Protein Kinase C Alpha (PKCα) overexpression leads to a better response to retinoid acid therapy through Retinoic Acid Receptor Beta (RARß) activation in mammary cancer cells.

PURPOSE: Retinoids have proved to be effective for hematologic malignancies treatment but till nowadays, their use as single agent for the solid tumor's management is still controversial. All-trans retinoic acid (ATRA), the main active metabolite of vitamin A, exerts non-genomic interactions with different members of the protein kinase C (PKC) family, recognized modulators of different tumor progression pathways. To determine whether a group of patients could become benefited employing a retinoid therapy, in this study we have evaluated whether PKCα expression (a poor prognosis marker in breast cancer) could sensitizes mammary cells to ATRA treatment. METHODS: PKCα overexpression was achieved by stable transfection and confirmed by western blot. Transfected PKC functionality was determined by nuclear translocation-induction and confocal microscopy. In vitro proliferation was evaluated by cell counting and cell cycle distribution was analyzed by flow cytometry. In vivo studies were performed to evaluate orthotopic tumor growth and experimental lung colonization. Retinoic acid response elements (RARE) and AP1 sites-dependent activity was studied by gene reporter assays and retinoic acid receptors (RARs) were measured by RT-qPCR. RESULTS: Our findings suggest that high PKCα levels improve the differentiation response to ATRA in a RAR signaling-dependent manner. Moreover, RARß expression appears to be critical to induce ATRA sensitization, throughout AP1 trans-repression. CONCLUSION: Here we propose that retinoids could lead a highly personalized anticancer treatment, bringing benefits to patients with aggressive breast tumors resulting from high PKCα expression but, an adequate expression of the RARß receptor is required to ensure the effect on this process.

Consequences of Vitamin A Deficiency: Immunoglobulin Dysregulation, Squamous Cell Metaplasia, Infectious Disease, and Death.

Vitamin A is an important regulator of immune protection, but it is often overlooked in studies of infectious disease. Vitamin A binds an array of nuclear receptors (e.g., retinoic acid receptor, peroxisome proliferator-activated receptor, retinoid X receptor) and influences the barrier and immune cells responsible for pathogen control. Children and adults in developed and developing countries are often vitamin A-deficient or insufficient, characteristics associated with poor health outcomes. To gain a better understanding of the protective mechanisms influenced by vitamin A, we examined immune factors and epithelial barriers in vitamin A deficient (VAD) mice, vitamin D deficient (VDD) mice, double deficient (VAD+VDD) mice, and mice on a vitamin-replete diet (controls). Some mice received insults, including intraperitoneal injections with complete and incomplete Freund's adjuvant (emulsified with PBS alone or with DNA + Fus-1 peptide) or intranasal inoculations with Sendai virus (SeV). Both before and after insults, the VAD and VAD+VDD mice exhibited abnormal serum immunoglobulin isotypes (e.g., elevated IgG2b levels, particularly in males) and cytokine/chemokine patterns (e.g., elevated eotaxin). Even without insult, when the VAD and VAD+VDD mice reached 3-6 months of age, they frequently exhibited opportunistic ascending bacterial urinary tract infections. There were high frequencies of nephropathy (squamous cell hyperplasia of the renal urothelium, renal scarring, and ascending pyelonephritis) and death in the VAD and VAD+VDD mice. When younger VAD mice were infected with SeV, the predominant lesion was squamous cell metaplasia of respiratory epithelium in lungs and bronchioles. Results highlight a critical role for vitamin A in the maintenance of healthy immune responses, epithelial cell integrity, and pathogen control.

Nuclear Receptors, Ligands and the Mammalian B Cell.

Questions concerning the influences of nuclear receptors and their ligands on mammalian B cells are vast in number. Here, we briefly review the effects of nuclear receptor ligands, including estrogen and vitamins, on immunoglobulin production and protection from infectious diseases. We describe nuclear receptor interactions with the B cell genome and the potential mechanisms of gene regulation. Attention to the nuclear receptor/ligand regulation of B cell function may help optimize B cell responses, improve pathogen clearance, and prevent damaging responses toward inert- and self-antigens.

Regulatory mechanism for the transmembrane receptor that mediates bidirectional vitamin A transport.

Vitamin A has diverse biological functions and is essential for human survival at every point from embryogenesis to adulthood. Vitamin A and its derivatives have been used to treat human diseases including vision diseases, skin diseases, and cancer. Both insufficient and excessive vitamin A uptake are detrimental, but how its transport is regulated is poorly understood. STRA6 is a multitransmembrane domain cell-surface receptor and mediates vitamin A uptake from plasma retinol binding protein (RBP). STRA6 can mediate both cellular vitamin A influx and efflux, but what regulates these opposing activities is unknown. To answer this question, we purified and identified STRA6-associated proteins in a native mammalian cell type that takes up vitamin A through STRA6 using mass spectrometry. We found that the major protein repeatedly identified as STRA6-associated protein is calmodulin, consistent with the cryogenic electron microscopy (cryo-EM) study of zebrafish STRA6 associated with calmodulin. Using radioactivity-based, high-performance liquid chromatography (HPLC)-based and real-time fluorescence techniques, we found that calmodulin profoundly affects STRA6's vitamin A transport activity. Increased calcium/calmodulin promotes cellular vitamin A efflux and suppresses vitamin A influx through STRA6. Further mechanistic studies revealed that calmodulin enhances the binding of apo-RBP to STRA6, and this enhancement is much more pronounced for apo-RBP than holo-RBP. This study revealed that calmodulin regulates STRA6's vitamin A influx or efflux activity by modulating its preferential interaction with apo-RBP or holo-RBP. This molecular mechanism of regulating vitamin A transport may point to new directions to treat human diseases associated with insufficient or excessive vitamin A uptake.

Role of Vitamins A and D in BCR-ABL Arf-/- Acute Lymphoblastic Leukemia.

The effects of vitamin A and/or vitamin D deficiency were studied in an Arf-/- BCR-ABL acute lymphoblastic leukemia murine model. Vitamin D sufficient mice died earlier (p = 0.003) compared to vitamin D deficient (VDD) mice. Vitamin A deficient (VAD) mice fared worst with more rapid disease progression and decreased survival. Mice deficient for vitamins A and D (VADD) had disease progression similar to VAD mice. Regulatory T cells, previously shown to associate with poor BCR-ABL leukemia control, were present at higher frequencies among CD4+ splenocytes of vitamin A deficient vs. sufficient mice. In vitro studies demonstrated 1,25-dihydroxyvitamin D (1,25(OH)2VD3) increased the number of BCR-ABL ALL cells only when co-cultured with bone marrow stroma. 1,25(OH)2VD3 induced CXCL12 expression in vivo and in vitro in stromal cells and CXCL12 increased stromal migration and the number of BCR-ABL blasts. Vitamin D plus leukemia reprogrammed the marrow increasing production of collagens, potentially trapping ALL blasts. Vitamin A (all trans retinoic acid, ATRA) treated leukemic cells had increased apoptosis, decreased cells in S-phase, and increased cells in G0/G1. ATRA signaled through the retinoid X receptor to decrease BCR-ABL leukemic cell viability. In conclusion, vitamin A and D deficiencies have opposing effects on mouse survival from BCR-ABL ALL.

The mitochondrial PKCδ/retinol signal complex exerts real-time control on energy homeostasis.

The review focuses on the role of vitamin A (retinol) in the control of energy homeostasis, and on the manner in which certain retinoids subvert this process, leading potentially to disease. In eukaryotic cells, the pyruvate dehydrogenase complex (PDHC) is negatively regulated by four pyruvate dehydrogenase kinases (PDKs) and two antagonistically acting pyruvate dehydrogenase phosphatases (PDPs). The second isoform, PDK2, is regulated by an autonomous mitochondrial signal cascade that is anchored on protein kinase Cδ (PKCδ), where retinoids play an indispensible co-factor role. Along with its companion proteins p66Shc, cytochrome c, and vitamin A, the PKCδ/retinol complex is located in the intermembrane space of mitochondria. At this site, and in contrast to cytosolic locations, PKCδ is activated by the site-specific oxidation of its cysteine-rich activation domain (CRD) that is configured into a complex RING-finger. Oxidation involves the transfer of electrons from cysteine moieties to oxidized cytochrome c, a step catalyzed by vitamin A. The PKCδ/retinol signalosome monitors the internal cytochrome c redox state that reflects the workload of the respiratory chain. Upon sensing demands for energy PKCδ signals the PDHC to increase glucose-derived fuel flux entering the KREBS cycle. Conversely, if excessive fuel flux surpasses the capacity of the respiratory chain, threatening the release of damaging reactive oxygen species (ROS), the polarity of the cytochrome c redox system is reversed, resulting in the chemical reduction of the PKCδ CRD, restoration of the RING-finger, refolding of PKCδ into the inactive, globular form, and curtailment of PDHC output, thereby constraining the respiratory capacity within safe margins. Several retinoids, notably anhydroretinol and fenretinide, capable of displacing retinol from binding sites on PKCδ, can co-activate PKCδ signaling but, owing to their extended system of conjugated double bonds, are unable to silence PKCδ in a timely manner. Left in the ON position, PKCδ causes chronic overload of the respiratory chain leading to mitochondrial dysfunction. This review explores how defects in the PKCδ signal machinery potentially contribute to metabolic and degenerative diseases.

Strategies to achieve adequate vitamin A intake for young children: options for Cameroon.

Meeting children's vitamin A (VA) needs remains a policy priority. Doing so efficiently is a fiscal imperative and protecting at-risk children during policy transitions is a moral imperative. Using the Micronutrient Intervention Modeling tool and data for Cameroon, we predict the impacts and costs of alternative VA intervention programs, identify the least-cost strategy for meeting targets nationally, and compare it to a business-as-usual (BAU) strategy over 10 years. BAU programs effectively cover ∼12.8 million (m) child-years (CY) and cost ∼$30.1 m; ∼US$2.34 per CY effectively covered. Improving the VA-fortified oil program, implementing a VA-fortified bouillon cube program, and periodic VA supplements (VAS) in the North macroregion for 3 years effectively cover ∼13.1 m CY at a cost of ∼US$9.5 m, or ∼US$0.71 per CY effectively covered. The tool then identifies a sequence of subnational policy choices leading from the BAU toward the more efficient strategy, while addressing VA-attributable mortality concerns. By year 4, fortification programs are predicted to eliminate inadequate VA intake in the South and Cities macroregions, but not the North, where VAS should continue until additional delivery platforms are implemented. This modeling approach offers a concrete example of the strategic use of data to follow the Global Alliance for VA framework and do so efficiently.

Investigation of miR-136-5p key target genes and pathways in lung squamous cell cancer based on TCGA database and bioinformatics analysis.

BACKGROUND: Lung squamous cell cancer (LUSC) is a common but challenging malignancy. It is important to illuminate the molecular mechanism of LUSC. Thus, we aim to explore the molecular mechanism of miR-136-5p in relation to LUSC. METHODS: We used the Cancer Genome Atlas (TCGA) database to investigate the expression of miR-136-5p in relation to LUSC. Then, we identified the possible miR-136-5p target genes through intersection of the predicted miR-136-5p target genes and LUSC upregulated genes from TCGA. Bioinformatics analysis was performed to determine the key miR-136-5p targets and pathways associated with LUSC. Finally, the expression of hub genes, correlation between miR-136-5p and hub genes, and expected significance of hub genes were evaluated via the TCGA and Genotype-Tissue Expression (GTEx) project. RESULTS: MiR-136-5p was significantly downregulated in LUSC patients. Glucuronidation, glucuronosyltransferase, and the retinoic acid metabolic process were the most enriched metabolic interactions in LUSC patients. Ascorbate and aldarate metabolism, pentose and glucuronate interconversions, and retinol metabolism were identified as crucial pathways. Seven hub genes (UGT1A1, UGT1A3, UGT1A6, UGT1A7, UGT1A10, SRD5A1, and ADH7) were found to be upregulated, and UGT1A1, UGT1A3, UGT1A6, UGT1A7, and ADH7 were negatively correlated with miR-136-5p. UGT1A7 and ADH7 were the most significantly involved miR-136-5p target genes, and high expression of these genes was correlated with better overall survival and disease-free survival of LUSC patients. CONCLUSIONS: Downregulated miR-136-5p may target UGT1A7 and ADH7 and participate in ascorbate and aldarate metabolism, pentose and glucuronate interconversions, and retinol metabolism. High expression of UGT1A7 and ADH7 may indicate better prognosis of LUSC patients.

Vitamin A and the epigenome.

The epigenetic phenomena refer to heritable changes in gene expression other than those in the DNA sequence, such as DNA methylation and histone modifications. Major research progress in the last few years has provided further proof that environmental factors, including diet and nutrition, can influence physiologic and pathologic processes through epigenetic alterations, which in turn influence gene expression. This influence is termed nutritional epigenetics, and one prominent example is the regulation of gene transcription by vitamin A through interaction to its nuclear receptor. Vitamin A is critical throughout life. Together with its derivatives, it regulates diverse processes including reproduction, embryogenesis, vision, growth, cellular differentiation and proliferation, maintenance of epithelial cellular integrity and immune function. Here we review the epigenetic role of vitamin A in cancer, stem cells differentiation, proliferation, and immunity. The data presented here show that retinoic acid is a potent agent capable of inducing alterations in epigenetic modifications that produce various effects on the phenotype. Medical benefits of vitamin A as an epigenetic modulator, especially with respect to its chronic use as nutritional supplement, should rely on our further understanding of its epigenetic effects during health and disease, as well as through different generations.

Oxidized frying oil and its polar fraction fed to pregnant mice are teratogenic and alter mRNA expressions of vitamin A metabolism genes in the liver of dams and their fetuses.

We previously observed a higher incidence of congenital malformations in the fetuses of dams fed an oxidized frying oil (OFO)-containing diet during pregnancy. In this study, we hypothesized that, during pregnancy, maternal ingestion of OFO, specifically the oxidized components (i.e. the polar fraction), modulates peroxisome proliferator-activated receptor (PPARα) or aryl hydrocarbon receptor (AhR) transactivity, altering the metabolism of retinoic acid (RA), a well-characterized morphogen, resulting in teratogenesis. Pregnant C57BL/6J mice were divided into four groups which, from d1 (conception) to d18, were fed a diet containing 10 g/100 g of fresh soybean oil (SO), OFO or the non-polar (NP) or polar (PO) fraction of OFO. Reporter assays testing the transactivity of PPARα and AhR showed that free fatty acids from OFO, specifically the PO fraction, up-regulated PPARα transactivity and down-regulated AhR transactivity. In vivo study showed that the PO fraction group had a significantly higher number of dead fetuses and resorptions per litter than the SO and NP fraction groups. The incidence of abnormalities in terms of gross morphology and skeletal ossification of the fetus was greatest in the PO fraction group, followed by the OFO group, both values being significantly higher than in the other two groups. Hepatic expression of genes encoding enzymes associated with RA synthesis and catabolism in dams and fetuses was differentially affected by PO fraction assault. We conclude that OFO-mediated teratogenesis is associated with disturbed RA metabolism in the dams and fetuses caused, at least in part, by modulation of PPARα and AhR transactivity by the oxidized components in OFO.

Two carotenoid oxygenases contribute to mammalian provitamin A metabolism.

Mammalian genomes encode two provitamin A-converting enzymes as follows: the ß-carotene-15,15'-oxygenase (BCO1) and the ß-carotene-9',10'-oxygenase (BCO2). Symmetric cleavage by BCO1 yields retinoids (ß-15'-apocarotenoids, C20), whereas eccentric cleavage by BCO2 produces long-chain (>C20) apocarotenoids. Here, we used genetic and biochemical approaches to clarify the contribution of these enzymes to provitamin A metabolism. We subjected wild type, Bco1(-/-), Bco2(-/-), and Bco1(-/-)Bco2(-/-) double knock-out mice to a controlled diet providing ß-carotene as the sole source for apocarotenoid production. This study revealed that BCO1 is critical for retinoid homeostasis. Genetic disruption of BCO1 resulted in ß-carotene accumulation and vitamin A deficiency accompanied by a BCO2-dependent production of minor amounts of ß-apo-10'-carotenol (APO10ol). We found that APO10ol can be esterified and transported by the same proteins as vitamin A but with a lower affinity and slower reaction kinetics. In wild type mice, APO10ol was converted to retinoids by BCO1. We also show that a stepwise cleavage by BCO2 and BCO1 with APO10ol as an intermediate could provide a mechanism to tailor asymmetric carotenoids such as ß-cryptoxanthin for vitamin A production. In conclusion, our study provides evidence that mammals employ both carotenoid oxygenases to synthesize retinoids from provitamin A carotenoids.

[Expression of genes involved in retinoic acid biosynthesis in human gastric cancer].

All-trans-retinoic acid (ATRA) is the main biologically active metabolite of retinol (vitamin A) that is required for the regulation of such processes as embryogenesis, tissue differentiation, proliferation, and others. Multiple alcohol, retinol and retinaldehyde dehydrogenases (ADHs, RDHs and RALDHs) as well as aldo-keto reductases (AKRs) catalyze the biosynthesis of retinoic acid in humans. For many normal and neoplastic tissues, the key ATRA-synthesizing enzymes remain unknown. We identified ATRA-generating genes that are expressed in normal and malignant gastric tissues using the transcriptomic database analysis. Quantitative changes in the expression levels of these genes in gastric cancer were determined by semi-quantitative RT-PCR and real-time PCR. Significant decreases in the mRNA levels of genes encoding enzymes that catalyze the reversible oxidation/reduction of retinol and retinaldehyde (ADH4, ADH1B, ADH1C, RDHL, AKR1B10, AKR1B1, and RDH12), as well as the oxidation of retinaldehyde (RALDH1) were revealed in most of the tumor samples. The sharp reduction in the expression levels of genes encoding the key enzymes that convert retinol and retinaldehyde to retinoic acid could lead to a significant decrease in the content of ATRA--the transcriptional regulator of many genes, which in turn can lead to a dysregulation of cell proliferation/differentiation and initiate cancer development.

Plasma carotenoid- and retinol-weighted multi-SNP scores and risk of breast cancer in the National Cancer Institute Breast and Prostate Cancer Cohort Consortium.

BACKGROUND: Dietary and circulating carotenoids have been inversely associated with breast cancer risk, but observed associations may be due to confounding. Single-nucleotide polymorphisms (SNPs) in ß-carotene 15,15'-monooxygenase 1 (BCMO1), a gene encoding the enzyme involved in the first step of synthesizing vitamin A from dietary carotenoids, have been associated with circulating carotenoid concentrations and may serve as unconfounded surrogates for those biomarkers. We determined associations between variants in BCMO1 and breast cancer risk in a large cohort consortium. METHODS: We used unconditional logistic regression to test four SNPs in BCMO1 for associations with breast cancer risk in 9,226 cases and 10,420 controls from the National Cancer Institute Breast and Prostate Cancer Cohort Consortium (BPC3). We also tested weighted multi-SNP scores composed of the two SNPs with strong, confirmed associations with circulating carotenoid concentrations. RESULTS: Neither the individual SNPs nor the weighted multi-SNP scores were associated with breast cancer risk [OR (95% confidence interval) comparing extreme quintiles of weighted multi-SNP scores = 1.04 (0.94-1.16) for ß-carotene, 1.08 (0.98-1.20) for α-carotene, 1.04 (0.94-1.16) for ß-cryptoxanthin, 0.95 (0.87-1.05) for lutein/zeaxanthin, and 0.92 (0.83-1.02) for retinol]. Furthermore, no associations were observed when stratifying by estrogen receptor status, but power was limited. CONCLUSIONS: Our results do not support an association between SNPs associated with circulating carotenoid concentrations and breast cancer risk. IMPACT: Future studies will need additional genetic surrogates and/or sample sizes at least three times larger to contribute evidence of a causal link between carotenoids and breast cancer.

Application of a novel hybrid study design to explore gene-environment interactions in orofacial clefts.

Orofacial clefts are common birth defects with strong evidence for both genetic and environmental causal factors. Candidate gene studies combined with exposures known to influence the outcome provide a highly targeted approach to detecting GxE interactions. We developed a new statistical approach that combines the case-control and offspring-parent triad designs into a "hybrid design" to search for GxE interactions among 334 autosomal cleft candidate genes and maternal first-trimester exposure to smoking, alcohol, coffee, folic acid supplements, dietary folate and vitamin A. The study population comprised 425 case-parent triads of isolated clefts and 562 control-parent triads derived from a nationwide study of orofacial clefts in Norway (1996-2001). A full maximum-likelihood model was used in combination with a Wald test statistic to screen for statistically significant GxE interaction between strata of exposed and unexposed mothers. In addition, we performed pathway-based analyses on 28 detoxification genes and 21 genes involved in folic acid metabolism. With the possible exception of the T-box 4 gene (TBX4) and dietary folate interaction in isolated CPO, there was little evidence overall of GxE interaction in our data. This study is the largest to date aimed at detecting interactions between orofacial clefts candidate genes and well-established risk exposures.

Comparative endocrine disruptive effects of contaminants in ringed seals (Phoca hispida) from Svalbard and the Baltic Sea.

We investigated variables related to thyroid, vitamin A and calcitriol homeostasis, immune function and tumour development in ringed seals (Phoca hispida) from the polluted Baltic Sea and a less polluted reference location at Svalbard, Norway. We also examined the relationships between the biological variables and the concentrations of persistent organic pollutants (POPs) and their hydroxylated (OH) metabolites. Our data show higher plasma concentrations of free triiodothyronine (T3), and ratios of free and total T3 in Baltic seals as compared to Svalbard seals. Baltic seals had also higher hepatic mRNA expressions of deiodinase-I, thyroid hormone receptor beta, retinoic acid receptor alpha, growth hormone receptor and interleukin-1beta compared to Svalbard seals. Levels of plasma retinol were lower in the Baltic seals as compared to Svalbard seals. No geographical difference was observed for other thyroid hormone levels and hepatic retinoid levels. Ratios of free and total T3 were positively correlated to OH-POPs in plasma. The results of the present study suggest that endocrine homeostasis may be affected by contaminant and metabolite exposure in the Baltic ringed seals with respect to circulating hormones and retinol and hepatic mRNA expressions. In addition, OH-POPs may putatively produce the disruption of thyroid hormone transport in plasma.

A human ALDH1A2 gene variant is associated with increased newborn kidney size and serum retinoic acid.

Nephron number varies widely between 0.3 and 1.3 million per kidney in humans. During fetal life, the rate of nephrogenesis is influenced by local retinoic acid (RA) level such that even moderate maternal vitamin A deficiency limits the final nephron number in rodents. Inactivation of genes in the RA pathway causes renal agenesis in mice; however, the impact of retinoids on human kidney development is unknown. To resolve this, we tested for associations between variants of genes involved in RA metabolism (ALDH1A2, CYP26A1, and CYP26B1) and kidney size among normal newborns. Homozygosity for a common (1 in 5) variant, rs7169289(G), within an Sp1 transcription factor motif of the ALDH1A2 gene, showed a significant 22% increase in newborn kidney volume when adjusted for body surface area. Infants bearing this allele had higher umbilical cord blood RA levels compared to those with homozygous wild-type ALDH1A2 rs7169289(A) alleles. Furthermore, the effect of the rs7169289(G) variant was evident in subgroups with or without a previously reported hypomorphic RET 1476(A) proto-oncogene allele that is critical in determining final nephron number. As maternal vitamin A deficiency is widespread in developing countries and may compromise availability of retinol for fetal RA synthesis, our study suggests that the ALDH1A2 rs7169289(G) variant might be protective for such individuals.

An essential set of basic DNA response elements is required for receptor-dependent transcription of the lecithin:retinol acyltransferase (Lrat) gene.

Lecithin:retinol acyltransferase (LRAT) is essential for vitamin A storage. Nuclear run-on assays demonstrated transcriptional regulation of the Lrat gene in vivo by all-trans-retinoic acid (RA) and other retinoids. Analysis of a 2.5 kb segment of rat genomic DNA revealed that the region approximately 300 bp upstream from the transcription start site (TSS) is necessary for high luciferase (Luc) reporter activity in HEK293T and HepG2 cells. Although this region lacks retinoid receptor binding elements, it responded to the nuclear receptors RARalpha, RARbeta or RARgamma, with RXRalpha, with and without ligand. Removal of -111 bp from the TSS, which is well conserved in human, rat and mouse genomes, completely eliminated activity. This region contains several basic elements (TATA box, SP3 site, AP-1 site, CAAT box), all of which were essential. Nuclear extracts from RA-treated cells exhibited enhanced binding. Therefore, this proximal region together with basal transcription factors may be sufficient to drive Lrat expression.

Disruption of ceruloplasmin and hephaestin in mice causes retinal iron overload and retinal degeneration with features of age-related macular degeneration.

Mechanisms of brain and retinal iron homeostasis have become subjects of increased interest after the discovery of elevated iron levels in brains of patients with Alzheimer's disease and retinas of patients with age-related macular degeneration. To determine whether the ferroxidase ceruloplasmin (Cp) and its homolog hephaestin (Heph) are important for retinal iron homeostasis, we studied retinas from mice deficient in Cp and/or Heph. In normal mice, Cp and Heph localize to Müller glia and retinal pigment epithelium, a blood-brain barrier. Mice deficient in both Cp and Heph, but not each individually, had a striking, age-dependent increase in retinal pigment epithelium and retinal iron. The iron storage protein ferritin was also increased in Cp-/-Heph-/Y retinas. After retinal iron levels had increased, Cp-/-Heph-/Y mice had age-dependent retinal pigment epithelium hypertrophy, hyperplasia and death, photoreceptor degeneration, and subretinal neovascularization, providing a model of some features of the human retinal diseases aceruloplasminemia and age-related macular degeneration. This pathology indicates that Cp and Heph are critical for CNS iron homeostasis and that loss of Cp and Heph in the mouse leads to age-dependent retinal neurodegeneration, providing a model that can be used to test the therapeutic efficacy of iron chelators and antiangiogenic agents.

Vitamin A controls epithelial/mesenchymal interactions through Ret expression.

Mutations or rearrangements in the gene encoding the receptor tyrosine kinase RET result in Hirschsprung disease, cancer and renal malformations. The standard model of renal development involves reciprocal signaling between the ureteric bud epithelium, inducing metanephric mesenchyme to differentiate into nephrons, and metanephric mesenchyme, inducing the ureteric bud to grow and branch. RET and GDNF (a RET ligand) are essential mediators of these epithelial-mesenchymal interactions. Vitamin A deficiency has been associated with widespread embryonic abnormalities, including renal malformations. The vitamin A signal is transduced by nuclear retinoic acid receptors (RARs). We previously showed that two RAR genes, Rara and Rarb2, were colocalized in stromal mesenchyme, a third renal cell type, where their deletion led to altered stromal cell patterning, impaired ureteric bud growth and downregulation of Ret in the ureteric bud. Here we demonstrate that forced expression of Ret in mice deficient for both Rara and Rarb2 (Rara(-/-)Rarb2(-/-)) genetically rescues renal development, restoring ureteric bud growth and stromal cell patterning. Our studies indicate the presence of a new reciprocal signaling loop between the ureteric bud epithelium and the stromal mesenchyme, dependent on Ret and vitamin A. In the first part of the loop, vitamin-A-dependent signals secreted by stromal cells control Ret expression in the ureteric bud. In the second part of the loop, ureteric bud signals dependent on Ret control stromal cell patterning.

Isolation and characterization of nucleolin gene as one of the vitamin A-responsive genes in airway epithelium by a palindromic primer-based mRNA differential display method.

A palindromic primer-based mRNA differential display method has been used to isolate various vitamin A-responsive genes from primary cultures of monkey tracheobronchial epithelial cells. This method, as compared with the original mRNA differential display (mDD) method described by Liang and Pardee, used only one arbitrarily designed primer instead of two in the polymerase chain reaction. The single-primer mDD method has several advantages over the two-primer mDD system, especially in the reamplification and the selection of 5'-end cDNA clone. To verify the usefulness of this approach, one of these differential display bands, M34, was initially chosen for further amplification and cloning. The clone derived from the M34 band has a DNA sequence with > 90% homology to the human nucleolin gene. Furthermore, DNA sequencing confirms that both 5' and 3' ends of the insert of M34 contain the invertly repetitive nucleotide sequence that was used to direct this cloning. Nucleolin is a multifunctional phosphoprotein that plays an important role in ribosome biogenesis and mRNA stability. Northern blot analysis demonstrated that in addition to the elevation by vitamin A, the level of nucleolin message is significantly higher in fetal than in adult tracheobronchial epithelial cultures. Furthermore, in situ hybridization demonstrated that the amount of nucleolin message is significantly higher in both basal and ciliated cell types than in mucous and intermediary cell types. These results support the feasibility that the single-primer mDD technique can be used to isolate vitamin A-responsive genes with a palindromic nature.