The retinoid receptors.

The retinoid receptors belong to a large superfamily of ligand-inducible transcription factors that include the steroid, vitamin D and thyroid hormone receptors, the peroxisome proliferator-activated receptor, the insect edysteroid receptor, and a number of orphan receptors whose ligands are unknown. All nuclear receptors have several well-characterized structural domains, including a conserved DNA-binding domain, and a ligand binding domain at the carboxyl terminus of the receptor. The RAR and RXR classes of nuclear retinoic acid receptors are each composed of alpha, beta and gamma subtypes with more than one isoform for each receptor subtype. Data from many investigators suggest there are RAR- and RXR-dependent gene pathways, and that the individual receptor subtypes may control distinct gene expression patterns. In addition, RXR has been found to heterodimerize with other nuclear receptors to form active transcriptional complexes, which influence the activity of a variety of gene pathways important in growth and differentiation. As a result, retinoids have been useful clinical agents in Dermatology and Oncology. However, upon prolonged exposure to retinoic acid, resistance to retinoids has often been encountered both in the clinical setting and in long-term cell culture (HL60R and RAC65 cells). In the latter case, retinoid resistance has been associated with a mutation in the RAR gene which transcribes a RAR receptor truncated at the C-terminal end. These mutated RAR receptors exhibit a reduced affinity for retinoic acid while retaining the ability to bind to a retinoic acid response element on DNA. As a result, these mutant receptors exhibit dominant-negative activity by binding to the DNA without activating transcription and by competing with other receptors for sites on the response element. In fact, dominant-negative activity may be very important in the development of many neoplastic diseases, including acute promyelocytic leukemia (APL), where a t(15;17) chromosomal translocation fuses the PML gene to the RAR gene, to produce a PML-RAR fusion protein in large excess in the cell. However, retinoid resistance in the patient is most probably the result of pharmacokinetic problems, whereby, with continuous retinoid treatment, the plasma levels of retinoic acid gradually decrease to below that required to maintain differentiation of leukemic cells in vivo. A major challenge for drug discovery is to design a drug which circumvents these pharmacokinetic problems either by designing novel drug delivery systems or by employing retinoids which do not bind to CRABP, such as 9-c-RA.(ABSTRACT TRUNCATED AT 400 WORDS)

The retinoid receptors.

The retinoid receptors belong to a large superfamily of ligand-inducible transcription factors that include the steroid, vitamin D and thyroid hormone receptors, the peroxisome proliferator-activated receptor, the insect edysteroid receptor, and a number of orphan receptors whose ligands are unknown. All nuclear receptors have several well-characterized structural domains, including a conserved DNA-binding domain, and a ligand binding domain at the carboxyl terminus of the receptor. The RAR and RXR classes of nuclear retinoic acid receptors are each composed of alpha, beta and gamma subtypes with more than one isoform for each receptor subtype. Data from many investigators suggest there are RAR- and RXR-dependent gene pathways, and that the individual receptor subtypes may control distinct gene expression patterns. In addition, RXR has been found to heterodimerize with other nuclear receptors to form active transcriptional complexes, which influence the activity of a variety of gene pathways important in growth and differentiation. As a result, retinoids have been useful clinical agents in Dermatology and Oncology. However, upon prolonged exposure to retinoic acid, resistance to retinoids has often been encountered both in the clinical setting and in long-term cell culture (HL60R and RAC65 cells). In the latter case, retinoid resistance has been associated with a mutation in the RAR gene which transcribes a RAR receptor truncated at the C-terminal end. These mutated RAR receptors exhibit a reduced affinity for retinoic acid while retaining the ability to bind to a retinoic acid response element on DNA. As a result, these mutant receptors exhibit dominant-negative activity by binding to the DNA without activating transcription and by competing with other receptors for sites on the response element. In fact, dominant-negative activity may be very important in the development of many neoplastic diseases, including acute promyelocytic leukemia (APL), where a t(15;17) chromosomal translocation fuses the PML gene to the RAR gene, to produce a PML-RAR fusion protein in large excess in the cell. However, retinoid resistance in the patient is most probably the result of pharmacokinetic problems, whereby, with continuous retinoid treatment, the plasma levels of retinoic acid gradually decrease to below that required to maintain differentiation of leukemic cells in vivo. A major challenge for drug discovery is to design a drug which circumvents these pharmacokinetic problems either by designing novel drug delivery systems or by employing retinoids which do not bind to CRABP, such as 9-c-RA.(ABSTRACT TRUNCATED AT 400 WORDS)

Expression of nuclear retinoic acid receptors in wild-type and mutant embryonal carcinoma PCC4.aza1R cells.

Retinoic acid (RA) induces differentiation of murine embryonal carcinoma PCC4.aza1R cells. In this study, the expression of nuclear retinoic acid receptors (RARs) in PCC4.aza1R cells is examined. Analyses of [3H]RA-labeled nuclear extracts prepared from PCC4.aza1R cells by size-exclusion high-performance liquid chromatography demonstrated the presence of a specific RA-binding activity that migrated with a molecular weight of approximately 50,000. More than 95% of this binding activity was associated with the nuclear fraction. In contrast to cytosolic retinoic acid-binding protein, the RARs bound RA analogues of the Ch-series very effectively. Northern blot analyses of total RNA with complementary DNA probes specific for RAR alpha, RAR beta, and RAR gamma showed that PCC4.aza1R cells contain predominantly transcripts encoding RAR alpha and RAR gamma; RAR beta transcripts were undetectable. Treatment of PCC4.aza1R cells with RA increased the levels of RAR beta mRNA in a dose- and time-dependent manner. The RA concentration for half-maximum induction of RAR beta mRNA was 1 nM. An increase in RAR beta mRNA was detectable as early as 2 h after the addition of RA. This increase was not abrogated by cycloheximide, suggesting that protein synthesis is not required for this response. The ability of several retinoids to increase RAR beta mRNA levels in PCC4.aza1R cells correlated well with their binding affinity to the RARs but not with their binding affinity to cytosolic retinoic acid-binding protein. Two mutant cell lines, PCC4(RA)-1 and (RA)-2, which do not undergo differentiation after RA treatment, contained levels of RAR-binding activity very similar to those of the parental cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Retinoic acid receptor belongs to a subclass of nuclear receptors that do not form "docking" complexes with hsp90.

We have recently reported that, in contrast to the glucocorticoid receptor, the thyroid hormone receptor does not bind to hsp90 when the receptor is translated in rabbit reticulocyte lysate [Dalman, F. C., Koenig, R. J., Perdew, G. H., Massa, E., & Pratt, W. B. (1990) J. Biol. Chem. 265, 3615-3618]. All of the steroid receptors that are known to bind hsp90 are recovered in the cytosolic fraction when hormone-free cells are ruptured in hypotonic buffer. In contrast, unliganded thyroid hormone receptors and retinoic acid receptors are tightly associated with nuclear components. In this paper, we translated the human estrogen receptor and the human retinoic acid receptor in reticulocyte lysate and then immunoadsorbed the [35S]methionine-labeled translation products with the 8D3 monoclonal antibody against hsp90. The estrogen receptor is bound to hsp90, as indicated by coimmunoadsorption, but the retinoic acid receptor is not. Translation and immunoadsorption of chimeric proteins containing the DNA binding domain of one receptor and the N-terminal and COOH-terminal segments of the other show that the DNA binding finger region of the estrogen receptor is neither necessary nor sufficient for hsp90 binding. These observations suggest that there are two classes within the steroid receptor family. In one class (e.g., glucocorticoid, mineralocorticoid, sex hormone, and dioxin receptors), the receptors bind to hsp90 and remain in some kind of inactive "docking" mode until hormone-triggered release of hsp90 occurs. In the retinoic acid/thyroid hormone class, the unligated receptors do not bind to hsp90, and the receptors appear to proceed directly to their high-affinity nuclear acceptor sites without entering the "docking" state.

Ectopic Hoxa-1 induces rhombomere transformation in mouse hindbrain.

Homeobox genes are expressed with a specific spatial and temporal order, which is essential for pattern formation during the early development of both invertebrates and vertebrates. Here we show that widespread ectopic expression of the Hoxa-1 (Hox 1.6) gene directed by a human beta-actin promoter in transgenic mice is embryolethal and produces abnormal phenotypes in a subset of domains primarily located in anterior regions. Interestingly, this abnormal development in the Hoxa-1 transgenic mice is associated with ectopic expression of the Hoxb-1 (Hox 2.9) gene in select hindbrain regions. At gestation day 9.5, two domains of strong Hoxb-1 expression are found in the anterior region of the hindbrains of Hoxa-1 transgenic embryos. One region represents the normal pattern of Hoxb-1 expression in rhombomere 4 and its associated migrating neural crest cells, while another major domain of Hoxb-1 expression consistently appears in rhombomere 2. Similar ectopic domains of beta-galactosidase activity are detected in dual transgenic embryos containing both beta-actin/Hoxa-1 transgene and a Hoxb-1/lacZ reporter construct. Expression of another lacZ reporter gene that directs beta-galactosidase activity predominantly in rhombomere 2 is suppressed in the Hoxa-1 transgenic embryos. We have also detected weaker and variable ectopic Hoxb-1 expression in rhombomeres 1, 3 and 6. No ectopic Hoxb-1 expression is detected in rhombomere 5 and the expression of Hoxa-3 and Krox-20 in this region is unchanged in the Hoxa-1 transgenic embryos. While no obvious change in the morphology of the trigeminal or facial-acoustic ganglia is evident, phenotypic changes do occur in neurons that emanate from rhombomeres 2 and 3 in the Hoxa-1 transgenic embryos. Additionally, alterations in the pattern of Hoxa-2 and Hoxb-1 expression in a subpopulation of neural crest cells migrating from the rhombomere 2 region are detected in these transgenics. Taken together, these data suggest that ectopic Hoxa-1 expression can reorganize select regions of the developing hindbrain by inducing partial transformations of several rhombomeres into a rhombomere-4-like identity.

All-trans-retinoic acid and hexamethylene bisacetamide (HMBA) regulate TGF-alpha and Hst-1/kFGF expression in differentiation sensitive but not in resistant human teratocarcinomas.

The multipotent human teratocarcinoma (TC) cell NTera-2 clone D1 (abbreviated NT2/D1) differentiates into a neuronal lineage after retinoic acid (RA) treatment and a distinct phenotype after hexamethylene bisacetamide (HMBA) treatment. We previously reported that RA treatment of NT2/D1 cells reduces cellular cloning efficiency and nude mouse tumorigenicity. This accompanied a loss of mRNA expression of transforming growth factor-alpha (TGF-alpha) and the fibroblast growth factor kFGF, also known as hst-1 (abbreviated hst-1/kFGF). This study extends prior work by reporting that the distinct phenotype induced by HMBA also decreases cloning efficiency, tumorigenicity, and TGF-alpha and hst-1/kFGF mRNA expression in NT2/D1 cells. These RNA findings were confirmed by measurements of growth factor protein in the conditioned media of inducer-treated and untreated NT2/D1 cells. In two established human TC lines refractory to the actions of RA, N2102ep and Tera-1, RA fails to decrease expression of either growth factor despite induction of its nuclear receptor, RAR-beta. However, HMBA induces morphologic maturation and down-regulation of these growth factors in N2102ep cells. This indicates that the loss of TGF-alpha and hst-1/kFGF expression serves as a new marker of differentiation in human TCs. To explore the effects of these growth factors on growth and differentiation of NT2/D1 cells, TGF-alpha or hst-1/kFGF protein was added following inducer treatment or no treatment. Neither growth factor blocked immunophenotypic differentiation, but both promoted the growth of uninduced NT2/D1 cells in cloning assays.(ABSTRACT TRUNCATED AT 250 WORDS)