14.5. HNF4, like RXRalpha a member of the nuclear receptor family, is abundantly expressed in fetal liver hepatocytes, and is competitive with retinoic acid receptors for occupancy of the Epo gene enhancer DR2 element. It is proposed that Epo expression is regulated during the E9.5-E11.5 phase of fetal liver erythropoiesis by RXRalpha and retinoic acid, and that expression then becomes dominated by HNF4 activity from E11.5 onward. This transition may be responsible for switching regulation of Epo expression from retinoic acid control to hypoxic control, as is found throughout the remainder of life (Makita, 2001).

Fusion and hypoplasia of the first two branchial arches, a defect typically observed in retinoic acid (RA) embryopathy, is generated in cultured mouse embryos upon treatment with BMS453, a synthetic compound that exhibits retinoic acid receptor ß (RARß) agonistic properties in transfected cells. By contrast, no branchial arch defects are observed following treatment with synthetic retinoids that exhibit RARalpha or RARgamma agonistic properties. The BMS453-induced branchial arch defects are mediated through RAR activation, since they are similar to those generated by a selective pan-RAR agonist -- they are prevented by a selective pan-RAR antagonist and cannot be mimicked by exposure to a pan-RXR agonist alone. They are enhanced in the presence of a pan-RXR agonist, and cannot be generated in Rarß-null embryos. Furthermore, they are accompanied, in the morphologically altered region, by ectopic expression of Rarß and of several other direct RA target genes. Therefore, craniofacial abnormalities characteristic of the RA embryopathy are mediated through ectopic activation of RARß/RXR heterodimers, in which the ligand-dependent activity of RXR is subordinated to that of RARß. Endodermal cells lining the first two branchial arches respond to treatment with the RARß agonist, in contrast to neural crest cells and ectoderm, which suggests that a faulty endodermal regionalization is directly responsible for RA-induced branchial arch dysmorphologies. Additionally, the first in vivo evidence is provided that the synthetic RARß agonist BMS453 exhibits an antagonistic activity on the two other RAR isotypes (Matt, 2003).

Mammalian RXR - Role in long-term potentiation

Hippocampal long-term potentiation (LTP) and long-term depression (LTD) are the most widely studied forms of synaptic plasticity thought to underlie spatial learning and memory. RARbeta deficiency in mice virtually eliminates hippocampal CA1 LTP and LTD. It also results in substantial performance deficits in spatial learning and memory tasks. Surprisingly, RXRgamma null mice exhibit a distinct phenotype in which LTD is lost whereas LTP is normal. Thus, while retinoid receptors contribute to both LTP and LTD, they do so in different ways. These findings not only genetically uncouple LTP and LTD but also reveal a novel and unexpected role for vitamin A in higher cognitive functions (Chiang, 1998).

Chromatin remodeling mediated by liganded and unliganded nuclear receptors