Association analysis of IL1A and IL1B variants in alopecia areata.

Alopecia areata is an inflammatory hair loss disease with a major genetic component. The disease is characterized by focal inflammatory lesions with perifollicular T-cell infiltrates, reflecting the role of local cytokine production in the development of patchy hair loss. IL-1 alpha and IL-1 beta are important inhibitors of hair growth in vitro. Their effect is opposed by the interleukin-1 receptor antagonist, IL-1ra. Genes of the IL-1 cluster are candidate genes in the pathogenesis of alopecia areata. To investigate the role of the IL-1 system in alopecia areata we examined three biallelic polymorphisms within the IL-1 gene cluster (IL1A+4845, IL1B+3954 and IL1B-511) in 165 patients and a large number of matched controls (n=1150). There was no significant association of IL1B-511 or IL1B+3954 genotypes with the overall dataset, or with disease severity or age at onset, in contrast with a previous report. The results suggested the possibility of an association with IL1A+4845 in the overall dataset [OR 1.39 (95% CI 1.00, 1.93)] although this was not statistically significant. This was due mainly to the contribution from mild cases of alopecia areata [OR 1.48 (0.96, 2.29)], suggesting that IL-1 alpha may have a particular role in the pathogenesis of this subgroup.

Cellular retinol-binding protein I is essential for vitamin A homeostasis.

The gene encoding cellular retinol (ROL, vitA)-binding protein type I (CRBPI) has been inactivated. Mutant mice fed a vitA-enriched diet are healthy and fertile. They do not present any of the congenital abnormalities related to retinoic acid (RA) deficiency, indicating that CRBPI is not indispensable for RA synthesis. However, CRBPI deficiency results in an approximately 50% reduction of retinyl ester (RE) accumulation in hepatic stellate cells. This reduction is due to a decreased synthesis and a 6-fold faster turnover, which are not related to changes in the levels of RE metabolizing enzymes, but probably reflect an impaired delivery of ROL to lecithin:retinol acyltransferase. CRBPI-null mice fed a vitA-deficient diet for 5 months fully exhaust their RE stores. Thus, CRBPI is indispensable for efficient RE synthesis and storage, and its absence results in a waste of ROL that is asymptomatic in vitA-sufficient animals, but leads to a severe syndrome of vitA deficiency in animals fed a vitA-deficient diet.

A retinol-binding protein receptor-mediated mechanism for uptake of vitamin A to postimplantation rat embryos.

The major form of dietary vitamin A in blood is retinol bound to retinol-binding protein (RBP), but it is not clear how maternal retinol reaches the embryo. We demonstrate here that uptake of radiolabeled retinol from holo-RBP to the visceral yolk sac of cultured rat embryos exhibits receptor-mediated characteristics (Km = 4.1 x 10(-7) M, Vmax = 3.0 x 10(-12) mol/sec per gram yolk sac protein) and also demonstrate the presence in the yolk sac endoderm of a protein previously described as an RBP receptor. Furthermore, transport of radiolabeled retinol from the yolk sac to the embryo proper can be markedly decreased by inhibition of visceral yolk sac RBP synthesis. Thus embryonic accumulation of retinol depends on a relay of retinol from maternal RBP to RBP synthesized in the yolk sac, via interaction with a visceral yolk sac RBP receptor.

Identification of a mechanism to localize generation of retinoic acid in rat embryos.

Vitamin A (retinol) is essential for normal mammalian development. However, its biological activity depends upon its conversion to retinoic acid (RA), a local mediator of cellular proliferation and differentiation. Previous studies have shown that embryonic RA is found specifically in tissues known to depend upon vitamin A for normal development and that its production follows uptake of maternal retinol. The aim of this study was to identify the mechanism for tissue-specific generation of RA in developing rat embryos. Here we show immunohistochemical localization of the retinol binding protein receptor, cellular retinol binding protein, retinol dehydrogenase and retinal dehydrogenase in rat embryos (presomitic to the 25-30 somite pair stage). These proteins are proposed to be responsible for cellular uptake of retinol, its intracellular transport and its conversion to RA. Thus, they potentially constitute the entire metabolic pathway from vitamin A to RA. All four proteins were detected specifically in tissues that are known to depend upon vitamin A for normal development including the yolk sac, heart, gut, notochord, somites, sensory placodes and the limb. Furthermore, our previous studies have demonstrated that uptake of retinol into the yolk sac depends upon a retinol binding protein receptor. Here we provide evidence that this mechanism functions also in the heart. Colocalization of cellular retinol binding protein, retinol and retinal dehydrogenase with the retinol binding protein receptor in tissues dependent upon vitamin A for normal development suggests that coordinate functioning of these proteins is responsible for cellular uptake of circulating retinol and its metabolism to RA. This is the first evidence of a tissue-specific mechanism for generation of RA from its precursor retinol in the developing embryo.

Developmental abnormalities in cultured mouse embryos deprived of retinoic by inhibition of yolk-sac retinol binding protein synthesis.

Presomitic and 3- to 12-somite pair cultured mouse embryos were deprived of retinoic acid (RA) by yolk-sac injections of antisense oligodeoxynucleotides for retinol binding protein (RBP). Inhibition of yolk-sac RBP synthesis was verified by immunohistochemistry, and the loss of activity of a lacZ-coupled RA-sensitive promoter demonstrated that embryos rapidly became RA-deficient. This deficiency resulted in malformations of the vitelline vessels, cranial neural tube, and eye, depending upon the stage of embryonic development at the time of antisense injection. Addition of RA to the culture medium at the time of antisense injection restored normal development implicating the role of RBP in embryonic RA synthesis. Furthermore, the induced RA deficiency resulted in early down-regulation of developmentally important genes including TGF-beta1 and Shh.

Epitope mapping of a monoclonal antibody that blocks the binding of retinol-binding protein to its receptor.

To define the receptor binding site of retinol-binding protein (RBP) we have generated monoclonal antibodies (mAbs) to human RBP and examined their ability to interfere with the receptor binding. MAbs to two conserved regions efficiently blocked the binding. No major conformational changes in the protein occurred upon mAb binding, since the mAbs could co-immunoprecipitate the RBP-transthyretin (TTR) complex. One blocking mAb showed reactivity to a synthetic peptide corresponding to one entrance loop of the retinol-binding pocket (amino acid residues 60-70). Thus, our results show that at least one of the entrance loops of the barrel of RBP is located in or close to the receptor binding site. It can also be concluded that the receptor and TTR binding sites involve different regions of RBP.

Retinol-binding protein mediates uptake of retinol to cultured human keratinocytes.

Retinol (vitamin A) circulates in the blood bound to retinol-binding protein (RBP). The process by which target cells acquire retinol is not fully elucidated, although a cell surface receptor for RBP has recently been identified. We show here that retinol is at least an order of magnitude more efficient at blocking the terminal differentiation of cultured normal human keratinocytes when administered as a complex with RBP than when administered free in solution. This inhibition of differentiation by RBP can be reversed by monoclonal antibody P142, reactive toward the RBP-binding membrane protein p63. These results demonstrate, at least in this in vitro system, the importance of the RBP receptor in the generation of a cellular response to retinol.

The retinal pigment epithelial membrane receptor for plasma retinol-binding protein. Isolation and cDNA cloning of the 63-kDa protein.

Retinol, a metabolic precursor of retinal and retinoic acid, is transported in plasma by the plasma retinol-binding protein (RBP). The cellular uptake of retinol from RBP is believed to involve a specific membrane receptor for RBP. In retinal pigment epithelium the RBP receptor appears to be an oligomeric protein complex, and we have previously identified a 63-kDa membrane protein as part of this receptor. The 63-kDa protein (p63) has now been isolated, and we have cloned the corresponding cDNA. In a data base search no sequences similar to p63 were identified. Hydropathy analyses of the 533 amino acids deduced from the cDNA sequence did not indicate an N-terminal signal sequence or obvious transmembrane regions. In vitro translation of synthetic mRNA encoding p63, in the presence of heterologous microsomes, verified that p63 does not become cotranslationally membrane-inserted. Transcripts for p63 are abundantly expressed in retinal pigment epithelium with no detectable expression in several other tissues. Southern blotting analysis of bovine and human genomic DNA revealed several hybridizing fragments suggesting a complex organization of the corresponding genes.

Characterization of a plasma retinol-binding protein membrane receptor expressed in the retinal pigment epithelium.

A specific membrane receptor for plasma retinol-binding protein (RBP) is expressed in the retinal pigment epithelium (RPE). When chemically cross-linking RBP to RPE membranes, an 86-kDa RBP.RBP receptor complex is formed, and a 63-kDa protein was identified as the RBP-binding membrane protein (Båvik, C.-O., Eriksson, U., Allen, R., and Peterson, P. (1991) J. Biol. Chem. 266, 14978-14985). To explore in more detail the characteristics of this membrane receptor, we have generated a monoclonal antibody, A52, to the 63-kDa protein (p63). A52 binds the 86-kDa RBP.RBP receptor complex and p63. Several lines of evidence suggest that p63 is not a regular integral membrane protein, and it occurs in different forms. One form is firmly attached to membranes, is part of a high molecular weight complex, and is able to bind RBP. The other form of p63 can be removed from membranes by treatment with an alkaline buffer and is unable to bind RBP. Both forms of p63 contain extensive hydrophobic domains and are found in the detergent phase upon extraction with Triton X-114. The expression of p63 is restricted to RPE, and immunohistochemical localization of tissue sections from bovine retina showed highest expression in the basolateral portion of RPE cells. Immunofluorescence localization, using isolated RPE cells, showed that p63 is exposed on the cell surface of newly isolated RPE cells.

Identification and partial characterization of a retinal pigment epithelial membrane receptor for plasma retinol-binding protein.

We have developed a membrane binding assay by which we have been able to characterize the interaction between 125I-labeled retinol-binding protein and its receptor in microsome fractions derived from retinal pigment epithelial cells. The binding of retinol-binding protein to the membranes was fast, with a dissociation constant in the range of 31-72 nM, and maximum binding occurred at neutral pH. Receptor binding sites were also found in microsome fractions of liver and kidney, whereas lung and muscle contained few, if any. Chemical cross-linking of retinol-binding protein to the microsomal membranes yielded a major molecular complex of Mr 86,000 upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The protein responsible for binding of retinol-binding protein was identified as a Mr 63,000 protein using a label transfer cross-linking technique. Further characterization demonstrated that the receptor for retinol-binding protein is a terminally glycosylated membrane protein noncovalently associated with a high molecular weight complex.

Characterization of the rat retinol-binding protein gene and its comparison to the three-dimensional structure of the protein.

Rat genomic DNA fragments bearing the retinol-binding protein (RBP) gene have been isolated and characterized. The gene spans 6.9 kilobases and contains six exons. The five intervening sequences range in size from 78 base pairs to 4.4 kilobase pairs with the first interrupting the 5' untranslated region. A comparison of the gene organization with the three-dimensional structure of RBP reveals that all translated exon transcripts closely correspond to discrete tertiary structural elements. Residues of the protein involved in the retinol binding are encoded by three separate exons. It has been proposed that the two regions displaying internal homology in the human RBP, both at the primary and tertiary structure levels, arose by a partial ancestral gene duplication. If such an event were involved, evidence for it at the nucleotide sequence and exon-intron organization levels has been obscured.

Cellular retinoid binding proteins.

The cellular retinol-binding protein (CRBP) and the cellular retinoic acid binding protein (CRABP) have similar physicochemical characteristics. The amino acid sequences of rat CRBP and bovine CRABP have been elucidated and they display 40% sequence identity. Both protein sequences appear to be evolutionarily highly conserved. The amino acid sequence of human CRBP, deduced from a cDNA-clone, is 96% identical to the rat CRBP sequence. CRBP and CRABP are members of a protein family, all members of which may bind hydrophobic ligands and interact with membrane components. All members of the protein family are probably related in tertiary structure and might interact with membrane components through two regions with a high probability for alpha-helix. The tissue distribution of CRBP and CRABP, together with their relation to lipid transporting proteins suggests that CRBP and CRABP are cellular transporting proteins for retinol and retinoic acid, respectively.

Cloning and sequencing of a full length cDNA corresponding to human cellular retinol-binding protein.

We have isolated and sequenced a cDNA clone corresponding to the human cellular retinol-binding protein (CRBP). The deduced amino acid sequence, which encompasses 134 amino acid residues, shows significant homology with several low molecular weight proteins which bind hydrophobic ligands. No homology to the plasma retinol-binding protein was observed. Southern and Northern blot analyses suggest that the CRBP gene is present in a single copy in the haploid genome and that it is transcribed in a single mRNA species.