Correlation of Vitamin D3 (Calcitriol) Serum Concentrations with Vitamin B12 and Folic Acid in Women Undergoing in vitro Fertilisation/Intracytoplasmatic Sperm Injection.

BACKGROUND: The important role of vitamin D3 in human health is well recognized. In this study, we measured serum concentrations of vitamin D3, vitamin B12 and B9 (folic acid) in 410 women undergoing in vitro fertilisation (IVF)/intracytoplasmatic sperm injection (ICSI) with dedicated focus on 3-month changes in consideration of patients' BMI. METHODS: Patients were of European origin and did not take any supplementation of D3. In preparing for pregnancy, patients took ≥4 weeks 400 µg folic acid combined with 9 µg vitamin B12 and 150 µg iodide as recommended. RESULTS: We found a significant 3-month quartile change of D3 serum concentrations (p < 0.0001) with maximum levels in autumn and lowest in spring. D3 correlated significantly with B12 (p = 0.035, ρ = 0.102) and folic acid (p < 0.0001, ρ = 0.191). BMIs however showed a negative correlation with B12 (p = 0.031, ρ = -0.105) and folic acid (p = 0.012, ρ = -0.125). CONCLUSIONS: Our results suggest a model in which the sun exposure during summer months enables storage of D3 followed by a slow release as a major factor to maintain D3 levels throughout the year. Finally, our data indicate that B12 and folic acid uptake might be influenced by vitamin D receptor and D3, where D3 and the BMI appear to have an indirect relationship - via B12 and folic acid.

Vitamin D3 modulates the innate immune response through regulation of the hCAP-18/LL-37 gene expression and cytokine production.

INTRODUCTION: The steroid hormone metabolite of vitamin D3, 1α,25-dihydroxyvitamin D3 (1,25D3), promotes osteogenic activity and regulates calcium and phosphate metabolism, which are actions regarded as classical vitamin D-regulated functions. Besides its role in these processes, 1,25D3 also seems implicated in the host defense against microbial/pro-inflammatory attacks. Low serum levels of vitamin D3 (vitamin D deficiency) are associated with osteoporosis and increased risk of fractures but also inflammatory diseases and their disease progression, presumably via mechanisms associated with 1,25D3-evoked modulation of the innate immune system. 1,25D3 has been reported to modulate many inflammatory responses, suggesting that it regulates multiple transcriptional targets within the inflammatory system. RESULTS: Experimental studies in various experimental systems show that 1,25D3 differentially regulates the production of pro-inflammatory cytokines and chemokines depending on cell type. Importantly, many reports show that 1,25D3 up-regulates expression of the human antimicrobial peptide hCAP-18/LL-37 gene. The hCAP-18/LL-37 gene seems indeed to be an important transcriptional target for 1,25D3. However, only limited evidence is presented showing that 1,25D3 consistently increases the amount of biologically active LL-37 peptide. CONCLUSION: In the present review, we discuss 1,25D3-induced down-regulation of cytokine/chemokine production and stimulation of hCAP-18/LL-37 gene expression which represent two very important pathways for 1,25D3-evoked regulation of the innate immune response.

Is There a Relationship between Nutrition, Facial Development, and Crowding of the Teeth?

Nutrition plays an important role, especially key vitamins D3 and K2 which are necessary for proper dentofacial development and food consistency influence on crowding and dental arches narrowing. Changes in our dentition and facial appearance are caused by changing our diet from primitive hunter gatherer to a more modern industrialized agriculture. Nutrition and its impact on epigeneticaly- mediated mechanisms continuously shape our phenotype which impacts overall health and can reverse the path for overall health and facial bone development. Orthodontics and nutrition both play a role in following nature's path to reestablishing facial balance and dental arches proportions to accommodate all 32 teeth.

Transfer of regulatory properties from tolerogenic to proinflammatory dendritic cells via induced autoreactive regulatory T cells.

Infectious tolerance is a term generally assigned to the process through which regulatory T cells (Tregs) transfer immunoregulatory properties to other T cells. In this study, we demonstrated that a similar process applies to human dendritic cells (DCs), albeit through a different mechanism. We induced and cloned proinsulin-specific Tregs using tolerogenic DCs and investigated mechanisms by which induced Ag-specific regulatory T cells (iaTregs) endorse the suppressive effects. iaTregs expressed FOXP3, programmed death-1, and membrane-bound TGF-ß and upregulated IL-10 and CTLA-4 after stimulation with the cognate Ag. The iaTregs suppressed effector T cells only when both encountered the cognate Ags on the same APCs (linked suppression). This occurred independently of IL-10, TGF-ß, programmed death-1, or CTLA-4. Instead, iaTregs used a granzyme B-mediated mechanism to kill B cells and monocytes, whereas proinflammatory DCs that resisted being killed were induced to upregulate the inhibitory receptors B7 (family) homolog 3 and ICOS ligand. These re-educated mature monocyte-derived dendritic cells (mDCs) suppressed effector T cells and induced IL-10-producing cells from the naive T cell pool. Our data indicated that human tolerogenic DCs confer infectious tolerance by inducing Ag-specific Tregs, which, in turn, re-educate proinflammatory mature DCs into DCs with regulatory properties.

The vitamin D analog, TX527, promotes a human CD4+CD25highCD127low regulatory T cell profile and induces a migratory signature specific for homing to sites of inflammation.

The use of hypocalcemic vitamin D analogs is an appealing strategy to exploit the immunomodulatory actions of active vitamin D in vivo while circumventing its calcemic side effects. The functional modulation of dendritic cells by these molecules is regarded as the key mechanism underlying their ability to regulate T cell reactivity. In this article, we demonstrate the capacity of the vitamin D analog, TX527, to target T cells directly. Microarray analysis of purified human CD3(+) T cells, cultured in the presence of TX527, revealed differential expression of genes involved in T cell activation, proliferation, differentiation, and migratory capacity. Accordingly, functional analysis showed a TX527-mediated suppression of the T cell proliferative capacity and activation status, accompanied by decreased expression of effector cytokines (IFN-γ, IL-4, and IL-17). Furthermore, TX527 triggered the emergence of CD4(+)CD25(high)CD127(low) regulatory T cells featuring elevated levels of IL-10, CTLA-4, and OX40 and the functional capacity to suppress activation and proliferation of effector T cells. Moreover, the vitamin D analog profoundly altered the homing receptor profile of T cells and their migration toward chemokine ligands. Remarkably, TX527 not only modulated skin-homing receptors as illustrated for the parent compound, but also reduced the expression of lymphoid organ-homing receptors (CD62L, CCR7, and CXCR4) and uniquely promoted surface expression of inflammatory homing receptors (CCR5, CXCR3, and CXCR6) on T cells. We conclude that TX527 directly affects human T cell function, thereby inhibiting effector T cell reactivity while inducing regulatory T cell characteristics, and imprints them with a specific homing signature favoring migration to sites of inflammation.

Peripubertal vitamin D(3) deficiency delays puberty and disrupts the estrous cycle in adult female mice.

The mechanism(s) by which vitamin D(3) regulates female reproduction is minimally understood. We tested the hypothesis that peripubertal vitamin D(3) deficiency disrupts hypothalamic-pituitary-ovarian physiology. To test this hypothesis, we used wild-type mice and Cyp27b1 (the rate-limiting enzyme in the synthesis of 1,25-dihydroxyvitamin D(3)) null mice to study the effect of vitamin D(3) deficiency on puberty and reproductive physiology. At the time of weaning, mice were randomized to a vitamin D(3)-replete or -deficient diet supplemented with calcium. We assessed the age of vaginal opening and first estrus (puberty markers), gonadotropin levels, ovarian histology, ovarian responsiveness to exogenous gonadotropins, and estrous cyclicity. Peripubertal vitamin D(3) deficiency significantly delayed vaginal opening without affecting the number of GnRH-immunopositive neurons or estradiol-negative feedback on gonadotropin levels during diestrus. Young adult females maintained on a vitamin D(3)-deficient diet after puberty had arrested follicular development and prolonged estrous cycles characterized by extended periods of diestrus. Ovaries of vitamin D(3)-deficient Cyp27b1 null mice responded to exogenous gonadotropins and deposited significantly more oocytes into the oviducts than mice maintained on a vitamin D(3)-replete diet. Estrous cycles were restored when vitamin D(3)-deficient Cyp27b1 null young adult females were transferred to a vitamin D(3)-replete diet. This study is the first to demonstrate that peripubertal vitamin D(3) sufficiency is important for an appropriately timed pubertal transition and maintenance of normal female reproductive physiology. These data suggest vitamin D(3) is a key regulator of neuroendocrine and ovarian physiology.

[Vitamin D in relation to frailty and locomotive syndrome].

Frailty is an extremely common and serious health problem in the elderly. Frailty has been described as "a biologic syndrome of decreased reserve and resistance to stressors, resulting from cumulative declines across multiple physiologic systems and causing vulnerability to adverse health outcomes" by Fried and colleagues. Frailty is associated with incident falls, functional limitation, disability, and mortality. There are many reports that vitamin D deficiency may play roles in diabetes mellitus, cancers, multiple sclerosis, and other autoimmune diseases, and was associated with poorer physical performance, falls and fractures, and a greater risk of nursing home admission. Recently, researches suggest that vitamin D may provide treatment and prevention from these diseases lead to frailty. Vitamin D is expected to be a treatment for frailty in an aging society.

[Vitamin D deficiency in Germany, is it a danger for increased morbidityand mortality?]. / Vitamin-D-Unterversorg ung in Deutschland. Gefahr für erhöhte Morbidität und Mortalität?

Vitamin D regulates the calcium-phosphate metabolism and thereby plays an important role for the integrity and functioning of bone, muscle and nerves. Studies have shown furthermore an influence on certain types of cancer, diabetes and cardiovascular diseases. Vitamin D is mainly produced by the skin. During exposure to sunlightthe precursor7-dehydrocholesterol is transformed to colecalciferol (vitamin D3). Smaller amounts are supplied by nutrition. In our latitude vitamin D synthesis takes only place during summertime. The vitamin stored in fat tissue in generally is not sufficient for the whole winter period and accordingly insufficiency is very frequent. In a cross-sectional study all over Germany (DeViD, 2007) only about 8% of the population was vitamin D sufficient in spring time.Two prospective studies (2008) proved a correlation between vitamin D supply and overall mortality. An amelioration of vitamin D supply can be achieved either by increasing sun exposure or daily oral intake of 800-2000 IU (=20-50 microg) colecalciferol.

Proteomics analysis reveals the effect of 1α,25(OH)2VD3-glycosides on development of early testes in piglets.

1α,25(OH)2VD3 is the most active form of VD3 in animals. It plays an important role in regulating mineral metabolism but also in reproduction. Testes are the main reproductive organs of male mammals. Our research aims to reveal the effect of 1α,25(OH)2VD3-glycosides on development of early testes in piglets. 140 weaned 21-day old piglets were selected. The piglets were randomly divided into four groups and were fed a commercial diet supplemented with 0, 1, 2 and 4 µg/kg of 1α,25(OH)2VD3, provided as 1α,25(OH)2VD3-glycosides. Sixty days after the start of the experiment, at piglet age 82 days, testes were harvested. The morphology and histology of early testicular development were assessed. In addition, the proteomic TMT/iTRAQ labelling technique was used to analyse the protein profile of the testes in each group. Western blotting was applied to verify the target of differentially abundant proteins (DAPs). The analysis of morphology and histology of testes showed that a certain concentration of 1α,25(OH)2VD3-glycosides had a positive and significant effect on testicular development. And the results of proteomics analysis showed that of the identified 132,715 peptides, 122,755 were unique peptides. 7852 proteins, of which 6573 proteins contain quantitative information. Screening for DAPs focused on proteins closely related to the regulation of testicular development such as steroid hormone synthesis, steroid biosynthesis, peroxisome and fatty acid metabolism pathways. These results indicated that 1α,25(OH)2VD3 is involved in the regulation of early testicular development in piglets. At the same time, these findings provide valuable information for the proteins involved in the regulation of testicular development, and help to better understand the mechanisms of 1α,25(OH)2VD3 in regulating the development of piglets' testes.

Lipopolysaccharide (LPS) partial structures inhibit responses to LPS in a human macrophage cell line without inhibiting LPS uptake by a CD14-mediated pathway.

Lipopolysaccharides (LPS) that lack acyloxyacyl groups can antagonize responses to LPS in human cells. Although the site and mechanism of inhibition are not known, it has been proposed that these inhibitory molecules compete with LPS for a common cellular target such as a cell-surface binding receptor. In the present study, we used an in vitro model system to test this hypothesis and to evaluate the role of CD14 in cellular responses to LPS. Cells of the THP-1 human monocyte-macrophage cell line were exposed to 1,25 dihydroxyvitamin D3 to induce adherence to plastic and expression of CD14, a binding receptor for LPS complexed with LPS-binding protein (LBP). The uptake of picograms of [3H]LPS (agonist) and enzymatically deacylated LPS [3H]dLPS (antagonist) was measured by exposing the cells to the radiolabeled ligands for short incubation periods. The amounts of cell-associated LPS and dLPS were then correlated with cellular responses by measuring the induction of nuclear NF-kappa B binding activity and the production of cell-associated interleukin (IL)-1 beta. We found that similar amounts of [3H]LPS or [3H]dLPS were taken up by the cells. The rate of cellular accumulation of the ligands was greatly enhanced by LBP and blocked by a monoclonal antibody to CD14 (mAb 60b), yet no cellular responses were induced by dLPS or dLPS-LBP complexes. In contrast, LPS stimulated marked increases of NF-kappa B binding activity and IL-1 beta. These responses were enhanced by LBP and inhibited by mAb 60b. dLPS and its synthetic lipid A counterpart, LA-14-PP (also known as lipid Ia, lipid IVa, or compound 406) strongly inhibited LPS-induced NF-kappa B and IL-1 beta, yet neither antagonist inhibited the uptake of LPS via CD14. dLPS did not inhibit NF-kappa B responses to tumor necrosis factor (TNF) alpha or phorbol ester. Our results indicate that (a) both stimulatory and nonstimulatory ligands can bind to CD14 in the presence of LBP; (b) the mechanism of inhibition by dLPS is LPS-specific, yet does not involve blockade of LPS binding to CD14; and (c) in keeping with previous results of others, large concentrations of LPS can stimulate the cells in the absence of detectable binding to CD14. The findings indicate that the site of dLPS inhibition is distal to CD14 binding in the LPS signal pathway in THP-1 cells, and suggest that molecules other than CD14 are important in LPS signaling.

[Roles of PPAR and p21WAF1/CIP1 in monocyte/macrophage differentiation: are circulating monocytes able to proliferate?]. / Rôles de PPAR et de p21WAF1/CIP1 dans la différenciation monocyte/macrophage: les monocytes circulants prolifèrent-ils?

Macrophages are involved in the immune and the inflammatory response. The deregulation of their physiological properties is associated with several pathologies such as atherosclerosis and some cancers. Cytokines action on this blood lineage modulates p21WAF1/CIP1 expression. It appears that this protein may play a role in the inflammation regulation through PPAR (peroxysome proliferator-activated receptors) transcription factors, strongly linked to lipid metabolism. It could also be involved in the control of the proliferation of monocytes/macrophages, even if these cells are classically described as devoided of any proliferative capacity.

[Vitamin D--an old vitamin in a new perspective]. / Vitamin D3, ein altes Vitamin im neuen Licht.

Vitamin D, is a secosteroid which, in its active form 1,25-(OH)2-Vitamin D3, has hormone activities. Most cells and tissues in the human body have vitamin D receptors that stimulate the nuclear transcription of various genes to alter cellular function. Vitamin D, appears to have an effect on numerous disease states and disorders, including osteoporosis, chronic musculoskeletal pain, diabetes (types 1 and 2), multiple sclerosis, cardiovascular disease, and cancers of the breast, prostate, and colon. According to many researchers there is currently a worldwide vitamin D deficiency in various populations, including infants, pregnant and lactating women, and the elderly. The prevalence of vitamin D, insufficiency in the general German population is high. Vitamin D in the food supply is limited and most often inadequate to prevent deficiencies. Supplemental vitamin D is likely necessary to avoid deficiency, especially in winter months. The estimated cost saving effect of improving vitamin D status in Germany might be up to 37.5 billion euros annually.

Vitamin D3 and its nuclear receptor increase the expression and activity of the human proton-coupled folate transporter.

Folates are essential for nucleic acid synthesis and are particularly required in rapidly proliferating tissues, such as intestinal epithelium and hemopoietic cells. Availability of dietary folates is determined by their absorption across the intestinal epithelium, mediated by the proton-coupled folate transporter (PCFT) at the apical enterocyte membranes. Whereas transport properties of PCFT are well characterized, regulation of PCFT gene expression remains less elucidated. We have studied the mechanisms that regulate PCFT promoter activity and expression in intestine-derived cells. PCFT mRNA levels are increased in Caco-2 cells treated with 1,25-dihydroxyvitamin D(3) (vitamin D(3)) in a dose-dependent fashion, and the duodenal rat Pcft mRNA expression is induced by vitamin D(3) ex vivo. The PCFT promoter region is transactivated by the vitamin D receptor (VDR) and its heterodimeric partner retinoid X receptor-alpha (RXRalpha) in the presence of vitamin D(3). In silico analyses predicted a VDR response element (VDRE) in the PCFT promoter region -1694/-1680. DNA binding assays showed direct and specific binding of the VDR:RXRalpha heterodimer to the PCFT(-1694/-1680), and chromatin immunoprecipitations verified that this interaction occurs within living cells. Mutational promoter analyses confirmed that the PCFT(-1694/-1680) motif mediates a transcriptional response to vitamin D(3). In functional support of this regulatory mechanism, treatment with vitamin D(3) significantly increased the uptake of [(3)H]folic acid into Caco-2 cells at pH 5.5. In conclusion, vitamin D(3) and VDR increase intestinal PCFT expression, resulting in enhanced cellular folate uptake. Pharmacological treatment of patients with vitamin D(3) may have the added therapeutic benefit of enhancing the intestinal absorption of folates.

Regulation of phosphate transport in proximal tubules.

Homeostasis of inorganic phosphate (P(i)) is primarily an affair of the kidneys. Reabsorption of the bulk of filtered P(i) occurs along the renal proximal tubule and is initiated by apically localized Na(+)-dependent P(i) cotransporters. Tubular P(i) reabsorption and therefore renal excretion of P(i) is controlled by a number of hormones, including phosphatonins, and metabolic factors. In most cases, regulation of P(i) reabsorption is achieved by changing the apical abundance of Na(+)/Pi cotransporters. The regulatory mechanisms involve various signaling pathways and a number of proteins that interact with Na(+)/P(i) cotransporters.

Role of the vitamin D3 pathway in healthy and diseased skin--facts, contradictions and hypotheses.

Irradiation of human keratinocytes with UVB (280-320 nm) in vitro and in vivo activates the metabolism of 7-dehydrocholesterol to hormonally active calcitriol. The production of calcitriol in the skin strongly depends on the photosynthesis of vitamin D(3) which is biologically inactive in the first instance. Vitamin D(3) serves as the starting substrate for two subsequent enzymatic hydroxylation steps in epidermal keratinocytes. Both the amount of vitamin D(3) and the activity of anabolic and catabolic vitamin D hydroxylases determine the cutaneous level of calcitriol. The hormonally active metabolite of vitamin D(3) regulates a huge number of genes in keratinocytes, and thus acts in an autocrine and/or paracrine manner. This local pathway of vitamin D(3) is unique, but its relevance for healthy and diseased skin is widely unknown, yet. Experimental findings implicate several questions: (1) Is UVB-induced formation of calcitriol involved in regulation of growth and differentaition of epidermal cells as well as immunological and skin protective processes? (2) What endogenous and exogenous factors including drugs affect the cutaneous vitamin D(3) pathway? From a therapeutical point of view, it has been known for a long time that topical application of calcitriol and its analogs can improve hyperproliferative skin diseases like psoriasis. In spite of many encouraging studies in recent years, the fields of the routinely therapeutical application of calcitriol or vitamin D analogs in dermatology (e.g. treatment of immunological, inflammatory, malignancies and infectious skin diseases) have not been intensified. Why is that?

Perinatal Undernutrition, Metabolic Hormones, and Lung Development.

Maternal and perinatal undernutrition affects the lung development of litters and it may produce long-lasting alterations in respiratory health. This can be demonstrated using animal models and epidemiological studies. During pregnancy, maternal diet controls lung development by direct and indirect mechanisms. For sure, food intake and caloric restriction directly influence the whole body maturation and the lung. In addition, the maternal food intake during pregnancy controls mother, placenta, and fetal endocrine systems that regulate nutrient uptake and distribution to the fetus and pulmonary tissue development. There are several hormones involved in metabolic regulations, which may play an essential role in lung development during pregnancy. This review focuses on the effect of metabolic hormones in lung development and in how undernutrition alters the hormonal environment during pregnancy to disrupt normal lung maturation. We explore the role of GLP-1, ghrelin, and leptin, and also retinoids and cholecalciferol as hormones synthetized from diet precursors. Finally, we also address how metabolic hormones altered during pregnancy may affect lung pathophysiology in the adulthood.

Vitamin D and the digestive system.

Target tissues of in vivo receptor binding and deposition of 1,25(OH)2 vitamin D3 and its oxygen analog OCT are reviewed in rats, mice, hamsters and zebra finch, identified with high-resolution microscopic autoradiography. Throughout the digestive system numerous sites with nuclear receptor binding of 3H-1,25(OH)2 vitamin D3 and 3H-OCT exist: in the oral region, epithelial cells of the oral cavity, tongue and gingiva, teeth odontoblast and ameloblast precursor pulp and stratum intermedium cells; in the parotid, submandibular and sublingual salivary glands, epithelial cells of striated ducts and granular convoluted tubules, intercalated ducts and acinar cells, as well as myoepithelial cells; in the stomach, neck mucous cells of gastric glands, endocrine cells of the antrum, and muscle cells of the pyloric sphincter; in the small and large intestine, absorptive and crypt epithelial cells; in the pancreas, predominantly islet B-cells. Perisinusoidal stellate (Ito) cells in the liver concentrate and retain variable amounts of radiolabeled compound in regions of their cytoplasm after administration of 3H-I,25(OH)2 vitamin D3 and 3H-25(OH) vitamin D3, probably sites of specific storage, similar to vitamin A. Submucosa in stomach and intestine also retain variable amounts of radiolabel, however unspecific with all compounds studied. In pilot studies with 3H-25(OH)2 vitamin D3 and 3H-24,25(OH)2 vitamin D3, no nuclear concentration was detectable. The reviewed data for vitamin D and its oxygen analogue OCT indicate genomic effects on multiple target tissues of the digestive system that involve cell proliferation and differentiation, endo- and exocrine secretion, digestion and absorption for maintaining optimal functions, with potentials for health prophylaxis and therapies.

Role of vitamin D glucosiduronate in calcium homeostasis.

Evidence has been presented suggesting the presence of vitamin D(3) 3beta-glucosiduronate and 1,25-dihydroxyvitamin D(3) glucosiduronate in rat bile. To evaluate the role of vitamin D glucosiduronates in calcium and phosphorus homeostasis, we synthesized vitamin D(3) 3beta-glucosiduronate and tested its biological activity in calcium- and vitamin D-deficient rats. After the intravenous administration of vitamin D(3) 3beta-glucosiduronate to rats maintained on a low calcium diet, there was an increase in duodenal calcium transport and an increase in serum calcium. Vitamin D(3) 3beta-glucosiduronate, however, was less active than equimolar amounts of vitamin D(3). At doses of less than 0.65-1 nmol per rat, the conjugate exhibited no activity. When vitamin D(3) 3beta-glucosiduronate was administered to vitamin D-deficient rats, 25-hydroxyvitamin D was detected in the serum; the increase in serum 25-hydroxyvitamin D levels was less than that observed after the administration of an equimolar amount of vitamin D(3). Vitamin D(3) 3beta-glucosiduronate showed no detectable activity in the induction of calcium binding protein in chick embryonic duodena, a system in which no endogenous steroid beta-glucuronidase activity is detectable. These data demonstrate that vitamin D(3) 3beta-glucosiduronate is biologically active in vivo and that the observed activity is due to hydrolysis of the conjugate to vitamin D(3). As vitamin D(3) 3beta-glucosiduronate is excreted in the bile of rats, it is possible that this conjugate is reutilized in vivo after hydrolysis to free vitamin D(3). These results suggest the existence of a mechanism for reutilization of the biliary products of vitamin D(3).

Chicken ovalbumin upstream promoter transcription factor (COUP-TF) dimers bind to different GGTCA response elements, allowing COUP-TF to repress hormonal induction of the vitamin D3, thyroid hormone, and retinoic acid receptors.

Alignment of natural chicken ovalbumin upstream promoter transcription factor (COUP-TF) response elements shows that, in addition to the predominant direct repeat of the GGTCA motif with a 2-bp spacing, there are other functional COUP elements with variations in the GGTCA orientation and spacing. We systematically analyzed the binding of in vitro-synthesized COUP-TFs and showed that COUP-TF is capable of binding to oligonucleotides containing both direct repeats and palindromes and with different spacings of the GGTCA repeats. Subsequently, we analyzed four possible mechanisms proposed to explain how COUP-TF could bind to these spatial variations of the GGTCA repeat. We demonstrated that the functional DNA-binding form of COUP-TF is a dimer which requires two GGTCA half-sites to bind DNA. We demonstrated that the COUP-TF dimer undergoes a remarkable structural adaptation to accommodate binding to these spatial variants of the GGTCA repeats. A functional consequence of the promiscuous DNA binding of COUP-TF is its ability to down-regulate hormonal induction of target gene expression by other members of the steroid-thyroid hormone receptor superfamily such as the vitamin D3, thyroid hormone, and retinoic acid receptors. Our data indicate that COUP-TF may have an important role in hormonal regulation of gene expression by these receptors.

A two-hit mechanism for vitamin D3-mediated transcriptional repression of the granulocyte-macrophage colony-stimulating factor gene: vitamin D receptor competes for DNA binding with NFAT1 and stabilizes c-Jun.

We previously described a control element in the granulocyte-macrophage colony-stimulating factor (GM-CSF) enhancer that is necessary and sufficient to mediate both transcriptional activation in response to T-cell stimuli and transcriptional repression by 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] through the vitamin D3 receptor (VDR). This DNA element is a composite site that is recognized by both Fos-Jun and NFAT1; it is directly bound by VDR in the absence of a retinoid X receptor as an apparent monomer, and it is bound in a unique tertiary conformation. We describe here the mechanism by which VDR elicits its transcriptional inhibitory effect. Firstly, VDR outcompetes NFAT1 for binding to the composite site. Overexpression of NFAT1 in vivo by transient transfection is able to relieve the 1,25(OH)2D3-dependent repression. Secondly, VDR stabilizes the binding of a Jun-Fos heterodimer to the adjacent AP-1 portion of the element. This appears to occur through a direct interaction between VDR and c-Jun, as demonstrated in vitro by direct glutathione S-transferase coprecipitation assays. In vivo, overexpression of c-Jun, but not c-Fos, leads to a rescue of the 1, 25(OH)2D3-mediated repression. Transfected FLAG-VDR bound to the NFAT1-AP-1 DNA binding element can be selectively precipitated from nuclear extracts that are made from cells treated with activating agents in the presence of 1,25(OH)2D3. VDR is not detected in the complex in the absence of the ligand. Thus, VDR acts selectively on the two components required for activation of this promoter/enhancer: it competes with NFAT1 for binding to the composite site, positioning itself adjacent to Jun-Fos on the DNA. Co-occupancy apparently leads to an inhibitory effect on c-Jun's transactivation function. These two events mediated by VDR effectively block the NFAT1-AP-1 activation complex, resulting in an attenuation of activated GM-CSF transcription.