A new heparin binding protein regulated by retinoic acid from chick embryo

A 19 KDa heparin binding protein was previously purified from chicken embryos. Essentially localized within basement membranes in early embryonic tissues, this protein is very rich in basic and cystein residues. Its N-terminal fragment is similar to corresponding fragment of two other proteins expressed during embryogenesis and postnatal period. Its synthesis and secretion are induced by retinoic acid in chicken myoblasts and fibroblasts. This new retinoic acid induced heparin binding protein (RI-HB) does stimulate neurite outgrowth and proliferation on PC12 cells. These results suggest that retinoic acid could regulate some aspect of differentiation and development by inducing the synthesis of a new family of growth and neurotrophic factors.     

Expression and function of a retinoic acid receptor in budding ascidians

 Retinoic acid is thought to induce transdifferentiation of multipotent epithelial stem cells in the developing buds of the ascidian Polyandrocarpa misakiensis. We isolated a cDNA clone from this species, named PmRAR, encoding a retinoic acid receptor (RAR) homologue. PmRAR clusters with other RARs on phylogenetic trees constructed by three different methods. Within the cluster, PmRAR is on a separate branch from all the subtypes of RARs, suggesting that RAR subtypes arose in the ancestral vertebrates after divergence of vertebrates and urochordates. The embryos of another ascidian species Ciona intestinalis were co-electroporated with a mixture of a PmRAR expression vector and a lacZ reporter plasmid containing vertebrate-type retinoic acid response elements. The expression of lacZ depended on the presence of both retinoic acid and PmRAR, suggesting that PmRAR is a functional receptor. PmRAR mRNA is expressed in the epidermis and mesenchyme cells of the Polyandrocarpa developing bud. The mRNA is not detectable in the mesenchyme cells in the adult body wall, but its expression can be induced by retinoic acid in vitro. These results suggest that the PmRAR is a mediator of retinoic acid signalling in transdifferentiation during asexual reproduction of protochordates. Received: 6 April 1998 / Accepted: 27 July 1998     

Inhibition by chlorogenic acid of haematin-catalysed retinoic acid 5,6-epoxidation.

Chlorogenic acid (3-O-caffeoylquinic acid) inhibited haematin- and haemoglobin-catalysed retinoic acid 5,6-epoxidation. Some other phenol compounds (caffeic acid and 4-hydroxy-3-methoxybenzoic acid) also showed inhibitory effects on the haematin- and haemoglobin-catalysed epoxidation, but salicylic acid did not. Of the above compounds, caffeic acid and chlorogenic acid were potent inhibitors compared with the other two, suggesting that the o-hydroquinone moiety of chlorogenic acid and caffeic acid is essential to the inhibition of the epoxidation. Although caffeic acid inhibited retinoic acid 5,6-epoxidation requiring the consumption of O2, formation of retinoic acid radicals was not inhibited on the addition of caffeic acid to the incubation mixture. The above results suggest that caffeic acid does not inhibit the formation of retinoic acid radicals but does inhibit the step of conversion of retinoic acid radical into the 5,6-epoxide.     

Molecular cloning of RI-HB, a heparin binding protein regulated by retinoic acid

RI-HB is an extracellular heparin binding protein regulated by retinoic acid and essentially expressed during embryogenesis. This study reports the cloning and sequencing of the cDNA that encodes RI-HB. The sequence of RI-HB contains 121 amino acid residues and is very rich in basic amino acids and cysteines. This sequence was compared to those of HBGAM and MK protein, two other heparin binding proteins exhibiting growth and/or neurotrophic activities. Northern blot analysis indicates that RI-HB mRNA is strongly expressed during early chicken embryogenesis and that it is induced by retinoic acid treatment of chicken fibroblasts and myotubes in culture.     

An ascidian homologue of the gonadotropin-releasing hormone receptor is a retinoic acid target gene

Transdifferentiation of the multipotent atrial epithelium is a key event during budding of the ascidian Polyandrocarpa misakiensis. The transdifferentiation is induced by mesenchyme cells that were stimulated by retinoic acid. The fluorescent differential display identified a few cDNA fragments for retinoic acid-inducible genes. One of the cDNA clones, named Pm-GnRHR, encoded a seven-pass transmembrane receptor similar to gonadotropin-releasing hormone receptors. Putative amino acid sequence showed high similarity to Ciona intestinalis GnRHRs and formed a cluster with other GnRHR proteins in a phylogenetic tree. The level of expression of the Pm-GnRHR mRNA increased during the early stage of bud development, suggesting that the Pm-GnRHR function is involved in some aspects of bud development.     

Disabled-2 mediates c-Fos suppression and the cell growth regulatory activity of retinoic acid in embryonic carcinoma cells

F9 embryonic stem cell-like teratocarcinoma cells are widely used to study early embryonic development and cell differentiation. The cells can be induced by retinoic acid to undergo endodermal differentiation. The retinoic acid-induced differentiation accompanies cell growth suppression, and thus, F9 cells are also often used as a model for analysis of retinoic acid biological activity. We have recently shown that MAPK activation and c-Fos expression are uncoupled in F9 cells upon retinoic acid-induced endodermal differentiation. The expression of the candidate tumor suppressor Disabled-2 is induced and correlates with cell growth suppression in F9 cells. We were not able to establish stable Disabled-2 expression by cDNA transfection in F9 cells without induction of spontaneous cell differentiation. Transient transfection of Dab2 by adenoviral vector nevertheless suppresses Elk-1 phosphorylation, c-Fos expression, and cell growth. In PA-1, another teratocarcinoma cell line of human origin that has no or very low levels of Disabled-2, retinoic acid fails to induce Disabled-2, correlating with a lack of growth suppression, although PA-1 is responsive to retinoic acid in morphological change. Transfection and expression of Disabled-2 in PA-1 cells mimic the effects of retinoic acid on growth suppression; the Disabled-2-expressing cells reach a much lower saturation density, and serum-stimulated c-Fos expression is greatly suppressed and disassociated from MAPK activation. Thus, Dab2 is one of the principal genes induced by retinoic acid involved in cell growth suppression, and expression of Dab2 alone is sufficient for uncoupling of MAPK activation and c-Fos expression. Resistance to retinoic acid regulation in PA-1 cells likely results from defects in retinoic acid up-regulation of Dab2 expression.     

Differential cellular distribution of retinoic acid during staurosporine potentiation of retinoic acid-induced granulocytic differentiation in human leukemia HL-60 cells

Pretreatment of cells with staurosporine, a protein kinase C (PKC) inhibitor, was found to potentiate the granulocytic differentiation induced by a brief (2 h) retinoic acid treatment. By cell cycle analysis, staurosporine was found to have little effect on the cell cycle. Retinoic acid was distributed equally in the nuclei (40%) and in the plasma membrane (40%) of staurosporine-pretreated cells while less than 20% of retinoic acid was found in the membrane of control non-staurosporine-pretreated cells during the retinoic acid-induced differentiation. These results indicate that the enhancing effect of staurosporine may be somehow associated with the localization of retinoic acid in the plasma membrane of the cell. -1This work is dedicated to Prof. Harris Busch (Baylor College of Medicine, Tex., USA) for his 33 wonderful years at Baylor and 50 years in medicine.     

Stimulation of premature retinoic acid synthesis in Xenopus embryos following premature expression of aldehyde dehydrogenase ALDH1.

In order for nuclear retinoic acid receptors to mediate retinoid signaling, the ligand retinoic acid must first be produced from its vitamin A precursor retinal. Biochemical studies have shown that retinal can be metabolized in vitro to retinoic acid by members of the aldehyde dehydrogenase enzyme family, including ALDH1. Here we describe the first direct evidence that ALDH1 plays a physiological role in retinoic acid synthesis by analysis of retinoid signaling in Xenopus embryos, which have plentiful stores of maternally derived retinal. The Xenopus ALDH1 gene was cloned and shown to be highly conserved with chick and mammalian homologs. Xenopus ALDH1 was not expressed at blastula and gastrula stages, but was expressed at the neurula stage. We used a retinoic acid bioassay to demonstrate that retinoic acid is normally undetectable in embryos from fertilization to the initial gastrula stage, but that a tremendous increase in retinoic acid occurs during neurulation when ALDH1 is first expressed. Overexpression of ALDH1 by injection of Xenopus embryos with mRNAs encoding the mouse, chick or Xenopus ALDH1 homologs induced high levels of retinoic acid detection during the blastula stage. Thus, premature expression of ALDH1 stimulates premature synthesis of retinoic acid. These findings reveal an important conserved role for ALDH1 in retinoic acid synthesis in vivo, and demonstrate that conversion of retinoids from the aldehyde form to the carboxylic acid form is a crucial regulatory step in retinoid signaling.     

Retinol forms retinoic acid via retinal.

Hepatic cytosol from normal deermice having cytosolic alcohol dehydrogenase (ADH+) also displays retinol dehydrogenase activity and converts retinol to retinoic acid, whereas cytosol from ADH- deermice lacks these enzyme activities and does not produce retinoic acid. Furthermore, microsomes from either strain do not convert retinol to retinoic acid. However, when cytosol from ADH- animals is added to the microsomes, retinoic acid is produced. The obligatory role of retinal as an intermediary step in retinoic acid formation is further shown by isotopic dilution of retinoic acid formed from labeled retinol upon addition of unlabeled retinal. Microsomal retinol dehydrogenase also catalyzes the reduction of retinal to retinol, thereby explaining the decrease in retinoic acid production from retinol in liver cytosol of ADH+ deermice when microsomes are added. Thus, the results of this study indicate that retinal is an obligatory intermediate in the hepatic production of retinoic acid from retinol and that cytosolic and microsomal retinol dehydrogenases play a key role in this process.     

Binding of retinoic acid by the inhibitory serpin protein C inhibitor.

The serpin superfamily includes inhibitors of serine proteases and noninhibitory members with other functions (e.g. the hormone precursor angiotensinogen and the hormone carriers corticosteroid-binding globulin and thyroxine-binding globulin). It is not known whether inhibitory serpins have additional, noninhibitory functions. We studied binding of (3)H-labeled hydrophobic hormones (estradiol, progesterone, testosterone, cortisol, aldosterone, and all-trans-retinoic acid) to the inhibitory serpins antithrombin III, heparin cofactor II, plasminogen activator inhibitor-1, and protein C inhibitor (PCI). All-trans-[(3)H]retinoic acid bound in a specific dose-dependent and time-dependent way to PCI (apparent K(d) = 2.43 microm, 0.8 binding sites per molecule of PCI). We did not observe binding of other hormones to serpins. Intact and protease-cleaved PCI bound retinoic acid equally well, and retinoic acid did not influence inhibition of tissue kallikrein by PCI. Gel filtration confirmed binding of retinoic acid to PCI in purified systems and suggested that PCI may also function as a retinoic acid-binding protein in seminal plasma. Therefore, our present data, together with the fact that PCI is abundantly expressed in tissues requiring retinoic acid for differentiation processes (e.g. the male reproductive tract, epithelia in various organs), suggest an additional biological role for PCI as a retinoic acid-binding and/or delivering serpin.     

The oxidation and decarboxylation of retinoic acid by horseradish peroxidase.

The decarboxylation of retinoic acid by horseradish peroxidase was investigated. A marked increase in the yield of products was obtained. However, the data indicated the reaction was a nonenzymatic, heme catalyzed peroxidation. Previously reported requirements for phosphate, oxygen and ferrous ion were eliminated when hydrogen peroxide was provided. Peroxide also eliminated the EDTA and cyanide induced inhibition of the phosphate dependent system. In the presence of hydrogen peroxide, horseradish peroxidase was not essential to the reaction; heme equivalent amounts of hemoglobin decarboxylated retinoic acid with equal facility. However, hemoglobin was ineffective in the absence of hydrogen peroxide. Attainment of 50--60% decarboxylation represented complete utilization of the available retinoic acid. Thus the products of the reaction can be divided into two groups, products of retinoic acid oxidation and products of an oxidative decarboxylation of retinoic acid.     

The cellular retinoic acid binding proteins

The two cellular retinoic acid binding proteins, CRABP I and CRABP II, belong to a family of small cytosolic lipid binding proteins and are highly conserved during evolution. Both proteins are expressed during embryogenesis, particularly in the developing nervous system, craniofacial region and limb bud. CRABP I is also expressed in several adult tissues, however, in contrast, CRABP II expression appears to be limited to the skin. It is likely that these proteins serve as regulators in the transport and metabolism of retinoic acid in the developing embryo and throughout adult life. It has been proposed that CRABP I sequesters retinoic acid in the cytoplasm and prevents nuclear uptake of retinoic acid. A role in catabolism of retinoic acid has also been proposed. Recent gene targeting experiments have shown that neither of the two CRABPs are essential for normal embryonic development or adult life. Examination of CRABP I expression at subcellular resolution reveals a differential cytoplasmic and/or nuclear localization of the protein. A regulated nuclear uptake of CRABP I implies a role for this protein in the intracellular transport of retinoic acid. A protein mediated mechanism which controls the nuclear uptake of retinoic acid may play an important role in the transactivation of the nuclear retinoic acid receptors.     

In vitro organogenesis of pancreas in Xenopus laevis dorsal lips treated with retinoic acid

Dorsal lips of Xenopus laevis may differentiate into pancreas after treatment with retinoic acid in vitro. The dorsal lip region is fated to be dorsal mesoderm and anterior endoderm. Dorsal lip cells isolated from stage 10 early gastrula differentiate into tissues such as notochord, muscle and pharynx. However, in the present study, dorsal lips treated with 10(-4) M retinoic acid for 3 h differentiated into pancreas-like structures accompanied by notochord and thick endodermal epithelium. Sections of the explants showed that some cells gathered and formed an acinus-like structure as observed under microscopes. In addition to the morphological changes, expressions of the pancreas-specific molecular markers, XIHbox8 and insulin, were induced in retinoic acid-treated dorsal lip explants. Therefore, it is suggested that retinoic acid may induce the dorsal lip cells to differentiate into a functional pancreas. However, continuous treatment with retinoic acid did not induce pancreas differentiation at any concentration. Dorsal lips treated with retinoic acid within 5 h after isolation differentiated into pancreas-like cells, while those treated after 15 h or more did not. The present study provided a suitable test system for analyzing pancreas differentiation in early vertebrate development.     

Inhibition and reversion of chondrogenesis by retinoic acid in rat limb bud cell cultures

Summary Mesenchyme cells derived from embryonic rat limb buds cultured at high density differentiated into chondrocytes. The degree of chondrogenesis was assessed by alcian blue staining, a stain specific for cartilage matrix. The addition of retinoic acid on day 1 of culture inhibited chondrogenesis in a dose-dependent fashion. When retinoic acid was added to the cultures on day 5, the cartilage nodules, consisting of newly differentiated cartilage cells, disappeared during the following 6 days. Coinciding with this process the histochemically demonstrable alkaline phosphatase activity, localized in the internodular areas, also disappeared. This indicated that retinoic acid not only inhibited chondrogenesis but also induced resorption of cartilage cells and that at least two cell types were affected, the cartilage cells and the cells bearing alkaline phosphatase.Actinomycin D and cycloheximide, inhibitors of RNA and protein synthesis, suppressed the retinoic acid effect in day 5 limb bud cell cultures. This result indicated that the effect of retinoic acid required RNA and protein synthesis and is compatible with the view that vitamin A may act in a hormone-like way.     

Unbinding of retinoic acid from the retinoic acid receptor by random expulsion molecular dynamics

Unbinding pathways of retinoic acid (RA) bound to retinoic acid receptor (RAR) have been explored by the random expulsion molecular dynamics (REMD) method. Our results show that RA may exit the binding site of RAR through flexible regions close to the H1-H3 loop and beta-sheets, without displacing H12 from its agonist position. This result may explain kinetic differences between agonist and antagonist ligands observed for other nuclear receptors. The extended and flexible structure of RA initiated a methodological study in a simplified two-dimensional model system. The REMD force should in general be distributed to all atoms of the ligand to obtain the most unbiased results, but for a ligand which is tightly bound in the binding pocket through a strong electrostatic interaction, application of the REMD force on a single atom is preferred.