Molecular cloning of cDNA encoding a second cellular retinoic acid-binding protein.
ABSTRACT: The vitamin A derivative retinoic acid is known to be a potent agent for control of differentiation and proliferation of epithelial cells and to exert profound effects on pattern formation during embryogenesis. Its action at the molecular level appears to be mediated by two distinct classes of proteins: a family of nuclear receptors that regulates gene transcription in a ligand-dependent fashion and a small cellular retinoic acid-binding protein (CRABP) for which a precise function remains to be elucidated. In this report, we describe the identification (by molecular cloning of its cDNA) of an isoform of CRABP, referred to as CRABP-II, expressed at high levels during mouse embryogenesis and in adult skin. We also show that CRABP-II mRNA levels are induced by at least 50-fold upon treatment of F9 teratocarcinoma cells with retinoic acid. The up-regulation of the gene encoding CRABP-II by its ligand suggests that CRABP-II might be involved in a feedback regulatory role in the mechanism of action of retinoic acid on cellular differentiation.
Project description:The physiological role of cellular retinoic acid-binding proteins (CRABPs) may be to influence the intracellular level of free retinoic acid in the cell. In the present study two isoforms of CRABP, CRABP-I and CRABP-II were partially characterized in various human Malpighian epithelia and in human cultured keratinocytes expressing various patterns of differentiation. We have developed a new sensitive radiobinding assay using a PAGE/autoradioblotting technique which effectively separates CRABP-I and CRABP-II. This method allows the simultaneous quantification of these proteins. We show that CRABP-I and -II have similar M(r) values (15,000), but differ in their dissociation constant towards retinoic acid (Kd of 16.6 nM and 50 nM respectively), in pI (4.86 and 5.13) and in their relative mobilities (RF) on PAGE under nondenaturating conditions (RF values 0.65 and 0.44). In addition, we show that CRABP-II is the major isoform expressed in human keratinocytes, in vivo as in vitro. Furthermore, we demonstrate that CRABP-II is actually the CRABP previously studied in epidermal cells by a PAGE assay (Siegenthaler & Saurat (1987) Eur. J Biochem. 166, 209-214) and whose levels are dramatically increased by retinoic acid and its analogues in human epidermis. Keratinocytes, in the absence of full terminal differentiation, as well as hyperplasia, such as cultured human differentiating keratinocytes, psoriatic plaques, and non-keratinized oral mucosa, contained high levels of CRABP-II. CRABP-I was not detected in cultured keratinocytes, whereas normal skin (at full terminal differentiation) expressed CRABP-I and CRABP-II at a ratio of approx. 1:1.4. This value was approx. 1:17 in lesional psoriatic skin and 1:8 in oral mucosa. These observations suggest that CRABP-I and -II are regulated differently in human keratinocytes. The sharp increases in CRABP-II levels are associated with an alteration in the differentiation programme, as well as with cell response to retinoic acid overload, whereas CRABP-I might be a marker for terminal differentiation.
Project description:Retinoic acid receptors (RARs) α, β and γ are key regulators of embryonic development. Hematopoietic differentiation is regulated by RARα, and several types of leukemia show aberrant RARα activity. We demonstrate that RARα plays an important role in cellular memory and imprinting by regulating the CpG methylation status of specific promoter regions. We used microarrays to identify genes, which display differential expression in F9 RARalpha knockout (RARaKO) cells relative to F9 wt cells. F9 teratocarcinoma cells (WT and RARaKO) were treated for 8 hours with either vehicle (EtOH) or all-trans Retinoic Acid (atRA) in the presence of the protein synthesis inhibitor Cycloheximide
Project description:Dr. Esko's laboratory focuses on the structure, function, and biosynthesis of glycoproteins and proteoglycans. This laboratory also works on the design and synthesis of small molecule inhibitors of glycosylation. Gene expression in F9 teratocarcinoma cells: Study of the differential expression of glycosyltransferases, sulfotransferases and proteoglycan core proteins in F9 teratocarcinoma cells before and after differentiation with retinoic acid/theophylline/cAMP. Published data indicated that differentiation of the cells induces the synthesis of anticoagulant heparin-like compounds and a large increase in overall glycosaminoglycan synthesis. Glyco-gene Chip microarray analysis of RNA samples (in triplicate) from the cells before and after differentiation to reveal factors that regulate the assembly process and how it leads to the generation of binding sites for glycan-binding proteins.
Project description:Rcd1, initially identified as a factor essential for the commitment to nitrogen starvation-invoked differentiation in fission yeast, is one of the most conserved proteins found across eukaryotes, and its mammalian homolog is expressed in a variety of differentiating tissues. Here we show that mammalian Rcd1 is a novel transcriptional cofactor and is critically involved in the commitment step in the retinoic acid-induced differentiation of F9 mouse teratocarcinoma cells, at least in part, via forming complexes with retinoic acid receptor and activation transcription factor-2 (ATF-2). In addition, antisense oligonucleotide treatment of embryonic mouse lung explants suggests that Rcd1 also plays a role in retinoic acid-controlled lung development.
Project description:Skin aging is an intricate biological process consisting of intrinsic and extrinsic alterations of epidermal and dermal structures. Retinoids play an important role in epidermal cell growth and differentiation and are beneficial to counteract skin aging. Cellular retinoic acid binding protein-II (CRABP-II) selectively binds <i>all trans</i>-retinoic acid, the most active retinoid metabolite, contributing to regulate intracytoplasmic retinoid trafficking and keratinocyte differentiation. Immunohistochemistry revealed a reduced epidermal and dermal CRABP-II expression in aged human and mouse skin. To better clarify the role of CRABP-II, we investigated age-related skin changes in CRABP-II knock-out mice. We documented an early reduction of keratinocyte layers, proliferation and differentiation rate, dermal and hypodermal thickness, pilosebaceous units and dermal vascularity in CRABP-II knock-out compared with wild-type mice. Ultrastructural investigation documented reduced number and secretion of epidermal lamellar bodies in CRABP-II knock-out compared with wild-type mice. Cultured CRABP-II knock-out-derived dermal fibroblasts proliferated less and showed reduced levels of TGF-? signal-related genes, Col1A1, Col1A2, and increased MMP2 transcripts compared with those from wild-type. Our data strongly support the hypothesis that a reduction of CRABP-II expression accelerates and promotes skin aging, and suggest CRABP-II as a novel target to improve the efficacy of retinoid-mediated anti-aging therapies.
Project description:The transforming growth factor beta (TGF-beta)-related products of the Xenopus Vg-1 and Drosophila decapentaplegic (DPP) genes have been implicated in the control of growth and differentiation during embryogenesis. We have isolated a mouse cDNA, Vgr-1, that encodes a polypeptide structurally related to Xenopus Vg-1. Sequence comparisons indicate that the Vgr-1 protein belongs to a family of DPP-like gene products within the TGF-beta superfamily. The levels of Vgr-1 RNA were determined in embryos and tissues isolated at various stages of development. A 3.5-kilobase mRNA increases throughout development and into adulthood in many tissues and in F9 teratocarcinoma cells differentiating into endoderm in response to retinoic acid and cAMP. The amino acid homologies and patterns of expression suggest that, like the DPP gene product, Vgr-1 plays a role at various stages of development.
Project description:Effect of all trans retinoic acid and the novel retinoid, ST1926, on the profile of gene expression in F9 teratocarcinoma sublines characterized by the presence or absence of the RAR gamma nuclear retinoic acid receptor
Project description:The objective of this study was to compare the properties of 9-cis and all-trans retinoic acid with respect to the induction of expression of retinoic acid receptor beta (RAR-beta) and cellular retinoic acid-binding protein (CRABP) II in human neuroblastoma SH SY 5Y cells. RAR-beta and CRABP II mRNA was induced by both all-trans and 9-cis retinoic acid in SH SY 5Y cells. Induction was rapid, detectable within 2-4 h, and inhibited by actinomycin D. Time-courses of induction for RAR-beta and CRABP II differed: RAR-beta mRNA levels reached a maximum 4-6 h after adding all-trans or 9-cis retinoic acid, whereas CRABP II mRNA levels increased over at least 18 h. These differences were attributed to the longer half-life of CRABP II mRNA (20 h) compared with RAR-beta mRNA (3.9 h). The dose-response characteristics of all-trans and 9-cis retinoic acid were different: all-trans was effective at nanomolar concentrations, whereas 10-fold higher levels of 9-cis retinoic acid were required to achieve comparable induction of RAR-beta and CRABP II. Conversely, at high concentrations, 9-cis retinoic acid gave a greater induction of RAR-beta and CRABP II than all-trans. The induction of RAR-beta and CRABP II by all-trans retinoic acid was maintained in the subsequent absence of all-trans retinoic acid, whereas induction by 9-cis retinoic acid was dependent on its continued presence in the culture medium. These results suggest that, at high concentrations, 9-cis retinoic acid may produce its transcriptional effects via retinoid X receptor (RXR) homodimers. This has implications for the cellular functions of 9-cis retinoic acid and its use as a biological response modifier.
Project description:We have cloned a cDNA and gene from the tobacco hornworm, Manduca sexta, which is related to the vertebrate cellular retinoic acid binding proteins (CRABPs). CRABPs are members of the superfamily of lipid binding proteins (LBPs) and are thought to mediate the effects of retinoic acid (RA) on morphogenesis, differentiation, and homeostasis. This discovery of a Manduca sexta CRABP (msCRABP) demonstrates the presence of a CRABP in invertebrates. Compared with bovine/murine CRABP I, the deduced amino acid sequence of msCRABP is 71% homologous overall and 88% homologous for the ligand binding pocket. The genomic organization of msCRABP is conserved with other CRABP family members and the larger LBP superfamily. Importantly, the promoter region contains a motif that resembles an RA response element characteristic of the promoter region of most CRABPs analyzed. Three-dimensional molecular modeling based on postulated structural homology with bovine/murine CRABP I shows msCRABP has a ligand binding pocket that can accommodate RA. The existence of an invertebrate CRABP has significant evolutionary implications, suggesting CRABPs appeared during the evolution of the LBP superfamily well before vertebrate/invertebrate divergence, instead of much later in evolution in selected vertebrates.
Project description:All-trans retinoic acid (RA) induces alkaline phosphatase (ALP) activity by 3-8-fold in murine F9 teratocarcinoma cells, in parallel with their differentiation towards primitive endoderm. The elevation of ALP activity is associated with increases in the amounts of liver/bone/kidney-type ALP protein and the respective transcript. These effects of RA are due to activation of ALP gene transcription rather than to an increase in the half-life of the mRNA. Induction of ALP mRNA does not require de novo protein synthesis, since it is not blocked by treatment with cycloheximide. Dibutyryl cyclic AMP, which is known to induce further differentiation of F9 cells from the primitive to the parietal endoderm, blocks the induction of ALP mRNA by RA.