Retinoic Acid Receptor Family Research Articles

Abstract P6-12-09: Retinoic acid receptor (RAR) signalling plays a role in neratinib (NER) resistance in HER2+ breast cancer (BC) cell lines

Abstract Introduction: NER is a pan-HER tyrosine kinase inhibitor (TKI) approved for the treatment of HER2+ BC in the adjuvant setting following trastuzumab and in combination with capecitabine for advanced disease. Resistance to small molecule TKIs like NER can develop in the clinic. Pre-clinical studies have highlighted that retinoic acid can inhibit BC growth and modulate HER2 signalling pathways. The RAR family of nuclear transcription factors consists of RARα, RARβ and RARγ. The synthetic retinoic acid fenretinide (FN) acts as a pan-RAR agonist, while AGN194310 (AG) acts as a pan-RAR antagonist. In order to investigate the anti-proliferative potential of co-targeting RAR and HER2 pathways in sensitive and resistant BC cell line models, we examined the effect of FN and AG in combination with NER in two HER2+, estrogen receptor-negative, trastuzumab-resistant cell lines HCC1569 and HCC1954, and their NER-resistant (NR) sub-lines HCC1569-NR and HCC1954-NR. Methods: HCC1569 and HCC1954 cell lines were cultured in RPMI/10% FCS at 370C/5% CO2. NR cell lines were generated by continuous exposure to 150nM NER for 6 months. 10 mM stock solutions of FN (H7779-Sigma), AG (SML2665-Sigma) and NER (supplied by Puma Biotechnology, Inc) were made in DMSO. Proliferation was measured as percentage growth versus DMSO control using an acid phosphatase based assay after 5 days drug exposure. The half-maximal inhibitory concentration (IC50) was calculated for each drug using CalcuSyn. The combination assays were performed using fixed ratios. The combination index (CI) values were calculated at the effective dose that inhibits 50% growth (ED50) using CalcuSyn. Values < 1 represent a synergistic effect, a value of 1 is additive and values > 1 represent an antagonistic effect. All data presented as the mean of biological triplicate experiments ± standard deviation. Results: This research found that the NR cell lines were >10-fold resistant to NER (HCC1569-NR IC50 0.44 ± 0.1 μM, HCC1954-NR IC50 0.198 ± 0.019 μM) compared to the parental HCC1569 (IC50 0.018 ± 0.015 μM) and HCC1954 (IC50 0.017 ± 0.001 μM) cell lines. Pan-RAR agonism by FN had a potent anti-proliferative effect in the HCC1569 (FN IC50 0.22 ± 0.02 μM) and the HCC1569-NR cell lines (FN IC50 0.28 ± 0.13 μM), with the HCC1954 and HCC1954-NR cell lines proving less sensitive (IC50 6.47 ± 1.3 μM and 1.9 ± 0.2 μM, respectively). When combined with NER, FN produced a strong antagonistic effect in the HCC1569 cell line (CI value: 15.63 ± 9.5) and a strong synergistic effect in the HCC1954 cell line (CI value: 0.42 ± 0.06). For the NR cell line models, the NER/FN combination proved synergistic (HCC1569-NR, CI value: 0.84 ± 0.46) or additive (HCC1954-NR, CI value: 0.97 ± 0.15). Next, we wanted to assess the impact of antagonising rather than activating RAR activity in our cell line models. All four cell lines were less sensitive to antagonist AG (IC50 >8μM for all cell lines) compared to FN. The addition of AG to NER resulted in responses diametrically opposed to the FN/NER combination. The AG/NER combination produced a strong synergistic effect in the HCC1569 cell line (CI value: 0.52 ± 0.17), an antagonistic effect in the HCC1954 cell line (CI value: 2.1 ± 0.4) and an antagonistic effect in both NR cell lines (HCC1954-NR, CI value: 2.69 ± 0.6 and HCC1569-NR, CI value: 1.58 ± 0.12). Conclusions: This pre-clinical study suggests involvement of the RAR signalling pathway in response to NER and the development of NR. Results also suggest pan-RAR agonism, rather than pan-RAR antagonism, as a potential therapeutic strategy to overcome NR. Further investigation is warranted to determine how targeting the RAR signalling pathway may assist in the treatment of HER2+ BC. Citation Format: Debbie O’Reilly, Lisa D. Eli, Alvin Wong, John Crown, Denis M. Collins. Retinoic acid receptor (RAR) signalling plays a role in neratinib (NER) resistance in HER2+ breast cancer (BC) cell lines [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P6-12-09.

The Pleiotropic Role of Retinoic Acid/Retinoic Acid Receptors Signaling: From Vitamin A Metabolism to Gene Rearrangements in Acute Promyelocytic Leukemia.

The family of retinoic acid receptors (RARs: RARα, -β, and -γ) has remarkable pleiotropy characteristics, since the retinoic acid/RARs pathway is involved in numerous biological processes not only during embryonic development, but also in the postnatal phase and during adulthood. In this review, we trace the roles of RA/RARs signaling in the immune system (where this pathway has both an immunosuppressive role or is involved in the inflammatory response), in hematopoiesis (enhancing hematopoietic stem cell self-renewal, progenitor cells differentiation or maintaining the bone marrow microenvironment homeostasis), and in bone remodeling (where this pathway seems to have controversial effects on bone formation or osteoclast activation). Moreover, in this review is shown the involvement of RAR genes in multiple chromosomal rearrangements generating different fusion genes in hematological neoplasms, with a particular focus on acute promyelocytic leukemia and its variant subtypes. The effect of different RARs fusion proteins on leukemic transformation, on patients’ outcome, and on therapy response is also discussed.

Activation of PPARδ: from computer modelling to biological effects.

PPARδ is a ligand-activated receptor that dimerizes with another nuclear receptor of the retinoic acid receptor family. The dimers interact with other co-activator proteins and form active complexes that bind to PPAR response elements and promote transcription of genes involved in lipid metabolism. It appears that various natural fatty acids and their metabolites serve as endogenous activators of PPARδ; however, there is no consensus in the literature on the nature of the prime activators of the receptor. In vitro and cell-based assays of PPARδ activation by fatty acids and their derivatives often produce conflicting results. The search for synthetic and selective PPARδ agonists, which may be pharmacologically useful, is intense. Current rational modelling used to obtain such compounds relies mostly on crystal structures of synthetic PPARδ ligands with the recombinant ligand binding domain (LBD) of the receptor. Here, we introduce an original computational prediction model for ligand binding to PPARδ LBD. The model was built based on EC50 data of 16 ligands with available crystal structures and validated by calculating binding probabilities of 82 different natural and synthetic compounds from the literature. These compounds were independently tested in cell-free and cell-based assays for their capacity to bind or activate PPARδ, leading to prediction accuracy of between 70% and 93% (depending on ligand type). This new computational tool could therefore be used in the search for natural and synthetic agonists of the receptor.

Melatonin in sperm biology: breaking paradigms.

Melatonin is a ubiquitous molecule, present in a wide range of organisms, and involved in multiple functions. Melatonin relays the information about the photoperiod to the tissues that express melatonin-binding sites in both central and peripheral nervous systems. This hormone has a complex mechanism of action. It can cross the cell plasma membrane and exert its actions in all cells of the body. Certain melatonin actions are mediated by receptors that belong to the superfamily of G-protein-coupled receptors (GPCRs), the MT1 and MT2 membrane. Melatonin can also bind to calmodulin as well as to nuclear receptors of the retinoic acid receptor family, RORα1, RORα2 and RZRβ. The purpose of this review is to report on recent developments in the physiological role of melatonin and its receptors. Specific issues concerning the biological function of melatonin in mammalian seasonal reproduction and spermatozoa are considered. The significance of the continuous presence of melatonin in seminal plasma with a fairly constant concentration is also discussed.

A highly conserved retinoic acid responsive element controlswt1aexpression in the zebrafish pronephros

The Wilms' tumor suppressor gene Wt1 encodes a zinc-finger transcription factor that plays an essential role in organ development, most notably of the kidney. Despite its importance for organogenesis, knowledge of the regulation of Wt1 expression is scarce. Here, we have used transgenesis in zebrafish harboring two wt1 genes, wt1a and wt1b, in order to define regulatory elements that drive wt1 expression in the kidney. Stable transgenic lines with approximately 30 kb of the upstream genomic regions of wt1a or wt1b almost exactly recapitulated endogenous expression of the wt1 paralogs. In the case of wt1b, we have identified an enhancer that is located in the far upstream region that is necessary and sufficient for reporter gene expression in the pronephric glomeruli. Regarding wt1a, we could also identify an enhancer that is located approximately 4 kb upstream of the transcriptional start site that is required for expression in the intermediate mesoderm. Interestingly, this intermediate mesoderm enhancer is highly conserved between fish and mammals, is bound by members of the retinoic acid receptor family of transcription factors in gel shift experiments and mediates responsiveness to retinoic acid both in vivo and in cell culture. To our knowledge, this is the first functional demonstration of defined regulatory elements controlling Wt1 expression in vivo. The identification of kidney-specific enhancer elements will help us to better understand the integration of extracellular signals into intracellular networks in nephrogenesis.

The role of retinoids and their receptors in metabolic disorders

AbstractRetinoid derivatives are potent regulators of a wide variety of cellular functions through the modulation of nuclear receptor activity. Three distinct families of retinoid receptors have been identified so far, namely retinoic acid receptors, retinoic acid X receptors, and retinoic acid‐related orphan receptors. This review focuses on retinoid signaling and the regulation of metabolic processes by the family of retinoic acid receptors in relation to the development of metabolic disorders.

The search for non‐chordate retinoic acid signaling: lessons from chordates

Signaling by retinoic acid (RA) is an important pathway in the development and homeostasis of vertebrate and invertebrate chordates, with a critical role in mesoderm patterning. Classical studies on the distribution of nuclear receptors of animals suggested that the family of RA receptors (RARs/NR1B) was restricted to chordates, while the family of RA X receptors (RXR/NR2B) was distributed from cnidarians to chordates. However, the accumulation of data from genome projects and studies in non-model species is questioning this traditional view. Here we discuss the evidence for non-chordate RA signaling systems in the light of recent advances in our understanding of carotene (pro-Vitamin A) metabolism and of the identification of potential RARs and members of the NR1 family in echinoderms and lophotrochozoan trematodes, respectively. We conclude, as have others before (Bertrand et al., 2004. Mol Biol Evol 21(10):1923-1937), that signaling by RA is more likely an ancestral feature of bilaterians than a chordate innovation.

Feedback Regulation of β,β-Carotene 15,15′-Monooxygenase by Retinoic Acid in Rats and Chickens

beta,beta-Carotene 15,15'-monooxygenase (formerly termed beta,beta-carotene 15,15'-dioxygenase, EC 1.13.11.21) catalyzes the conversion of provitamin A carotenoids to retinal in vertebrate tissues. In the present study, we investigated whether preformed vitamin A or beta-carotene and its direct metabolites can regulate the enzyme activity in vivo. We found dose-dependent decreases in intestinal beta,beta-carotene monooxygenase activity after oral administration to rats of retinyl acetate (up to -79%), beta-carotene (up to -79%), apo-8'-carotenal (up to -56%), all-trans retinoic acid (up to -88%), and 9-cis retinoic acid (up to -67%). Liver beta,beta-carotene 15,15'-monooxygenase (betaCMOOX) activity was not affected. Apo-12'carotenal and the retinoic acid receptor (RAR) alpha antagonist Ro 41-5253 significantly increased the intestinal enzyme activity by 55 and 94%, respectively. When beta-carotene was administered to rats pretreated with the two cytochrome P(450) (CYP) inducers, pentobarbital and naphthoflavone, the intestinal betaCMOOX activity increased by 39%. In a transcriptional study in chickens, treatment with retinoic acid resulted in low expression of the intestinal betaCMOOX. Our data suggest that retinoids and carotenoids might regulate betaCMOOX expression by a transcriptional feedback mechanism via interaction with members of the RAR family.

Cloning of cDNAs encoding retinoic acid receptors RARγ1, RARγ2, and a new splicing variant, RARγ3, from Ambystoma mexicanum and characterization of their expression during early development

To analyze retinoic acid (RA) receptor (RAR) expression during early development in the urodele embryo, we have isolated cDNAs for four members of the axolotl ( Ambystoma mexicanum) RAR family, namely RARα (NR1B1), aRARγ1 (NR1B3a), aRARγ2 (NR1B3b), and a new splicing variant of aRARγ2, aRARγ3 (NR1B3c), which contains an insertion of five hydrophobic amino acids in the C-terminal region of the DNA binding domain. The temporal expression pattern of the RARγ isoforms was established by RT-PCR using total RNA from embryos of different stages. The expression of aRARγ2 coincides with neurulation and is enhanced in the extremities of the embryo’s anteroposterior axis. The aRARγ3 is specifically expressed during gastrulation and early neurulation, whereas aRARγ1 is expressed later during organogenesis. Global aRARγ2 mRNA levels, as well as their spatio-temporal expression pattern in the neurula, were not affected by treatment with RA. These results show that several RARs are expressed in the axolotl embryo during early development, and reveal the existence of a new RARγ variant.

All-trans retinoic acid blocks the antiproliferative prodifferentiating actions of 1,25-dihydroxyvitamin D3 in normal human keratinocytes.

1,25-Dihydroxyvitamin D3 [1,25(OH)2D3] and all-trans retinoic acid (RA), the active metabolites of vitamins D and A respectively, regulate the proliferation and differentiation of keratinocytes. Both the vitamin D receptor (VDR) and the retinoic acid receptor family (RAR) bind to DNA response elements as heterodimers with the retinoic X receptor (RXR), suggesting that there are pathways of action that are shared by both compounds. Therefore, we examined the interactions of 1,25(OH)2D3 and RA upon the proliferation and differentiation of normal human keratinocytes (NHK) and of a squamous cell carcinoma cell line, SCC4. Although both 1,25(OH)2D3 and RA were each able to inhibit NHK proliferation in a dose-dependent manner, when they were administered in combination, proliferation was stimulated, suggesting mutual antagonism. In contrast, SCC4 cells proved insensitive in terms of proliferation to 1,25(OH)2D3 and to all but the highest concentration (10(-6) M) of RA. 1,25(OH)2D3 exerted a biphasic effect on transglutaminase (TGase) and involucrin (INV) mRNA levels, with maximal stimulation at 10(-9) M. RA inhibited TGase and INV mRNA levels and antagonized the stimulation by 1,25(OH)2D3. A similar pattern was observed for TGase protein, but, RA, which, by itself, reduced INV, markedly enhanced the ability of 1,25(OH)2D3 to raise INV levels, possibly by inhibiting 1,25(OH)2D3-stimulated TGase activity and cross-linking of soluble INV into the insoluble cornified envelope (CE). Thus, in NHK cells, RA antagonizes the antiproliferative prodifferentiating actions of 1,25(OH)2D3, but assessment of a single marker, such as INV protein, may be misleading.

All‐trans retinoic acid blocks the antiproliferative prodifferentiating actions of 1,25‐Dihydroxyvitamin D3 in normal human keratinocytes

1,25-Dihydroxyvitamin D3 [1,25(OH)2D3] and all-trans retinoic acid (RA), the active metabolites of vitamins D and A respectively, regulate the proliferation and differentiation of keratinocytes. Both the vitamin D receptor (VDR) and the retinoic acid receptor family (RAR) bind to DNA response elements as heterodimers with the retinoic X receptor (RXR), suggesting that there are pathways of action that are shared by both compounds. Therefore, we examined the interactions of 1,25(OH)2D3 and RA upon the proliferation and differentiation of normal human keratinocytes (NHK) and of a squamous cell carcinoma cell line, SCC4. Although both 1,25(OH)2D3 and RA were each able to inhibit NHK proliferation in a dose-dependent manner, when they were administered in combination, proliferation was stimulated, suggesting mutual antagonism. In contrast, SCC4 cells proved insensitive in terms of proliferation to 1,25(OH)2D3 and to all but the highest concentration (10−6 M) of RA. 1,25(OH)2D3 exerted a biphasic effect on transglutaminase (TGase) and involucrin (INV) mRNA levels, with maximal stimulation at 10−9 M. RA inhibited TGase and INV mRNA levels and antagonized the stimulation by 1,25(OH)2D3. A similar pattern was observed for TGase protein, but, RA, which, by itself, reduced INV, markedly enhanced the ability of 1,25(OH)2D3 to raise INV levels, possibly by inhibiting 1,25(OH)2D3-stimulated TGase activity and cross-linking of soluble INV into the insoluble cornified envelope (CE). Thus, in NHK cells, RA antagonizes the antiproliferative prodifferentiating actions of 1,25(OH)2D3, but assessment of a single marker, such as INV protein, may be misleading. J. Cell. Physiol. 174:1–8, 1998. © 1998 Wiley-Liss, Inc.

Retro-retinoids in regulated cell growth and death.

Vitamin A serves as a prohormone from which three classes of active metabolites are derived: the aldehydes, the carboxylic acids, and the retro-retinoids. Although these three classes are united under the rubric of signal transduction, they act by different molecular mechanisms: the 11-cis-retinaldehydes combine with opsin to form the universal visual pigments and the retinoic acids form ligands for transcription factors, whereas the retro-retinoids, as shown here, intersect with signal transduction at a cytoplasmic or membrane site. The retro-retinoid, anhydroretinol (AR), has long been known to act as a growth inhibitor in lymphocytes, whereas 14-hydroxy-4,14-retro-retinol (14-HRR) is required for normal lymphocyte proliferation. A mutually reversible relationship exists between these two retro-retinoids as one can reverse the effects of the other when given in pharmacological doses. The common explanation for reversible inhibition is competition for a shared receptor. We now provide evidence that when AR is given to T cells unmitigated by 14-HRR, rapid cell death can occur. The circumstances are closely related to nonclassical forms of apoptosis: within 2 h of AR administration the T cells undergo widespread morphological changes, notably surface blebbing and ballooning and, inevitably, bursting. In contrast, nuclear changes are comparatively mild, as indicated by absence of chromatin condensation and overt DNA cleavage to discrete nucleosomal fragments, although DNA nicks are readily discernible by terminal deoxynucleotidyl transferase assay. What further distinguishes the AR-induced form of apoptosis from classical ones is a lack of requirements of messenger RNA and protein synthesis, suggesting that the events leading to cell death are primarily initiated and play themselves out in the cytoplasm. This view is further reinforced by the finding that herbimycin A can prevent the onset of programmed cell death. The importance of our findings is that they strongly suggest a second messenger role for vitamin A metabolites in the cytoplasmic realm that has not been seen previously. These findings are entirely compatible with a general notion that in a cell requiring multiple coordinated signals for survival, the provision of an unbalanced signal can initiate programmed cell death. Collectively, our data also challenge the paradigm that retinoids (outside vision) solely mediate their function via the steroid/ retinoic acid receptor family of nuclear transcription factors. Instead, a mode of action in the cytoplasmic realm akin to one attributed to other small lipophilic second messenger molecules, such as diacyl glycerol or ceramide, may apply to retro-retinoids.

Cell-Type-Specific Expression of the Platelet-Derived Growth Factor α Receptor: a Role for GATA-Binding Protein

Platelet-derived growth factor alpha receptor (PDGF alpha R) is a transmembrane tyrosine kinase receptor for all three existing PDGF isoforms, AA, AB, and BB. Transcripts of PDGF alpha R are detected as early as in fertilized mouse eggs and throughout adulthood in a time- and space-specific manner, thereby suggesting an important role of PDGFs in mammalian development. In this study, we have investigated the mechanism involved in cell-type-specific PDGF alpha R gene expression during early embryonic development. Using F9 embryonic carcinoma cells as an in vitro study model, we identified a differentiation-dependent enhancer element within the PDGF alpha R promoter that controlled receptor expression during parietal endoderm cell differentiation induced by retinoic acid and dibutyryl cyclic AMP treatment. The differentiation-dependent enhancer element sequence bore no resemblance to consensus DNA-binding sites of either the retinoic acid receptor family or the cyclic AMP-responsive element-binding protein family. It was composed of two identical 12-bp direct repeats separated by a 17-bp insert sequence enriched in C and A nucleotides. Although only a single repeat was needed to form specific DNA-protein complexes with factors present in F9 parietal endoderm cell extracts, both repeats together were necessary to display cell-type-specific enhancing activity. Mutational analysis revealed that the protein-binding sites within the repeat sequences were identical to GATA-binding sites. In this study, we provided evidence to suggest that a member of the GATA transcription factor family (GATA-4) is responsible for parietal endoderm-specific PDGF alpha R expression.

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.

Evidence that class IV alcohol dehydrogenase may function in embryonic retinoic acid synthesis.

The signaling effects of the vitamin A derivative retinoic acid are mediated by the retinoic acid receptor family (Kastner et al., 1994; Mangelsdorf et al., 1994). Recent studies on mice carrying retinoic acid receptor mutations have demonstrated that retinoic acid is essential for development of several embryonic tissues including the craniofacial region, eye, heart, and other organs (Lohnes et al., 1994; Mendelsohn et al., 1994). However, little is known about the enzymatic mechanism involved in the correct spatial and temporal regulation of retinoic acid synthesis from retinol (vitamin A) during embryonic development.

Postnatal rat lung retinoic acid receptor (RAR) mRNA expression and effects of dexamethasone on RAR β mRNA

Retinoids exert multiple effects upon lung differentiation and growth. Although the mechanisms involved are presently poorly understood, increasing evidence points to a central role of nuclear retinoic acid receptors (RAR). The purpose of this study was to determine RAR mRNA expression profile during postnatal alveolarization, compared with the expression in prenatal and adult rat lung, and to describe the effects of dexamethasone (DEX) and oxygen on postnatal lung RAR gene expression. Total RNA was isolated from lungs of Sprague-Dawley rats on prenatal day 19, on postnatal days 1, 3, 7, 10, and 14 of life, and from adults. One subgroup of littermate pups was treated with DEX daily for 3 or 7 days. In a second experiment, rats were exposed to room air or to 95% oxygen for 72 hours, and received either DEX or saline. Northern hybridization showed that the levels of all RAR subtypes in fetal lung were 45% or less of levels at postnatal day 1. The 3.7 kb RAR alpha transcript levels were lower than day 1 on days 10 and 14 (relative to day 1, day 10 = 0.54 +/- 0.05; day 14 = 0.54 +/- 0.08), but there was no change in a 2.7 kb RAR alpha transcript over this time period. By contrast, RAR beta mRNA levels were significantly higher at days 3, 10, and 14 compared with day 1 (day 3 = 1.79 +/- 0.19; day 10 = 1.41 +/- 0.14; day 14 = 1.53 +/- 0.05). Similarly, RAR gamma mRNA expression levels were higher on day 10 (1.45 +/- 0.09), but by day 14 there was no difference from day 1. Adult lung 3.7 kb RAR alpha, 2.7 kb RAR alpha, and RAR gamma were lower than day 1, but RAR beta was significantly greater (3.7 alpha = 0.52 +/- 0.05; 2.7 alpha = 0.49 +/- 0.26; gamma = 0.74 +/- 0.06; beta = 1.63 +/- 0.22). Treatment with DEX prevented the rise in RAR beta mRNA occurring on day 3 and significantly lowered (0.65 +/- 0.06) the amount of RAR beta mRNA in day 7 lung. Exposure of rat pups to oxygen caused an increase in RAR beta mRNA (1.21 +/- 0.03). DEX treatment again decreased RAR beta mRNA in both control (0.55 +/- 0.06) and oxygen-exposed pups (0.67 +/- 0.12). In addition, 2.7 kb RAR alpha mRNA was decreased with the combination of DEX and oxygen exposure (0.63 +/- 0.06). The differential gene expression profiles and the response to DEX and oxygen of the various members of the RAR family suggest that each subtype may have a specific role during the period of alveolarization in rat lung.

Overview of Retinoid Metabolism

Substantial and exciting progress has been made in understanding the cytoplasmic metabolism and the nuclear functions of vitamin A. Keys to this understanding have come from studies of the plasma and cytoplasmic proteins that bind retinol and retinoic acid, thereby regulating retinoid concentration and specifying the enzymatic reactions through which retinol is absorbed, stored as esterified retinol or oxidized to retinoic acid. In the nucleus, the hormone-like activity of retinoic acid is transduced by a family of retinoic acid receptors that regulate a large number of retinoic acid-responsive genes. The coordination of cytoplasmic retinoid metabolism and the nuclear effects of retinoic acid or other retinoid metabolites is now recognized as critical to embryonic development and the control of cellular differentiation throughout life.

Multiple members of the retinoic acid receptor family are capable of mediating the granulocytic differentiation of HL-60 cells.

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.

Multiple members of the retinoic acid receptor family are capable of mediating the granulocytic differentiation of HL-60 cells.

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.

The molecular biology of retinoic acid receptors: orphan from good family seeks home.

A variety of c-DNAs coding for nuclear retinoic acid receptors (RARs) have recently been cloned. These receptors are members of the steroid/thyroid receptor superfamily and are believed to act as ligand-inducible transactivating factors; retinoic acid induces changes in receptor configuration that allows DNA binding and increased gene transcription from specific genes to occur. The retinoic acid receptor family itself may consist of up to 20 separate receptors each with a specific distribution and ligand binding characteristics. The RAR-gamma in the adult is found almost exclusively in the skin but other receptors which are found in a variety of other tissues are also present in skin. Associations of cutaneous disease states with receptor mutants have not yet been reported although some cases of leukaemia may be secondary to retinoic acid receptor gene rearrangements. A variety of approaches to identify the biological function of these receptors based on recombinant DNA technology are already underway.