Molecular determinants of visual pigment function.

Mutagenesis studies and comparisons of natural variants of rhodopsin and related visual pigments have led to new insights concerning photoreceptor function. The studies identify domains important for receptor folding, the residues that set the wavelength of absorption for the ligand 11-cis retinal, and residues, that when mutated, trigger the cell death of photoreceptors.     

Retinoid binding-proteins redirect retinoid metabolism: biosynthesis and metabolism of retinoic acid

Many tissues and cell types, starting early in embryogenesis, convert retinol (vitamin A) into an active form, all-trans-retinoic acid. This article will discuss a current model of retinol and retinoic acid metabolism that integrates the various reactions which maintain retinoic acid homeostasis, and will also integrate the enzymology with the functions of cellular retinoid binding proteins. These conserved, high-affinity binding proteins enjoy widespread expression throughout all vertebrates and throughout most vertebrate tissues. The binding proteins limit access to retinol and retinoic acid to select enzymes and serve as substrates and affecters of retinoid metabolism.     

Formation of vitamin A in a freshwater fish. Isolation of retinoic acid.

The intestines of freshly caught Saccobranchus fossilis (a freshwater fish that contains dehydroretinol) became free from carotenoids and from vitamin A when the fish were starved for about 20 days. When beta-carotene was administered to such fish, retinoic acid could be isolated from the intestines after approx. 4h. When lutein was administered to such fish, dehydroretinol and 3-hydroxyretinol could be isolated from the intestines after approx. 5h.     

Regulation of retinal dehydrogenases and retinoic acid synthesis by cholesterol metabolites

Retinoic acid (RA) constitutes the major active ingredient of vitamin A and is required for various biological processes. The tissue RA level is maintained through a cascade of metabolic reactions where retinal dehydrogenases (RALDHs) catalyze the terminal reaction of RA biosynthesis from retinal, a rate-limiting step. We showed that dietary supplement of cholesterol enhanced the expression of RALDH1 and 2 genes and the cellular RA content in vital organs such as brain, kidney, liver and heart. Consistently, the cholesterol-lowering agent (pravastatin sodium) downregulated the expression of RALDH1 and 2 genes in several organs especially the liver and in cultured liver cells. Further, cholesterol metabolites, predominantly the oxysterols, the natural ligands for liver X receptor (LXR), induced these genes via upregulation of sterol regulatory element binding protein-1c (SREBP-1c) that bound to the regulatory regions of these genes. Knockdown of LXRalpha/beta or SREBP-1c downregulated the expression of RALDH genes, which could be rescued by re-expressing SREBP-1c, suggesting SREBP-1c as a direct positive regulator for these genes. This study uncovered a novel crosstalk between cholesterol and RA biosynthesis.     

Effect of all-trans retinoic acid on the barrier function in human retinal pigment epithelial cells

To investigate the effects of all-trans retinoic acid (atRA) on the barrier function in human retinal pigment epithelial cells, ARPE-19 cells were cultured on the filters as monolayer with atRA being added in the apical side. The change of epithelial permeability was observed from the measurement of transepithelial electrical resistance (TER), permeability assay, and Western Blot analysis. We discovered that atRA promoted the epithelial barrier function in vitro, and its bioavailability regulates the epithelial barrier, which is accompanied by altering expression of tight junctions (TJ)-associated proteins. Our study indicates that atRA provides barrier-positive elements to the RPE cell.     

A role for retinoic acid in regulating the regeneration of deer antlers

Deer antlers are the only mammalian organs that can be repeatedly regenerated; each year, these complex structures are shed and then regrow to be used for display and fighting. To date, the molecular mechanisms controlling antler regeneration are not well understood. Vitamin A and its derivatives, retinoic acids, play important roles in embryonic skeletal development. Here, we provide several lines of evidence consistent with retinoids playing a functional role in controlling cellular differentiation during bone formation in the regenerating antler. Three receptors (alpha, beta, gamma) for both the retinoic acid receptor (RAR) and retinoid X receptor (RXR) families show distinct patterns of expression in the growing antler tip, the site of endochondral ossification. RAR alpha and RXR beta are expressed in skin ("velvet") and the underlying perichondrium. In cartilage, which is vascularised, RXR beta is specifically expressed in chondrocytes, which express type II collagen, and RAR alpha in perivascular cells, which also express type I collagen, a marker of the osteoblast phenotype. High-performance liquid chromatography analysis shows significant amounts of Vitamin A (retinol) in antler tissues at all stages of differentiation. The metabolites all-trans-RA and 4-oxo-RA are found in skin, perichondrium, cartilage, bone, and periosteum. The RXR ligand, 9-cis-RA, is found in perichondrium, mineralised cartilage, and bone. To further define sites of RA synthesis in antler, we immunolocalised retinaldehyde dehydrogenase type 2 (RALDH-2), a major retinoic acid-generating enzyme. RALDH-2 is expressed in the skin and perichondrium and in perivascular cells in cartilage, although chondroprogenitors and chondrocytes express very low levels. At sites of bone formation, differentiated osteoblasts which express the bone-specific protein osteocalcin express high levels of RALDH2. The effect of RA on antler cell differentiation was studied in vitro; all-trans-RA inhibits expression of the chondrocyte phenotype, an effect that is blocked by addition of the RAR antagonist Ro41-5253. In monolayer cultures of mesenchymal progenitor cells, all-trans-RA increases the expression of alkaline phosphatase, a marker of the osteoblastic phenotype. In summary, this study has shown that antler tissues contain endogenous retinoids, including 9-cis RA, and the enzyme RALDH2 that generates RA. Sites of RA synthesis in antler correspond closely with the localisation of cells which express receptors for these ligands and which respond to the effects of RA.     

Control of retinoic acid receptor heterodimerization by ligand-induced structural transitions. A novel mechanism of action for retinoid antagonists

Heterodimerization of retinoic acid receptors (RARs) with 9-cis-retinoic receptors (RXRs) is a prerequisite for binding of RXR.RAR dimers to DNA and for retinoic acid-induced gene regulation. Whether retinoids control RXR/RAR solution interaction remains a debated question, and we have used in vitro and in vivo protein interaction assays to investigate the role of ligand in modulating RXR/RAR interaction in the absence of DNA. Two-hybrid assay in mammalian cells demonstrated that only RAR agonists were able to increase significantly RAR interaction with RXR, whereas RAR antagonists inhibited RXR binding to RAR. Quantitative glutathione S-transferase pull-down assays established that there was a strict correlation between agonist binding affinity for the RAR monomer and the affinity of RXR for liganded RAR, but RAR antagonists were inactive in inducing RXR recruitment to RAR in vitro. Alteration of coactivator- or corepressor-binding interfaces of RXR or RAR did not alter ligand-enhanced dimerization. In contrast, preventing the formation of a stable holoreceptor structure upon agonist binding strongly altered RXR.RAR dimerization. Finally, we observed that RAR interaction with RXR silenced RXR ligand-dependent activation function. We propose that ligand-controlled dimerization of RAR with RXR is an important step in the RXR.RAR activation process. This interaction is dependent upon adequate remodeling of the AF-2 structure and amenable to pharmacological inhibition by structurally modified retinoids.     

Metabolism of retinoic acid in vivo in the vitamin A-deficient rat.

Sample preparation and high-pressure liquid-chromatography separation methods useful for the study of retinoic acid metabolism are reported. The sample preparation procedure does not cause significant degradation of retinoic acid, and the gradient high-pressure liquid-chromatography separation method gives excellent separation of the major metabolites of retinoic acid. These methods were used to examine the metabolites of retinoic acid in blood, trachea and lung, testes, kidneys and small intestine of vitamin A-deficient rats dosed subcutaneously with 2 micrograms of [11,12-3H] retinoic acid. At 6h after dosing, a total of eight metabolites of retinoic acid produced in vivo were found in the tissues examined. Of these, four were found in most of the epithelial tissues examined, and therefore may be of interest as possible active metabolites in the epithelial functions of vitamin A.