Identification of a second human retinoic acid receptor

We have previously described a human complementary DNA that encodes a novel protein which is homologous to members of the steroid/thyroid nuclear receptor multigene family. This novel protein (hap for hepatoma) exhibits strong homology with the human retinoic acid receptor (RAR) which has been recently characterized. To test the possibility that the hap protein might also be a retinoid receptor, a chimaeric receptor was created by replacing the putative DNA binding domain of hap with that of the human oestrogen receptor (ER). The resulting hap-ER chimaera was then tested for its ability to trans-activate an oestrogen-responsive reporter gene (vit-tk-CAT) in the presence of possible receptor ligands. Here we show that retinoic acid (RA) at physiological concentrations is effective in inducing the expression of this reporter gene by the hap-ER chimaeric receptor. This demonstrates the existence of two human retinoic acid receptors designated RAR-alpha and RAR-beta.     

A human retinoic acid receptor which belongs to the family of nuclear receptors

A cDNA encoding a protein that binds retinoic acid with high affinity has been cloned. The protein is homologous to the receptors for steroid hormones, thyroid hormones and vitamin D3, and appears to be a retinoic acid-inducible trans-acting enhancer factor, suggesting that the molecular mechanisms of the effect of retinoids (vitamin A) on embryonic development, differentiation and tumour cell growth are similar to those described for other members of this nuclear receptor family.     

Identification of a novel retinoic acid receptor in regenerative tissues of the newt

In urodele amphibians, the progenitor cells that regenerate amputated limbs (known as the blastema) normally replace only the missing structures. After systemic delivery of retinoic acid (RA), more proximal structures are also formed, indicating that RA can control position specification in the proximal-distal axis of the regenerating limb. According to dose and experimental context, retinoids can also re-specify the anteroposterior axis of the limb, induce deletions of skeletal elements, or block re-growth completely. To study the molecular basis of these morphogenetic effects, we screened complementary DNA libraries of newt regenerative tissues (limbs and tails) for hormone nuclear receptors activated by RA. Two functional retinoic acid receptors (RARs) were identified, one of which is the newt homologue of the human alpha-receptor (RAR alpha). The second receptor, called RAR delta, is novel. Sequence analysis suggests that the composite newt RAR previously reported is chimaeric, consisting of 5'RAR-beta-like and 3' RAR delta clones. We conclude that multiple RARs are expressed during limb regeneration in amphibians and suggest that receptor heterogeneity may underlie the different effects of retinoids on limb morphogenesis.     

Homodimer formation of retinoid X receptor induced by 9-cis retinoic acid.

Retinoid response pathways are mediated by two classes of receptors, the retinoic acid receptors (RARs) and the retinoid X receptors (RXRs). A central question is whether distinct response pathways are regulated by these two classes of receptors. The observation that the stereoisomer 9-cis-retinoic acid binds with high affinity to RXRs suggested that this retinoid has a distinct role in controlling RXR activity, but it was almost simultaneously discovered that RXRs function as auxiliary receptors for RARs and related receptors, and are essential for DNA binding and function of those receptors. Hence, although RARs seem to operate effectively only as heterodimeric RAR/RXR complexes, RXRs themselves apparently function predominantly, if not exclusively, as auxiliary receptors. Here we report that 9-cis-retinoic acid induces RXR homodimer formation. Our results demonstrate a new mechanism for retinoid action by which a ligand-induced homodimer mediates a distinct retinoid response pathway.     

Spatial and temporal expression of the retinoic acid receptor in the regenerating amphibian limb

Retinoic acid is known to have dramatic effects on vertebrate limb pattern in development and regeneration, supporting a model in which a gradient of retinoic acid serves as a morphogen to differentially supply positional information to a developing limb. The discovery of a retinoic acid receptor (RAR) and its homology to the steroid and thyroid hormone receptors provided a potential molecular mechanism for limb morphogenesis. One prediction of this model is that the receptor must be expressed in the developing and regenerating limb anlage. We investigated the expression of the RAR in the adult newt, Notophthalmus viridescens, whose amputated limbs are capable of regenerating and upon which retinoic acid can act to alter pattern. We report the cloning of cDNAs encoding a functional newt RAR and the localization of high and uniform levels of RAR mRNA specifically in the regenerating cells that control limb pattern. These results indicate that the morphogenic field is established through differential activation of pre-existing retinoic acid receptors rather than differential expression of the RAR gene.     

9-cis retinoic acid stereoisomer binds and activates the nuclear receptor RXR alpha.

Vitamin A (retinol) and its natural derivatives are required for many physiological processes. The activity of retinoids is thought to be mediated by interactions with two subfamilies of nuclear retinoic acid receptors, RAR and RXR. The RARs bind all-trans retinoic acid (t-RA) with high affinity and alter gene expression as a consequence of this direct ligand interaction. RXR alpha is activated by t-RA, yet has little binding affinity for this ligand. t-RA may be converted to a more proximate ligand that directly binds and activates RXR alpha, and we have developed a method of nuclear receptor-dependent ligand trapping to test this hypothesis. Here we report the identification of a stereoisomer of retinoic acid, 9-cis retinoic acid, which directly binds and activates RXR alpha. These results suggest a new role for isomerization in the physiology of natural retinoids.     

Hepatitis B virus integration in a cyclin A gene in a hepatocellular carcinoma

Hepatitis B virus (HBV) DNA frequently integrates into the genome of human primary liver cancer cells, but the significance of this integration in liver carcinogenesis is still unclear. Here we report the cloning of a single HBV integration site in a human hepatocellular carcinoma at an early stage of development, and of its germline counterpart. The normal locus was found to be transcribed into two polyadenylated messenger RNA species of 1.8 and 2.7 kilobases. We have isolated a complementary DNA clone from a normal adult human liver cDNA library which has an open reading frame with a coding capacity for a protein of 432 amino acids and relative molecular mass 48,536. The strong homology of the C-terminal half of the protein to the A-type cyclins of clam and Drosophila identifies it as a human cyclin A. The cyclin A gene has several exons, and the HBV integration occurs within an intron. As cyclins are important in the control of cell division, the disruption of a cyclin A gene by viral insertion might contribute to tumorigenesis.     

Hepatitis B virus DNA integration in a sequence homologous to v-erb-A and steroid receptor genes in a hepatocellular carcinoma

Hepatitis B virus (HBV) is clearly involved in the aetiology of human hepatocellular carcinoma (HCC) and the finding of HBV DNA integration into human liver DNA in almost all HCCs studied suggested that these integrated viral sequences may be involved in liver oncogenesis. Several HBV integrations in different HCCs and HCC-derived cell lines have been analysed after molecular cloning without revealing any obvious role for HBV. From a comparison of a HBV integration site present in a particular HCC with the corresponding unoccupied site in the non-tumorous tissue of the same liver, we now report that HBV integration places the viral sequence next to a liver cell sequence which bears a striking resemblance to both an oncogene (v-erb-A) and the supposed DNA-binding domain of the human glucocorticoid receptor and human oestrogen receptor genes. We suggest that this gene, usually silent or transcribed at a very low level in normal hepatocytes, becomes inappropriately expressed as a consequence of HBV integration, thus contributing to the cell transformation.     

Sequential activation of HOX2 homeobox genes by retinoic acid in human embryonal carcinoma cells

RETINOIC acid had been implicated as a natural morphogen in chicken and frog embryogenesis, and is presumed to act through the gene regulatory activity of a family of nuclear receptors. Homeobox genes, which specify positional information in Drosophila and possibly in vertebrate embryogenesis, are among the candidate responsive genes. We previously reported that retinoic acid specifically induces human homeobox gene (HOX) expression in the embryonal carcinoma cell line NT2/D1. We now show that the nine genes of the HOX2 cluster are differentially activated in NT2/D1 cells exposed to retinoic acid concentrations ranging from 10(-8) to 10(-5) M. Genes located in the 3' half of the cluster are induced at peak levels by 10(-8) M retinoic acid, whereas a concentration of 10(-6) to 10(-5) M is required to fully activate 5' genes. At both high and low retinoic acid concentrations, HOX2 genes are sequentially activated in embryonal carcinoma cells in the 3' to 5' direction.     

A new neuronal marker identified by phosphorylcholine-binding myeloma proteins.

The difficulty of working with the intact brain in vivo has led to the increasing use of nerve cell cultures in neurobiology. However, dissociated cells cannot be unambiguously identified by morphological criteria before the third week in culture, for it is not until then that the basic morphology and size of neurones become stable so that these and other cell types can be easily distinguished. However, cultured neurones can be identified by various cytochemical techniques based on (1) the detection of neurotransmitters or receptors for transmitters, (2) the presence of the Thy 1 antigen and the receptor for tetanus toxin, which are present on the membrane of most neurones, and (3) the presence in neurones of neurone-specific enolase (NSE), a cytoplasmic enzyme, which can only be identified on fixed specimens. Furthermore, other cell types in culture can also be specifically labelled. For instance, antisera to galactocerebroside bind selectively to oligodendrocytes, and antibodies to a neural tumour bind selectively to Schwann cells. We report here the selective interaction of phosphorylcholine-binding myeloma proteins (PC-BMP) with mouse neurones in culture and in suspension. Phosphorylcholine (PC) is found as part of lecithin and sphingomyelin molecules in variable amounts in eukaryotic and prokaryotic membranes, including plasma membranes.     

四株肺癌细胞系的维甲酸受体表达的研究

目的：观察来源于不同组织学类型的人肺癌细胞系（肺巨细胞癌细胞系PLA-801,肺鳞状上皮细胞癌细胞系C-57,个旧肺腺癌细胞系GLC-82和肺腺癌细胞系PC-84045）的维甲酸受体的含量和分布特征。方法：作ELISA和免疫组化显色计算机图象分析方法,对4株具有不同侵袭潜能,不同组织学类型的肺癌细胞系的维甲酸受体（RARs、RAR和RXRs）进行检测。结果：4株细胞系的RARβ的表达极少,甚至缺如;RARs主要分布在癌细胞胞浆中,但RXRs主要位于细胞核内,表达水平较高;RARs和RXRs在PLA-801细胞中表达最低。结论：肺癌中普遍存在着RAR的表达分布异常,这些改变可能与肺癌的异常分化有关,亦可能是肺癌细胞对维甲酸诱导分化不敏感的重要原因。RXRs较高水平的表达且主要位于核内,提示用9-顺式维甲酸来诱导肺癌的分化效果可能会较好。     

Evidence that Hensen's node is a site of retinoic acid synthesis.

Hensen's node of amniotes, like the Spemann organizer of amphibians, can induce a second body axis when grafted into a host embryo. The avian node, as well as several midline structures originating from it (notochord, floor plate), can also induce digit pattern duplications when grafted into the chick wing bud. We report here that the equivalent of Hensen's node from mouse is an effective inducer of digits in the chick wing bud. Tissues anterior and posterior to the node also evoke pattern duplications, but with a significantly lower efficiency. The finding that the murine node operates in an avian wing bud suggests that the same inducing agent(s) function in both primary and secondary embryonic fields and have been conserved during vertebrate evolution. Digit pattern duplications are also evoked by local administration of all-trans-retinoic acid. This similarity raises the possibility that Hensen's node is a source of retinoic acid. The mouse node is capable of synthesizing retinoic acid from its biosynthetic precursor all-trans-retinol at a substantially higher rate than either anterior or posterior tissues.