Retinoid-regulated gene expression in neural development

The discovery and development of information surrounding the retinoic acid receptors (RAR and RXR) has ushered in a new era in understanding the molecular mechanism of action of vitamin A in embryonic development and cellular differentiation. The mechanisms involved in the regulation of gene expression by the retinoids is at least partially known and involves binding of the RAR and RXR to retinoic acid response elements. Additional factors, including coregulatory proteins, associated regulatory elements, and cell-specific factors, may also be involved in determining the specificity of retinoid-regulation of gene expression during development. During embryogenesis, retinoids are required for the development of the posterior hindbrain and its associated structures, as well as for the survival and differentiation of certain classes of neurons and neural crest cell derivatives. At least some of the effects of retinoid on hindbrain development are related to the regulation of Hox gene expression. Additional retinoid-regulated genes have been implicated in nervous system development, and the manner in which they lead to phenotypic changes during embryogenesis remains to be determined.     

Pattern of retinoid-induced teratogenic effects: possible relationship with relative selectivity for nuclear retinoid receptors RAR alpha, RAR beta, and RAR gamma

Retinoic acid, an oxidative metabolite of vitamin A, is involved in the control of many biological processes including embryonic development. Excess as well as deficiency of retinoids were found to be teratogenic. The effects of retinoids in normal as well as abnormal development may be mediated by two members of retinoid receptors, the RAR's and RXR's, which exhibit a specific temporal and spatial expression during development. The significance of the retinoid receptors was investigated here by studying the teratogenic effects of retinoid ligands with relative selectivity for binding and transactivation of the retinoic acid receptors RAR alpha, RAR beta and RAR gamma. Pregnant NMRI mice were administered 5 or 15 mg/kg of CD 336 (Am 580) (alpha-ligand), CD 2019 (beta-ligand), CD 437 (gamma-ligand) or 37.5 mg/kg all-trans-retinoic acid in 25% Cremophor EL on day 8.25 or day 11 of gestation by gastric intubation. External, visceral and skeletal malformations were observed on day 18 of gestation. The order of teratogenic potency was: alpha-ligand > beta-ligand > gamma-ligand. In addition, these retinoids also produced a different spectrum of defects. The alpha-ligand induced the most varied defects including severe ear, mandible, and limb malformations. The beta-ligand induced defects of the urinary system and liver in greater frequency than expected from its relative potency. The gamma-ligand preferentially induced ossification deficiencies and defects of the sternebrae and vertebral body. Our results show that these three retinoids, which were previously demonstrated to exhibit retinoid-like activities in several systems, exert differing teratogenic activities, in regard to both potency and regioselectivity: we hypothesize that the relative selectivity for binding and transactivation of the three retinoic acid receptors could possibly be related to the differences of teratogenic effects observed in this study. The low potency of the gamma-ligand may lead the way to interesting new retinoids with improved therapeutic ratio.     

Effects of amide and amine plasma-treated ePTFE vascular grafts on endothelial cell lining in an artificial circulatory system

Vitamin A requirement for early embryonic development is clearly evident in the gross cardiovascular and central nervous system abnormalities and an early death of 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. We demonstrate that 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. Retinoids provided to the vitamin A-deficient embryo up to the 5-somite stage of development, but not later, 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. Our studies provide strong evidence for a narrow retinoid-requiring developmental window during early embryogenesis, in which the presence of bioactive retinoids and their receptors is essential for a subsequent normal embryonic development.     

1,25-dihydroxyvitamin D3 and 9-cis-retinoic acid act synergistically to inhibit the growth of LNCaP prostate cells and cause accumulation of cells in G1

Recent studies have suggested that the active metabolite of vitamin D3, 1,25-dihydroxyvitamin D3, can inhibit the growth and/or induce the differentiation of a variety of cell types and that these characteristics might be useful in the treatment of some cancers. Retinoids also promote the differentiation and inhibit the growth of some cells. That the vitamin D receptor acts as a heterodimer with the retinoid X receptor (RXR) suggests that there may be functional interactions between 1,25-dihydroxyvitamin D3 and retinoids. In this study, we show that the combination of 1,25-dihydroxyvitamin D3 and 9-cis retinoic acid synergistically inhibits the growth of LNCaP prostate cancer cells. That this effect is mediated by RXR rather than retinoic acid receptors was shown using RXR- and retinoic acid receptor-specific ligands. The vitamin D3 analog, EB1089, inhibited growth more effectively than 1,25-dihydroxyvitamin D3 and also acted synergistically with 9-cis-retinoic acid. These treatments caused cells to accumulate in the G1 phase of the cell cycle, suggesting that 1,25-dihydroxyvitamin D3 can regulate one or more factors critical for the G1/S transition.     

Metabolism to a response pathway selective retinoid ligand during axial pattern formation

We report identification of 9-cis-4-oxo-retinoic acid (9-cis-4-oxo-RA) as an in vivo retinoid metabolite in Xenopus embryos. 9-Cis-4-oxo-RA bound receptors (RARs) alpha, beta, and gamma as well as retinoid X receptors (RXRs) alpha, beta, and gamma in vitro. However, this retinoid displayed differential RXR activation depending on the response pathway used. Although it failed to activate RXRs in RXR homodimers, it activated RXRs and RARs synergistically in RAR-RXR heterodimers. 9-Cis-4-oxo-RA thus acted as a dimer-specific agonist. Considering that RAR-RXR heterodimers are major functional units involved in transducing retinoid signals during embryogenesis and that 9-cis-4-oxo-RA displayed high potency for modulating axial pattern formation in Xenopus, metabolism to 9-cis-4-oxo-RA may provide a mechanism to target retinoid action to this and other RAR-RXR heterodimer-mediated processes.     

Interaction between retinoic acid and vitamin D signaling pathways

The nuclear signaling pathways for retinoids and vitamin D differ in the specificity of the respective receptors for response elements. Two pathways for the action of both retinoic acid receptors (RARs) and vitamin D receptors (VDRs) have been identified, one being retinoid X receptor (RXR)-dependent and the other being RXR-independent. Moreover, RXRs were found to function as homodimers. In several steps we converted the retinoid specific response element of the human retinoic acid receptor beta promoter into the vitamin D/retinoic acid response element of the human osteocalcin promoter. We found that VDR homodimers only bind to the motif RGGTGA. The extended osteocalcin element also contains an imperfect direct repeat based on the motif RGGTGA spaced by three nucleotides, which is bound by RXR homodimers and activated by 9-cis-retinoic acid. The responsiveness of the osteocalcin element to all-trans-retinoic acid is mediated neither by RAR homodimers nor by RAR-RXR heterodimers. However, a VDR-RAR heterodimer binds to the osteocalcin response element and mediates activation by all-trans-retinoic acid. This heterodimer also binds to pure retinoid response elements, but it does not mediate activation by vitamin D alone. In combination with all-trans-retinoic acid, however, vitamin D enhances VDR-RAR heterodimer-mediated gene expression. This finding suggests a direct interaction between nuclear signaling by retinoic acid and vitamin D increasing the combinatorial possibilities for gene regulation by the nuclear receptors involved.     

Domains of retinoid signalling and neurectodermal expression of zebrafish otx1 and goosecoid are mutually exclusive

Retinoid signalling plays an important role in embryonic pattern formation. Excess of retinoic acid during gastrulation results in axial defects in vertebrate embryos, suggesting that retinoids are involved in early anteroposterior patterning. To study retinoid signalling in zebrafish embryos, we developed a novel method to detect endogenous retinoids in situ in embryos, using a fusion protein of the ligand inducible transactivation domain of a retinoic acid receptor and a heterologous DNA binding domain. Using this method, we show that retinoid signalling is localized in zebrafish embryos in the region of the embryonic shield, and towards the end of gastrulation in a posterior dorsal domain. To investigate the relationships between the spatial distribution of retinoid signalling and the regulation of retinoid target genes, we studied the downregulation by retinoic acid of two genes expressed in anterior regions of the embryo, goosecoid and otx1. These experiments show that expression of both genes is strongly downregulated in the anterior neurectoderm of zebrafish embryos treated with retinoic acid, whereas mesendodermal expression is only mildly affected. Interestingly, a significant downregulation of goosecoid expression by retinoic acid was observed only during midgastrulation but not in earlier stages. In agreement with these results, spatial expression of goosecoid and otx1 does not overlap with the region of retinoid signalling in the late gastrula. Our data support the hypothesis that a localized retinoid signal is involved in axial patterning during early development, at least in part through the repression of anterior genes in posterior regions of the embryo. Furthermore, our data suggest that the action of retinoids is spatially as well as temporally regulated in the developing embryo.     

Contribution of retinoic acid receptor gamma to retinoid-induced craniofacial and axial defects

Exogenous retinoic acid (RA) administered during mouse embryogenesis can alter the pattern of the axial skeleton during two developmental periods: an early window (7 to 8.5 days post-coitum; dpc) and a late window (9.5 to 11.5 dpc). Treatment during the early window results in vertebral homeotic transformations (predominantly posteriorizations) concomitant with rostral shifts in Hox gene expression, while treatment at the later window results in similar transformations without detectable alterations in Hox gene expression patterns. Mice null for retinoic acid receptor gamma (RAR gamma) exhibit axial defects, including homeosis of several vertebrae, therefore establishing a role for this receptor in normal axial specification RAR gamma null mutants are also completely resistant to RA-induced spina bifida, which occurs in wildtype embryos treated at 8.5-9.0 dpc, suggesting that this receptor specifically transduces at least a subset of the teratogenic effects of retinoids. To further investigate the role of RAR gamma in RA-induced defects during the early and late windows of retinoid-sensitive vertebral patterning, RAR gamma heterozygotes were intercrossed, pregnant females treated with vehicle or RA at 7.3, 10.5 or 11.5 dpc and full-term fetuses assessed for skeletal defects. Relative to wildtype littermates, RAR gamma null mutants treated at 7.3 dpc were markedly resistant to RA-induced embryolethality, craniofacial malformations, and neural tube defects. Furthermore, while RAR gamma null mutants were modestly resistant to certain vertebral malformations elicited by RA treatment at 7.3, they exhibited more pronounced resistance following treatment at 10.5 and 11.5 dpc. Moreover, several of the vertebral defects inherent to the RAR gamma null phenotype were abolished by RA treatment specifically at 10.5 dpc, suggesting that RAR alpha and/or RAR beta isoforms may substitute for certain RAR gamma functions, and that RAR gamma may elicit its normal effects on vertebral morphogenesis at this developmental stage.     

Ligand induction of retinoic acid receptors alters an acute infection by murine cytomegalovirus

Here we report that administration of retinoids can alter the outcome of an acute murine cytomegalovirus (MCMV) infection. We show that a crucial viral control element, the major immediate-early enhancer, can be activated by retinoic acid (RA) via multiple RA-responsive elements (DR2) that bind retinoid X receptor-retinoic acid receptor (RAR) heterodimers with apparent dissociation constants ranging from 15 to 33 nM. Viral growth is dramatically increased upon RA treatment of infected tissue culture cells. Using synthetic retinoid receptor-specific agonists and antagonists, we provide evidence that RAR activation in cells is required for mediating the response of MCMV to RA. Oral administration of RA to infected immunocompetent mice selectively exacerbates an infection by MCMV, while cotreatment with an RAR antagonist protects against the adverse effects of RA on MCMV infection. In conclusion, these chemical genetic experiments provide evidence that an RAR-mediated pathway can modulate in vitro and in vivo infections by MCMV.     

Expression of retinoic acid receptor-beta sensitizes prostate cancer cells to growth inhibition mediated by combinations of retinoids and a 19-nor hexafluoride vitamin D3 analog

Retinoids and analogs of vitamin D3 may achieve greater in vivo applications if the toxic side effects encountered at pharmacologically active doses could be alleviated. These seco-steroid hormones often act in concert, and therefore, we attempted to dissect these interactions by isolating combinations of receptor-selective retinoids and a potent vitamin D3 analog [1alpha,25(OH)2-16ene-23-yne-26,27,F6-19nor-D3, code name LH] that were potent inhibitors of prostate cancer cell growth at low, physiologically safer doses. Using a panel of prostate cancer cell lines representing progressively more transformed phenotypes, we found that the LNCaP cell line (least transformed) was either additively or synergistically inhibited in its clonal growth by LH and various naturally occurring and receptor-selective retinoids, the most potent combination being with a retinoic acid receptor (RAR)betagamma-selective retinoid (SR11262). The effect was not found with either PC-3 (intermediate transformation) or DU-145 (most transformed). We also undertook RT-PCR to examine the subtypes of RARs present, and we found that PC-3 and DU-145 did not express RARbeta. Stable expression of RARbeta into the RARbeta-negative PC-3 cells resulted in increased sensitivity to SR11262 and LH proportional to the amount of RARbeta expressed. This study indicates that RARbeta may play an important role in synergistically controlling cell proliferation, and expression is lost with increased prostate cancer cell transformation. Simultaneous administration of a potent vitamin D3 analog and receptor-selective retinoids may have therapeutic potential for the treatment of androgen-dependent and -independent prostate cancer.     

A selective retinoid with high activity against an androgen-resistant prostate cancer cell type

Retinoic acid (RA) and its natural and synthetic analogs, the retinoids, regulate many biological processes, including development, differentiation, cell growth, morphogenesis, metabolism and homeostasis. Retinoid effects are mediated by specific nuclear receptors, the RARs and RXRs. Because of their ability to control cell growth and induce differentiation, retinoids are being examined for the prevention and treatment of several cancers. The majority of retinoids so far analyzed and available inhibit primarily cell proliferation and tumor progression but cannot eliminate cancer cells. In addition, the beneficial effects of the natural retinoids are undermined by undesirable side effects, possibly due to indiscriminate activation of all retinoid receptor subtypes and response pathways. Here, we show that a synthetic retinoid, CD-271, that activates selectively the RAR gamma subtype in a given context, shows increased anti-proliferative activity against certain carcinoma cells over all-trans-retinoic acid (tRA). CD-271 exhibits enhanced activity against DU-145 prostate adenocarcinoma cells through apoptosis-inducing activity, while tRA does not. The selective anti-cancer cell action appears to be receptor-mediated as an RAR antagonist reverses the inhibition. This profile was not seen with other selective retinoids, such as RAR alpha-selective agonists, anti-AP-1 compounds and a non-apoptosis inducing RAR gamma agonist. Our data point to a specific role for RAR gamma in controlling the growth of the prostate, consistent with previous RAR gamma gene knockout data. The identified retinoid represents a new class of compounds with potential for the treatment of prostate cancer.     

Involvement of alcohol dehydrogenase, short-chain dehydrogenase/reductase, aldehyde dehydrogenase, and cytochrome P450 in the control of retinoid signaling by activation of retinoic acid synthesis

The effects of vitamin A (retinol) on growth and development are mediated by the active metabolite retinoic acid which controls a nuclear receptor signaling pathway. While elegant work on the retinoic acid receptor family has focused attention upon how the receptor controls this pathway, there now exists a relatively large gap in our understanding of how retinol is activated to form the ligand. During vertebrate embryogenesis and in adult organs retinoic acid is detected in a distinct spatiotemporal pattern, suggesting that it is produced from retinol in a regulated fashion. Enzymes involved in retinol and retinal metabolism are likely candidates for regulators of tissue retinoic acid levels. Members of the alcohol dehydrogenase and short-chain dehydrogenase/reductase enzyme families catalyze the reversible interconversion of retinol and retinal, the rate-limiting step, whereas members of the aldehyde dehydrogenase and cytochrome P450 enzyme families catalyze the irreversible oxidation of retinal to retinoic acid. The identification of enzymes likely to catalyze retinol oxidation in vivo has been particularly controversial, and this is made even more difficult by the reversible nature of this reaction. Taking into account enzymatic properties and coenzyme preferences, a case can be made that class IV alcohol dehydrogenase catalyzes retinol oxidation to provide retinal for retinoic acid synthesis, whereas microsomal retinol dehydrogenase (a short-chain dehydrogenase/reductase) catalyzes the reduction of retinal to retinol to promote retinoid storage. Further studies on these enzyme families will allow this layer of control in the retinoid signaling pathway to be understood.     

Ion-pair high-performance liquid chromatography for determining disaccharide composition in heparin and heparan sulphate

Rats deficient in vitamin A express low levels of P4502C7 mRNA in the liver. Administration of all-trans retinoic acid (at-RA) or growth hormone (GH) to deficient animals only partially restored the expression whereas the combined treatment returned the P4502C7 mRNA levels to that observed in normal rats. That a retinoid is the predominant inducer of P4502C7 at the cellular level is evident from studies performed with primary hepatocytes, but it became clear that GH is a prerequisite for the vitamin A effect in vivo. The at-RA induction of P4502C7 mRNA in primary rat hepatocytes was inhibited by ketoconazole, an inhibitor of P450 activity, and by cycloheximide, blocking ongoing protein synthesis. In contrast, the at-RA induction of RAR-beta2 mRNA was not affected by any of these compounds. This could indicate previously not recognized mechanisms of at-RA action. Interestingly, at-4-oxo-RA, an at-RA metabolite formed by a P450 catalyzed reaction, also induced P4502C7 mRNA. Induction of P4502C7 mRNA by the retinoic acid receptor (RAR) selective agonist TTNPB indicated that this pathway is preferred over the retinoid X receptor (RXR) pathway. In addition, analysis of RA metabolites in liver cell extracts revealed the formation of several as yet unidentified metabolites. The formation of some of these metabolites was inhibited by ketoconazole and they could therefore constitute potential inducers of CYP2C7. We suggest that metabolism of at-RA, possibly by a P450 enzyme, is an important step in the at-RA induction of P4502C7.     

The histostructure of the adrenal gland in the Campbell hamster after extirpation of a specific midventral gland]

The receptors for retinoic acid (RA) and for 1 alpha,25-dihydroxyvitamin D3 (VD), RAR, RXR, and VDR are ligand-inducible members of the nuclear receptor superfamily. These receptors mediate their regulatory effects by binding as dimeric complexes to response elements located in regulatory regions of hormone target genes. Sequence scanning of the tumor necrosis factor-alpha type 1 receptor (TNF alpha RI) gene identified a 3' enhancer region composed of two directly repeated hexameric core motifs spaced by 2 nucleotides (DR2). On this novel DR2-type sequence, but not on a DR5-type RA response element, VD was shown to act through its receptor, the vitamin D receptor (VDR), as a repressor of retinoid signalling. The repression appears to be mediated by competitive protein-protein interactions between VDR, RAR, RXR, and possibly their cofactors. This VDR-mediated transrepression of retinoid signaling suggests a novel mechanism for the complex regulatory interaction between retinoids and VD.     

Retinoic acid receptor/retinoid X receptor heterodimers can be activated through both subunits providing a basis for synergistic transactivation and cellular differentiation

The receptor for 9-cis-retinoic acid, retinoid X receptor (RXR), forms heterodimers with several nuclear receptors, including the receptor for all-trans-retinoic acid, RAR. Previous studies have shown that retinoic acid receptor can be activated in RAR/RXR heterodimers, whereas RXR is believed to be a silent co-factor. In this report we show that efficient growth arrest and differentiation of the human monocytic cell line U-937 require activation of both RAR and RXR. Also, we demonstrate that the allosteric inhibition of RXR is not obligatory and that RXR can be activated in the RAR/RXR heterodimer in the presence of RAR ligands. Remarkably, RXR inhibition by RAR can also be relieved by an RAR antagonist. Moreover, the dose response of RXR agonists differ between RXR homodimers and RAR/RXR heterodimers, indicating that these complexes are pharmacologically distinct. Finally, the AF2 activation domain of both subunits contribute to activation even if only one of the receptors is associated with ligand. Our data emphasize the importance of signaling through both subunits of a heterodimer in the physiological response to retinoids and show that the activity of RXR is dependent on both the identity and the ligand binding state of its partner.