RARβ Gene Research Articles

Retinoic Acid Differentially Regulates Retinoic Acid Receptor-Mediated Pathways in the HEP3B Cell Line

Retinoic acid (RA) up-regulates retinoic acid receptor β (RARβ) gene expression in a variety of cell lines. Whether up-regulation of the RARβ gene reflects increased activity in a RARβ-mediated biological process is unclear since RARβ tends to heterodimerize with retinoid x receptor (RXR). In F9 teratocarcinoma cell line, RA-induced differentiation is accompanied by increased expression of the RARβ, RXRα, and α-fetoprotein (AFP) genes. Previously, we have shown that the RA-mediated regulation of the AFP gene is through RXRα homodimers. In contrast to F9 cells, Hep3B is unique in that the AFP gene is down-regulated by RA in a manner reminiscent of down-regulation of AFP in postfetal liver. In this paper, we have examined the RA-mediated regulation of the RAR, RXR, peroxisome proliferator-activated receptor (PPAR), and AFP genes in Hep3B cells. RA induced the expression of RARα, β, and γ mRNA in Hep3B cells. However, the expression of RXRα mRNA was down-regulated, and the levels of RXRβ and RXRγ mRNA remained unchanged after RA treatment. In addition, the expression of the PPARα, β, and γ genes was also unchanged. Gel retardation assays demonstrated that RA decreased the overall binding of nuclear receptors to the RA and PPAR response elements. By super-shift assays using specific anti-RAR and -RXR antibodies, RA treatment decreased the amount of RXRα while increasing the amount RARβ bound to retinoic acid response element-DR1 (direct repeat with spacer of one nucleotide), indicating the levels of RAR/RXR heterodimer, RXR/RXR homodimer, or RAR/RAR homodimers were altered upon RA treatment of Hep3B cells. In addition, the RA-mediated reduction of RXRα in part results in down-regulation of the AFP gene. Our data indicates that RA exerts its effects by differentially regulating its own receptor gene expression.

Retinoid-induced differentiation of neuroblastoma: Comparison between LG69, an RXR-selective analogue and 9-cis retinoic acid

The aim of this study was to investigate in vitro the effects of all-trans retinoic acid (RA), 9-cis RA and the RXR-selective analogue, LG69, on the morphological differentiation, proliferation and gene expression of neuroblastoma cells. Three different cell lines were cultured with the retinoid for either 9 continuous days or for 5 days followed by 4 days without the retinoid and morphological differentiation was assessed both qualitatively and quantitatively. SH SY 5Y cell proliferation was examined by measuring cell numbers after exposure to the retinoids and RAR-β gene expression was examined by Northern blot analysis. Morphological differentiation was more effectively induced by all-trans and 9-cis RA than by LG69. SH SY 5Y cells, when treated with 9-cis RA for only 5 of the 9 days of culture, underwent apoptosis, but this was not seen with 9 days continuous exposure nor with LG69. Inhibition of SH SY 5Y cell proliferation by all-trans or 9-cis RA was dose-dependent, but LG69 had little effect. Conversely, LG69 induced higher expression of RAR-β than all-trans RA, but less than that produced by 9-cis RA. These data suggest that 9-cis RA as a single agent is the most effective modulator of neuroblastoma behaviour and may be the most appropriate therapeutic agent.

Specific Activation of Retinoic Acid Receptors (RARs) and Retinoid X Receptors Reveals a Unique Role for RARγ in Induction of Differentiation and Apoptosis of S91 Melanoma Cells

Retinoic acid (RA) and 9-cis-RA induce growth arrest and differentiation of S91 melanoma cells. RA activates retinoic acid receptors (RARs), whereas 9-cis-RA activates both RARs and retinoid X receptors (RXRs). Both classes of receptors function as ligand-dependent transcription factors. S91 melanoma cells contain mRNA for RXRalpha, RXRbeta, RARalpha, RARgamma, and RARbeta in low levels. Among these, only RARbeta gene transcription is induced by retinoids. However, at present the individual role(s) for each RXR and RAR isoform in these processes is unclear. We assessed the function of all isoforms in the S91 melanoma model by using RXR and RAR isoform-specific retinoids to study their effects on cell growth, RARbeta expression, and differentiation. Activation of each of the endogenous RXR or RAR isoforms induces RARbeta gene expression, and blocks cellular proliferation. However, only the RARgamma-ligands cause additional differentiation toward a melanocytic phenotype, which coincides with substantial apoptosis well before morphological changes are apparent. Apoptosis is completely dependent on de novo protein synthesis but cannot be induced by changes in activities of AP-1, protein kinase C, and protein kinase A, nor can it be blocked by the presence of the antioxidant glutathione. These results argue against a specific role for RARbeta, but suggest that RARgamma has a critical role in a genetic switch between melanocytes and melanoma, and induction of ligand-dependent apoptosis.

Restricted expression and retinoic acid-induced downregulation of the retinaldehyde dehydrogenase type 2 (RALDH-2) gene during mouse development

Retinaldehyde dehydrogenase type 2 (RALDH-2) was identified as a major retinoic acid generating enzyme in the early embryo. Here we report the expression domains of the RALDH-2 gene during mouse embryogenesis, which 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. At the headfold stage, mesodermal expression is restricted to posterior regions up to the base of the headfolds. 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 RARβ 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.

Genomic Footprinting of Retinoic Acid Regulated Promoters in Embryonal Carcinoma Cells

Retinoic acid (RA) treatment of embryonal carcinoma (EC) cells initiates a cascade of alterations in gene regulation, leading to their differentiation into various cell types. In P19 EC cells RA treatment stimulates induction of the RARβ gene, while it represses Oct3/4 gene expression. Here we present dimethylsulfate-based genomic footprinting analyses of these two genes. We found that the RARβ promoter is not occupied prior to RA treatment, but following RA treatment all regulatory elements in this promoter become occupied. On the other hand, the Oct 3/4 promoter is occupied at all three known elements before RA treatment, but this occupancy is coordinately lost following the treatment. Thus, factor occupancy coincides with expression of the genes. It is likely that the presence of factor binding or its absence revealed here represents a mechanism of the regulated expression of these genesin vivo.Our results demonstrate the power of genomic footprinting for studying regulatory events for transcriptionin vivo.In contrast, within vitroprotein–DNA binding assay, factors for both promoters are present in these cells regardless of RA treatment. It has been shown that RA receptor (RAR) and retinoid X receptor (RXR), by heterodimerization, mediate the RA action in EC cells. To elucidate the role of RAR/RXR heterodimers in the RARβ promoter occupancyin vivo,genomic footprinting has been performed in P19 cells stably expressing dominant negative mutants of RXR. Two such mutants, lacking either the DNA binding domain or the C-terminal activation domain, inhibit RA induction of the RARβ gene in these cells. RA-induced factor occupancy is also markedly inhibited at all elements in the RARβ promoter in these cells. Our results show that binding of liganded RAR/RXR heterodimers to RARE is required for other factors to gain access to their respective elements in the promoter.

Modulation of the Retinoic Acid and Retinoid X Receptor Signaling Pathways in P19 Embryonal Carcinoma Cells by Calreticulin

Calreticulin is a widely expressed calcium binding protein that can bind to an amino acid sequence motif, KXGFFKR, which is present in the cytoplasmic domain of all integrin α-subunits. Closely related sequences, KXFFKR and KXFFRR, are encoded in the DNA-binding domain of all members of the steroid/thyroid/retinoid receptor superfamily and it has recently been demonstrated that calreticulin inhibits their activity bothin vitroandin vivo.Here we present novel evidence that calreticulin can interfere directly with the retinoic acid (RARs) and retinoid X (RXRs) receptor pathways. Calreticulin exhibits the ability to inhibit DNA-binding activity of both heterodimeric RAR/RXR and homodimeric RXR complexesin vitro.Inhibition of RXR binding to DNA is achieved with a concentration of calreticulin that is approximately fourfold lower than that required for inhibition of RAR/RXR binding to a cognate binding site. Coprecipitation experiments suggest a direct protein:protein interaction between calreticulin and retinoid receptors. Stable overexpression of calreticulin in P19 embryonal carcinoma cells significantly decreases the rapid activation of the endogenous RA-responsive RARβ gene, abrogates the ability of endogenous RAR/RXR complexes to bind to DNA, and inhibits the emergence of the RA-induced differentiated phenotype. These data demonstrate that calreticulin can interfere with the two distinct retinoid signaling pathways through a mechanism likely involving direct protein:protein interactions and that disruption of the retinoid signal alters biological processesin vivo.

THE EFFECT OF RETINOIC ACID AND DEXAMETHASONE ON THE mRNA EXPRESSION OF THE RETINOIC ACID RECEPTOR-β IN THE RAT. † 1833

Retinol is essential for normal epithelial cell differentiation and maintenance of epithelial cell integrity. Retinoic acid (RA), the active metabolite of retinol, influences gene expression via binding to nuclear retinoic acid receptors (RAR). In vitamin A-deficient adult rats, lung RAR-β gene expression is induced by retinoic acid (Proc Natl Acad Sci 1991; 88:8272). Recently, RAR-β gene expression has been shown to be decreased by dexamethasone (DEX) in neonatal rats (Pediatr Pulmonol 1995; 20:234). We hypothesized that RA and DEX interact to influence RAR-β gene expression. To test this hypothesis, adult, female, vitamin A-sufficient rats were treated with RA 100 μg. DEX 2 μg/g body weight, both RA and DEX, or cottonseed oil or saline as controls. The animals were sacrificed 6 hours after treatment; lung and liver tissue were then obtained for northern blot RNA analysis using 32P labeled cDNA probes specific for RAR-β (two isoforms, β1 = 3.3 kb andβ2 = 3.0 kb) and G3PDH. Densitometry was performed and results are expressed in arbitrary densitometry units. The results (mean ±SD) are shown in the table.

Vitamin A is involved in estrogen-induced cell proliferation but not in cytodifferentiation of the chicken oviduct

We examined vitamin A-deficient chicks to determine whether vitamin A affects the estrogen-induced development of the chick oviduct. When oviduct development was stimulated for 5 days with the synthetic estrogen, diethylstilbestrol, the wet weight of the oviduct in vitamin A-deficient chicks was only half that in control chicks. The DNA content in this tissue showed that the decreased oviduct weight in the vitamin A-deficient chicks was caused by the decreased proliferation of oviduct cells. However, the estrogen-induced expression of the ovalbumin gene was not affected by the vitamin A deficiency, suggesting that estrogen-induced cytodifferentiation is not affected by vitamin A. To clarify the vitamin A action on estrogen-induced development in the oviduct, transcripts of nuclear estrogen receptor (ER) and all-trans-retinoic acid (RAR alpha, beta and gamma) receptors, which exert the effects of estrogen and vitamin A, were measured. The ER, RAR alpha and RAR beta genes, but not that of RAR gamma, were expressed during oviduct development, indicating that estrogen and vitamin A may control the expression of target genes through their cognate receptors. Thus, we have shown that vitamin A is involved in estrogen-induced cell proliferation but not in cytodifferentiation of the chicken oviduct.

Retinoid receptors in human lung cancer and breast cancer

Retinoids, the natural and synthetic vitamin A derivatives, are known to inhibit the proliferation of lung cancer and breast cancer cells and the growth of carcinogen-induced bronchogenic squamous cell carcinoma and mammary tumors, and have been used as chemoprevention agents against both types of cancer. However, clinical trials of retinoids in patients with advanced lung cancer and breast cancer have not been successful. In studying how retinoid sensitivity is lost in cancer cells, we have found that lack of the retinoic acid receptor β (RARβ) gene expression and its abnormal regulation by retinoic acid (RA) are common features in human lung cancer and breast cancer cells. The absence and abnormal RA regulation of RARβ correlates with the loss of anti-proliferation effect of RA in hormone-independent breast cancer cells, and is due to different abnormalities found in cancer cells. Furthermore, expression of RARβ gene in hormone-independent breast cancer cells restores their RA sensitivity. These data demonstrate that RARβ can mediate the growth inhibitory effect of RA and suggest that the lack of RARβ may contribute to retinoid resistance in certain cancer cells.

Mice lacking all isoforms of retinoic acid receptor β develop normally and are susceptible to the teratogenic effects of retinoic acid

Retinoic acids (RA) are vitamin A derivatives essential for normal embryonic development and viability of vertebrates. The RA signal is mediated by two distinct classes of receptors, RA receptors (RARs) and retinoid X receptors (RXRs). The RAR family is composed of three genes: RARα, β, and γ. The expression of RARβ gene is spatially and temporally restricted in certain structures in the developing embryo, suggesting that RARβ could play specific roles during morphogenesis. Four isoforms of the RARβ gene (β1–β4) are generated by differential usage of promoters and alternative splicing. It has recently been demonstrated that the RARβ2 isoform is dispensable for normal development. To ascertain the function of all RARβ isoforms in vivo, we have generated a mutation that disrupts all isoforms of the RARβ gene in the mouse by gene targeting in embryonic stem cells. Mice homozygous for the mutation are viable and fertile with no externally apparent abnormalities. During development, 1 11 RARβ mutant embryos showed fusion of the ninth and tenth cranial ganglia on both sides of the hindbrain. However, no obvious alterations in the spatial pattern of expression of Hoxb-1, Hoxb-4 and Hoxb-5 were observed in day 9.5 p.c. embryos. The RARβ null mutation did not alter the pattern or extent of the limb and craniofacial malformations induced by RA excess, suggesting that RARβ may not be mandatory to mediate the observed teratological effects of RA in these structures. These experiments demonstrate that RARβ isoforms are not absolutely required for embryonic development and provide additional support to the concept of functional redundancy among members of the RAR family.

Retinoic Acid and Mouse Skin Morphogenesis. II. Role of Epidermal Competence in Hair Glandular Metaplasia

Retinoic acid (RA) has marked effects on mouse upper-lip skin morphogenesis, leading to the development of glomerular gland instead of hair vibrissa follicle, but does not apparently change the dorsal pelage hair developmental program. In order to test the hypothesis that an up-regulation of the β retinoic acid nuclear receptor (RARβ) may be implicated in the alteration of the dermal-epidermal interactions which occur during cutaneous appendage development, RA-treated and untreated skin explants, controls as well as heterotopic recombinants, were made among nasal, upper-lip, and dorsal mouse embryonic tissues. They were analyzed by in situ hybridization with RARβ 35S-labeled probe after 48 hr of in vitro culture as well as by identification of the morphological phenotype of cutaneous appendages after 6 additional days of culture on the chick chorioallantoic membrane. The results show that only mesenchyme from the facial region can express the RARβ gene either normally or after RA treatment, depending on its nasal or upper-lip origin. However, the RARβ up-regulation is unrelated to hair glandular metaplasia, which depends both on a glandular bias of the upper-lip epidermis and on the weakening of hair follicle-inducing dermal properties. The latter occurs in both the upper-lip and dorsal dermis as a consequence of RA treatment.

Differential expression of RAR-ß and RXR-γ transcripts in cultured cranial neural crest cells.

In situ hybridization reveals that RAR-β and RXR-γ genes in mesencephalic neural crest cultures are independently regulated. RAR-β transcripts are found in all cells, with a slight increase in signal/cell with time in culture. In contrast, the distribution of RXR-γ transcript is initially uniform but becomes increasingly heterogeneous, so that after 72 h in culture, a significant proportion of cells lack transcripts while a small subpopulation contains very high levels of message. These differences in the behaviour of the RARβ and RXRγ genes in vitro can be related to differences in their expression patterns in vivo.

Induction of RAR-β2 Gene Expression in Embryos and RAR-β2 Transactivation by the Synthetic Retinoid Ro 13-6307 Correlates with Its High Teratogenic Potency

Vitamin A (retinol), its metabolite all-trans retinoic acid (RA), and many synthetic analogs (retinoids) express variable potencies as teratogens. Although biological activities of retinoids are mediated by nuclear RA receptors (RARs) and retinoid X receptors (RXRs), it is not known if any of these receptors mediate teratogenicity, and if the potency also depends on the nature of the ligand-receptor interactions. Previous evidence has implicated that one specific isoform, RAR-β2, does play a role in mediating retinoid teratogenicity. Here, we employed an aromatic retinoid with a triene side chain, Ro 13-6307, to study its interactions with RAR-β2 since its teratogenicity is much higher and its accessibility to the embryo is much lower than RA. A fully teratogenic dose of Ro 13-6307 (10 mg−kg) given to pregnant mice preferentially elevated the level of RAR-β2 mRNA in susceptible embryonic regions (maximal induction, 10- to 12-fold above control in limb buds) in a manner comparable to a fully teratogenic dose of all-trans RA (100 mg−kg). Using the RAR-β2 promoter linked to a reporter gene in cotransfection experiments, the efficacy of Ro 13-6307 and RAR-β2 in transcription transactivation was found to be 30-40 times greater than all-trans RA. Since the teratogenic potency of Ro 13-6307 is estimated from a previous study to be 44-fold greater than all-trans RA, we suggest that the teratogenicity of this synthetic retinoid is generally proportional to its ability to enhance receptor function.

Genomic organization of the human retinoic acid receptor β2

Recently three isoforms of the mouse retinoic acid receptor (mRARβ1, mRARβ2, mRARβ3) have been described, generated from the same gene (Zelent et al., 1991). The isoforms differ in their 5′-untranslated (5′-UTR) and A region, but have identical B to F regions. The N-terminal variability of mRARβ1 β3 is encoded in the first two exons (El and E2), while exon E3 includes N-terminal sequences of the mRARβ2 isoform. We have determined the structure of the human RARβ2 gene, using a genomic library from K562 cells. The open reading frame is split into eight exons: E3 contains sequences for the N-terminal A region and E4 to E10 encode the common part of the receptor, including the DNA-binding domain and ligand-binding domain. Corresponding to other nuclear receptors, both ‘zinc-forgers’ of the DNA-binding domain are encoded separately in two exons and the ligand-binding domain is assembled from five exons.

Position-dependence of retinoic acid receptor-β gene expression in the chick limb bud

Retinoic acid and 3,4-didehydroretinoic acid are metabolites of vitamin A that can induce duplications and other malformations when locally applied to the anterior margin of the chick limb bud. There is evidence that they may be natural signaling substances in the limb bud. Both compounds are thought to act by binding to ligand-dependent transcription factors that belong to the steroid/thyroid hormone nuclear receptor superfamily. In situ hybridization analyses show that in the mesenchyme of the chick wing bud between embryonic stages 20 and 27, retinoic acid receptor-β (RAR-β) transcripts are restricted to the proximal region of the bud and are present at highest levels in the region of the limb bud mesenchyme that contributes to the shoulder. We have performed grafting experiments in order to examine whether RAR-β gene expression in limb bud mesenchyme cells is cell-autonomous or whether it is dependent upon the cell's position within the limb bud. When tissue from the proximal region of the stage 22 wing bud, which contains high levels of RAR-β transcripts, was grafted to the distal tip of the bud, RAR-β transcripts were undetectable in the graft 6 hr later. When tissue from the distal tip of the bud was grafted to a proximal site, most of the grafts exhibited a slight increase in the level of RAR-β transcripts, which was detectable 6 hr after grafting. However, the levels of RAR-β transcripts in these grafts never approached those found in the proximal core of the bud. These data indicate that RAR-β gene expression in the chick wing bud is position-dependent in that it is repressed at the distal tip of the bud and partially activated by grafting distal tissue to a proximal site. However, accumulation of RAR-β transcripts to high levels appears to be a characteristic of mesenchyme that was initially specified to form proximal structures.

Expression of nuclear retinoic acid receptors in rat adipose tissue

Expression of nuclear retinoic acid receptors (RAR-α, β and γ) was examined by Northern blots in rat lung, liver, and adipose tissue. Three transcripts of approximate sizes 7.3, 3.7, and 2.8 Kb were detected in adipose tissue. In contrast to adipose tissue only 3.7 and 2.8 Kb mRNA species were detected in liver and lung. Two RAR-β gene transcripts (3.3 and 3.0 Kb) were expressed in adipose tissue, liver, and lung. RAR-γ mRNA (3.36 Kb) was detected in adipose tissue and lung. The distribution of three RARs in adipose tissue suggests the physiological role of retinoic acid in the regulation of specific genes via RARs in adipocytes.

Retinoic acid-induced glandular metaplasia in mouse skin is linked to the dermal expression of retinoic acid receptor β mRNA

The distribution of transcripts of nuclear (RARα, RARβ, and RARγ) and cytosolic (CRABP) retinoic acid receptors was analyzed in 13.5-day mouse embryo upper-lip skin, cultured in vitro for 48 hr with or without added retinoic acid. The results show a significant up-regulation of the transcription of the RARβ gene concomitant with the initiation of an alteration of hair vibrissae follicle development, leading, after transfer for 8 days to the chick embryo chorioallantoic membrane, to an exocrine-type gland morphogenesis.

Transcriptional regulation of retinoic acid receptor β in retinoic acid-sensitive and -resistant P19 embryocarcinoma cells

As in other embryocarcinoma (EC) cell lines retinoic acid (RA) rapidly induces expression of the nuclear retinoic acid receptor (RAR) β in murine P19 EC cells, while RARα is expressed constitutively. In the RA-resistant P19 EC-derived RAC65 cells, however, there is no such induction and an aberrant (smaller) RARα transcript is expressed. RAR γ 1 is expressed at low levels in both cell lines. To study the regulation of the RARβ gene and the possible involvement of RARα protein in transcriptional activation of the RARβ gene we transfected these cells with a construct containing a 1.6 kb promoter fragment of the human RARβ gene fused to the CAT gene. Upon transient assays in P19 EC cells CAT activity is enhanced rapidly by RA, to more than 100-fold in a concentration-dependent fashion. On the contrary no activity can be observed in the RA-resistant RAC65 cells; however, co-transfection of hRARα, hRARβ or hRAR γ 1 restores the RA-dependent induction of CAT activity. These results clearly show that RARα and RAR γ 1 can transactivate the RARβ gene; that RARβ can stimulate its own expression and that resistance to RA in RAC65 cells is probably due to the altered RARα transcript present in these cells.

Mouse and human retinoic acid receptor beta 2 promoters: sequence comparison and localization of retinoic acid responsiveness.

The retinoic acid receptor beta (RAR beta) gene is a member of the family of retinoic acid/thyroid hormone receptor genes, encoding retinoic acid-inducible transcription factors. To study regulation of the RAR beta gene, genomic clones containing the mouse and human retinoic acid receptor beta 2 (RAR beta 2) promoters were isolated and approximately 1.5 kb of upstream and downstream sequences relative to the transcriptional start site were completely sequenced. Both the mouse and human RAR beta 2 promoters are highly homologous around the transcription initiation site, with perfect conservation of the TATA box and retinoic acid responsive element (RARE). Promoter activation studies in P19 EC cells show, that the RARE in both the human and mouse promoters confers RA-responsiveness to the RAR beta 2 promoter. However, sequences located immediately upstream of the RARE also confer RA-inducibility to both the mouse and human RAR beta 2 gene promoters. This region contains conserved consensus sequences for a TPA-responsive element (TRE) and cAMP-responsive element (CRE), suggesting that in addition to regulation by RA receptors other transcription factors regulate RAR beta 2 expression in EC cells. Furthermore, the availability of the mouse RAR beta 2 promoter should facilitate studies for transgene expression and gene targeting experiments in embryonic stem cells.

Expression of retinoic acid receptor genes in neural crest-derived cells during mouse facial development

Retinoic acid (RA) is known as a teratogen that induces abnormalities in facial structures which are made up mainly of neural crest-derived mesenchyme. We investigated expression patterns of RA receptor (RAR) genes (subtypes α, β, γ) during mouse facial development. The expression of the rarβ gene is specific for the mesenchyme around developing eyes and nose, whereas the RARγ gene is expressed in the mesenchyme differentiating to facial cartilages and bones. In contrast, the RARα gene is expressed weakly and uniformly over the facial region. These results suggest that crucial roles of endogenous RA in facial development depend on differential functions of the RAR subtypes.