Vitamin D. Introduction. Synthesis of Vitamin D. Advanced article. Carsten Carlberg, School of Medicine, Institute of Biomedicine, University of

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1 Carsten Carlberg, School of Medicine, Institute of Biomedicine, University of Eastern Finland, Kuopio, Finland Advanced article Article Contents Introduction Synthesis of Vitamin D Evolutionary View on Vitamin D Genomic Actions of Vitamin D Vitamin D Response In Vivo Online posting date: 16 th February 2018 The fat-soluble secosteroid vitamin is synthesised in UV (ultraviolet)-b exposed human skin. It regulates calcium and phosphorus homeostasis and thus plays an important role in bone mineralisation. Moreover, vitamin contributes to the control of cellular growth and differentiation as well as to the responsiveness of the innate and adaptive immune system. At insufficient sun exposure, the molecule is a true vitamin and needs to be taken up via diet or by direct supplementation with pills. During the past years, the need of sufficient vitamin synthesis acted an evolutionary driver for skin lightening of anatomically modern humans migrating out of Africa. The vitamin metabolite 1α,25-dihydroxyvitamin is the exclusive high-affinity ligand of the transcription factor vitamin D receptor and has a direct effect on the expression of >1000 genes. Thus, vitamin is a modulator of the human epigenome and transcriptome in more than half of all human tissues and cell types. Introduction More than 100 years ago, exposure to sunlight was an efficient therapy of two rather different disorders: rickets, a childhood disease of bone malformation, and tuberculosis, an infectious disease caused by intracellular bacteria. Later on, it was discovered that both diseases are promoted by a deficiency in vitamin (also called cholecalciferol), a molecule that human skin can synthesise under the exposure of UV-B (ultraviolet-b) radiation (Holick, 1981). Thus, the sunshine cure of rickets and tuberculosis led to endogenous production of vitamin in the patients, so that they were overcoming their deficiency. Moreover, cod liver oil is known since long time to have excellent antirickets properties. In 1922, it was understood that cod liver oil contains a rather els subject area: Biochemistry How to cite: Carlberg, Carsten (February 2018) Vitamin D. In: els. John Wiley & Sons, Ltd: Chichester. DOI: / a high concentration of a molecule that was termed vitamin D, simply because it was the fourth vitamin to be named (McMollum et al., 1922). At that time, it had not been realised that humans can synthesise the molecule in their skin, that is it is not a vitamin in its strict sense. In 1971, the metabolite 1α,25-dihydroxyvitamin (1,25(OH) 2, also called calcitriol), which is produced primarily in the kidneys, was identified as the biologically active form of vitamin (Norman et al., 1971). 1,25(OH) 2 binds and activates the transcription factor vitamin D receptor (VDR), which was finally cloned in 1988 (Baker et al., 1988). The link to rickets let initial vitamin D research focus on its role in calcium and phosphorus homeostasis and bone formation. However, since some 35 years it is also known that 1,25(OH) 2 can control the growth of cancer cells (Colston et al., 1982) suggesting that it has a potent anticancer action. At the same time, a role of 1,25(OH) 2 in the differentiation of cells of the immune system was discovered (Abe et al., 1981). Later on, many evidences accumulated demonstrating that vitamin D serves as an important regulator of the response of both the innate and the adaptive immune system (Chun et al., 2014). This finally provided a cellular and molecular explanation for the cure of tuberculosis by sunlight exposure. For obtaining full benefits of the disease-preventive potential of vitamin, the molecule or its metabolites should circulate in sufficiently high concentrations in the bloodstream. In contrast, today s widespread vitamin D deficiency contributes to a number of serious disorders including musculoskeletal diseases, which are, in addition to rickets, osteomalacia, fracture risk, risk of falls and sarcopenia, and nonmusculoskeletal disorders, such as increased risks for cancers, cardiovascular disease, hypertension, all-cause mortality, infectious and autoimmune diseases (Holick, 2007). Synthesis of Vitamin D Under UV-B ( nm) exposure, the cholesterol precursor 7-dehydrocholesterol converts to pre-vitamin (most efficient are nm) and then isomerises in a nonenzymatic reaction into vitamin (Figure 1). Cholesterol biosynthesis is an evolutionary very old pathway and, for example, phytoplankton produces vitamin since more than 500 million years (Tremezaygues et al., 2006). The same reaction takes place in human skin, when it is exposed to sufficient doses of UV-B, that is humans els 2018, John Wiley & Sons, Ltd. 1

2 UV-B Skin H H OH H OH CYP2R1 CYP27B1 HO Vitamin (the vitamin) HO 25(OH) (the most abundant form) HO OH 1,25(OH) 2 (the hormone) Diet VDR Oily fish Mushroom (D 2 ) Figure 1 Vitamin D synthesis pathway. Vitamin is synthesised endogenously in the skin under essential catalysis by UV-B or taken up by diet. In the liver, vitamin is converted by the enzyme CYP2R1 into the circulating form 25(OH) and then in the kidneys (and in many additional tissues and cell types) by the enzyme CYP27B1 into the high-affinity VDR ligand 1,25(OH) 2. can produce vitamin on their own when staying in the sun. However, only at latitudes below 37 N, UV-B radiation is sufficient for a whole year vitamin synthesis, that is in European winter months there is no chance of endogenous vitamin production even when staying in the sun. Lifestyle changes, such as predominant activities indoors and textile coverage outdoors, during the last hundreds of years as well as the migration out of Africa to less sun-exposed geographic regions, such as Central and Northern Europe, over the past years, significantly reduced the average endogenous vitamin production in human. In consequence, nowadays a large proportion of the human population depends on external vitamin supply. Accordingly, for deficient persons vitamin is a vitamin in its original sense. Since only oily fish, egg yolk and some mushrooms (when exposed to UV-B) contain reasonable amounts of vitamin andvitamind 2 (ergocalciferol, carrying in the side chain a double bond between carbons 22 and 23 and a methyl group on carbon 24), respectively, average human diet is not an efficient source of vitamin D (Bendik et al., 2014). As a consequence, some countries decided for direct fortification of dietary products, such as milk, margarine and juices, with vitamin.inthe United States, the Institute of Medicine (IOM) recommends for both bone and overall health 25-hydroxyvitamin (25(OH), also called calcifediol) serum levels of 50 nm (Institute of Medicine, 2011). Accordingly, individuals are classified as (1) severely deficient (<12.5 nm), (2) deficient ( nm), (3) insufficient (25 50 nm) and (4) sufficient (>50 nm). Vitamin D sufficiency should be reached by a daily supplementation with vitamin doses of μg ( IU) for children and μg ( IU) for adults. However, these recommendations are under debate, as the US Endocrine Society suggests 25(OH) serum concentrations of at least 75 nm and daily supplementations with 25 μg (1000 IU) vitamin or more (Holick et al., 2011). Prolonged sun exposure of human skin does not lead to excess production of vitamin, but to high oral vitamin intakes can have toxic effects (Vieth, 2006), such as hypercalcaemia. These elevated serum calcium levels are caused by increased intestinal calcium absorption and mobilisation of calcium from bone, which can result in soft tissue calcification. Therefore, the upper limit for daily vitamin supplementation was set to 100 μg (4000 IU). However, it is not likely that symptoms of toxicity will occur at daily vitamin intakes below 250 μg ( IU). Biologically, vitamin is an inert molecule and needs to be enzymatically converted in a two-step reaction to the nuclear hormone 1,25(OH) 2 ( index.php/pathway:wp1531) (Figure 1). The cytochrome P450 (CYP) enzyme CYP2R1, which is primarily expressed in the liver, adds a hydroxyl group at carbon 25 of vitamin creating 25(OH) (Henry, 2011). This molecule has the rather long half-life of 2 3 weeks in human serum and is the most abundant form of vitamin D in the human body. Therefore, 25(OH) serum levels are used as biomarker for the vitamin D status (Hollis, 2005). 2 els 2018, John Wiley & Sons, Ltd.

3 The mitochondrial enzyme CYB27B1 hydroxylates 25(OH) at carbon 1 producing 1,25(OH) 2 (Figure 1). Signal transduction pathways stimulated by parathyroid hormone (PTH) and low levels in calcium and phosphate induce CYP27B1 gene expression in the kidneys. In contrast, 1,25(OH) 2 as well as fibroblast growth factor 23 (FGF23), which is secreted by bone osteoblasts and osteocytes in response to increasing serum phosphate levels, downregulate the CYP27B1 gene (Henry, 2011). Circulating endocrine levels of 1,25(OH) 2 primarily derive from proximal tubule cells of the kidneys, while a number of human tissues, such as macrophages of the innate immune system, keratinocytes of the skin and osteoblasts within bones, can produce the molecule in a paracrine and autocrine manner. Vitamin and its metabolites are transported in the bloodstream by the α-globulin carrier protein vitamin D-binding protein (DBP), so that there is only a small fraction of free vitamin D in serum. Moreover, the enzyme CYP24A1 hydroxylates 25(OH) and 1,25(OH) 2 at carbon 24 and initiates in this way the degradation of the molecules. Importantly, the CYP24A1 gene is one of the most responsive primary vitamin D targets. It is expressed primarily in the kidneys, but is found also in a number of cancer cells. Thus, because of tight control of its synthesis and degradation, serum levels of 1,25(OH) 2 are some 800 times lower than that of 25(OH). Evolutionary View on Vitamin D The historically first role of vitamin was to act as a chemical sunscreen. Phyto- and zooplankton use the photochemical reaction of vitamin production as a protection mechanism against UV-B-induced DNA (deoxyribonucleic acid) damage. Since the marine food chain starts with plankton, vitamin accumulates in the liver of many deep-water fish, such as cod. This explains why cod liver oil is rich in vitamin. When animal species evolved and some moved from calcium-rich water to the calcium-poor terrestrial environment, the control of calcium homeostasis became a speciality of the developing vitamin D endocrinology (Bouillon and Suda, 2014). This also explains why only vertebrates have a full vitamin D endocrine system, formed by plasma transport proteins, metabolising enzymes and a high-affinity receptor. Later on, endocrinology of vitamin D got also involved in modulating the response of the immune system as well as cellular growth and differentiation. The evolutionary precursors of the nuclear receptor VDR were ligand-independent transcription factors being primarily involved in the control of cellular metabolism (Escriva et al., 2004). In a multistep process, these ancestral nuclear receptors learned to bind and to be activated by metabolic compounds, such as bile acids in the case of the VDR precursor (Makishima et al., 2002). Further evolution of the ligand-binding domain (LBD) of this receptor molecule resulted in a ligand-binding pocket (LBP) that accommodates with high specificity and affinity 1,25(OH) 2 (Carlberg and Molnár, 2012). Anatomically modern humans, which developed some years ago in East Africa, had dark skin, in order to prevent UV-mediated degradation of the circulating methyl-group donor folate. Despite dark skin, the intensive sun exposure at the equator allowed sufficient vitamin synthesis, as witnessed by average 25(OH) serum levels of 119 nm in traditionally living Maasai people in Tanzania and Kenya (Luxwolda et al., 2012). Since humans had more than years about the same lifestyle as Maasai, their physiology and biochemistry adapted to this rather high vitamin D status. After the migration of modern humans to less sunny regions in Asia and Europe, which started some years ago, the need of sufficient endogenous vitamin production caused an evolutionary pressure for genetic adaptation in form of gradual skin lightening (Hochberg and Templeton, 2010). This process took some years, which in an evolutionary scale is very fast. In contrast, the immigration of light skin Europeans to the Americas and Australia and the involuntary transfer of dark skin Africans to the Americas within the last 500 years were too quick for an efficient genetic adaptation. For example, dark skin persons living in the United Kingdom or in Canada do not have sufficient endogenous vitamin production at these latitudes. In general, many humans live nowadays at latitudes to which their skin colour is not adapted. Moreover, because of colder climate and cultural traditions, most humans cover nearly their entire body with textile. This is an additional important contribution to the low endogenous vitamin production and causes a rather high rate in vitamin D deficiency even in sunny geographic regions, such as on the Arabic peninsula. Individuals differ in their genomes primarily via single nucleotide variations (SNVs) resulting in phenotypic and physiological differences. Some of these SNVs relate to the vitamin D status, that is to the individual s serum 25(OH) levels, such as rs (in the GC gene encoding for DBP), rs (near the DHCR7 (7-dehydrocholesterol reductase) gene) and rs (close to the CYP2R1gene) (Wang et al., 2010).The rather low rate of endogenous vitamin synthesis of contemporary humans parallels with many disease-promoting lifestyle changes, such as reduced physical activity and increased body mass index. However, evolution selects for benefits that result in a higher number of offspring reaching a reproductive age, but it does not protect from aging-related diseases. Thus, the higher vitamin D status in the past protected humans against infectious diseases, such as tuberculosis, rather than against disorders that normally occur at higher age, such as cancer and cardiovascular disease. Genomic Actions of Vitamin D The nuclear receptor VDR is the only protein encoded by the human genome that binds 1,25(OH) 2 at subnanomolar concentrations (Haussler et al., 1997), that is basically all physiological effects of vitamin D are mediated by this receptor. VDR is one of the some 1600 human transcription factors and shows highest expression in metabolic tissues, such as kidneys, intestine and bone ( However, low to moderate VDR expression levels are also found in more than half of the 400 tissues and cell types that form the human body, that is all of these are responsive to vitamin D. els 2018, John Wiley & Sons, Ltd. 3

4 C25 (a) (b) 1,25(OH) 2 LBP VDR LBD Figure 2 Structural view. The structure of 1,25(OH) 2 (a) and of VDR s LBD (b). The LBP is indicated in red. Please note that both structures are in different scale. VDR is a member of the nuclear receptor superfamily; other well-known members of which are the receptors for the steroid hormones cortisol and oestradiol, glucocorticoid receptor (GR) and oestrogen receptor (ER) (Evans, 2005). The molecular mechanisms of nuclear receptors are very similar. For example, VDR, ER and GR have a structurally conserved LBDs, each of which contains in its lower part an LBP that is perfectly adapted in its size and shape for accommodating the respective specific ligand (Nagy and Schwabe, 2004) (Figure 2). The origin of anatomically modern humans at the equator also implies that the human genome is evolutionary rather adapted to a constant vitamin D status than to level changes between summer and winter. Thus, while the levels of cortisol and oestradiol change on a daily (in all humans) and monthly basis (in premenopausal females), respectively, the endocrinology of vitamin D aims on a constant hormone level. Another highly conserved domain of nuclear receptors is their DNA-binding domain (DBD). The VDR DBD recognises the hexameric sequence RGKTSA (R = AorG,K= GorT,S= C or G) in the major groove of genomic DNA. In vitro experiments indicated that VDR preferentially forms heterodimers with the nuclear receptor retinoid X receptor (RXR) on a direct repeat of two hexameric motifs spaced by three nucleotides (DR3) (Carlberg et al., 1993; Sone et al., 1991). This was confirmed by genome-wide analysis of VDR binding sites in six human cell types, but only for approximately 15% of all detected sites (Tuoresmäki et al., 2014). Accordingly, there must be additional mechanisms how VDR associates with genomic DNA C1 C3 (Carlberg and Campbell, 2013; Carlberg, 2017). Interestingly, the genome-wide VDR binding pattern is rather cell-specific, which has an impact on the panel of vitamin D target genes in VDR expressing tissues and cell types (Carlberg, 2014; Campbell, 2014). In the past, vitamin D and its metabolite 1,25(OH) 2 were best characterised for their physiological role in calcium homeostasis and bone formation. However, to date most genome-wide data are available for the actions vitamin D in cells of the haematopoietic system, such as peripheral blood mononuclear cells (PBMCs), monocytes and macrophages (Carlberg, 2014). This emphasises the role of VDR and vitamin D in the control of innate and adaptive immunity. Genomic DNA is always wrapped around nucleosomes forming chromatin. The default stage of chromatin is densely packed heterochromatin preventing the access of transcription factors and most other nuclear proteins to genomic DNA (Beisel and Paro, 2011). Thus, chromatin has an intrinsic repressive potential conserving the epigenome of a differentiated cell. Per cell type this leaves only some chromatin sites, being primarily located at promoter and enhancer regions, accessible to transcription factors (ENCODE-Project-Consortium et al., 2012). The epigenome can be modulated on the level of DNA methylation, histone acetylation and methylation and three-dimensional chromatin organisation and is controlled by chromatin modifying and remodelling enzymes that can read, write or erase posttranslational chromatin marks and reposition nucleosomes, respectively. VDR colocates with a number of nuclear proteins, such as the pioneer transcription factor PU.1 and the chromatin organiser CTCF (Carlberg, 2017). Moreover, VDR is a component of a large protein complex containing chromatin modifying and remodelling enzymes, corepressor, coactivator and mediator proteins (Molnár, 2014). In this way, VDR and its ligand 1,25(OH) 2 can modulate many aspects of the epigenome, such as patterns of posttranslational histone modifications and accessible chromatin (Seuter et al., 2016). This means that vitamin has a direct effect on the epigenome. Vitamin D-triggered changes in the epigenome can serve as a first step in modulating the transcriptome of a cell (Carlberg, 2014). A high vitamin D status correlates with the availability of 1,25(OH) 2 in the nuclei of VDR expressing tissues and cell types. Therefore, the vitamin D status of an individual should have an impact on his/her epigenome and subsequently on the transcriptome. Interestingly, VDR is also located in the cytoplasm and can interact with membrane invaginations (caveolae). In this way, vitamin can stimulate signal transduction pathways that are mediated by mitogen-activated protein kinase and cyclic adenosine monophosphate. These rapid, nongenomic actions of vitamin were found in the intestine, vascular smooth muscle and pancreatic β-cells. Vitamin D Response In Vivo Vitamin D intervention studies often focus on the evaluation of the health status of the study participants via questionnaires, medical examination and/or serum biochemistry. In contrast, the 4 els 2018, John Wiley & Sons, Ltd.

5 5-month vitamin intervention trial VitDmet (NCT ) measured from PBMCs isolated from 71 elderly prediabetic study participants at start and end of the study mrna (messenger ribonucleic acid) expression of 24 vitamin D target genes (Carlberg et al., 2013). There was a significant correlation between mrna expression changes and variations in 25(OH) serum levels (Vukic et al., 2015), that is the vitamin D target genes served as biomarkers for functional consequences of changes in the vitamin D status. Moreover, from more than 100 clinical and biochemical parameters that had been determined in the VitDmet trial only 12, such as PTH serum levels, displayed significant correlations (Saksa et al., 2015). The in total 36 vitamin D-dependent parameters allowed to determine how well the study participants respond to vitamin supplementation. In fact, the tested persons showed an individual response to vitamin, referred to a vitamin D response index (Carlberg, 2016), which allowed to segregate them into high, mid and low responders. In the VitDbol intervention trial (NCT ), healthy human adults were treated once with a vitamin bolus (2000 μg) and samples were taken already 1 and 2 days after onset of the study (Vukic et al., 2015). Also this experimental approach allowed the determination of the vitamin D response index and segregated the study participants into high, mid and low responders (Carlberg and Haq, 2018). Interestingly, the dynamic response to vitamin, that is a comparison of vitamin D-triggered parameters at two or more time points, does not correlate with the vitamin D status as determined in other vitamin D trials. The vitamin D response index suggests for each individual a vitamin supplementation protocol that will direct to a personal optimal vitamin D status. This concept may help to solve the scientific dispute about recommended 25(OH) serum levels and amounts of daily vitamin supplementation. Moreover, the inclusion of vitamin D response index measurements in the stratification of study cohorts may be most appropriate, in order to challenge observational studies suggesting that high serum concentrations of vitamin protect against cardiovascular disease, diabetes, colorectal cancer and all-cause mortality. In conclusion, the response to vitamin supplementation varies considerably between individuals and depends on many parameters, such as baseline vitamin D status, adiposity and genotype. Glossary 1,25(OH) 2 The biologically most active form of vitamin D, also called calcitriol, and the only high affinity (k D 0.1 nm) ligand of the transcription factor VDR (Figure 2). Epigenome The genome-wide analysis of epigenetics and changes in gene functions that are heritable but do not involve changes in the genome. Very dynamic, varies from one cell type to the other and can respond to various signalling pathways. Genome The complete haploid DNA sequence of an organism comprising all coding genes and far larger noncoding regions. With the exception of cancer cells, the genome (3260 Mb) of all of the 400 tissues and cell types that form a human individual is identical and constant over time. Transcriptome The complete set of all transcribed RNA molecules of a tissue or cell type; significantly differs between tissues and depends on extra- and intracellular signals. VDR An endocrine member of the nuclear receptor superfamily and is structurally (Figure 2) and functionally comparable with the nuclear receptors for oestrogen, or cortisol. The transcription factor is the only high affinity target of 1,25(OH) 2, that is basically all biological actions of vitamin D are mediated by the VDR. Vitamin D response index The dynamic molecular response to vitamin D being calculated based on a comparison of vitamin D-triggered parameters, such as mrna expression of vitamin D target genes in PBMCs or serum levels of parathyroid hormone protein, at two or more time points in relation to changes in 25(OH) serum levels. Vitamin D status Determined based on the serum concentration of 25(OH) being the most abundant vitamin D metabolite. For good bone health and overall health, 25(OH) serum concentrations are recommended to be throughout year above 50 nm (US Institute of Medicine) or 75 nm (US Endocrine Society). References Abe E, Miyaura C, Sakgami H, et al. (1981) Differentiation of mouse myeloid leukemia cells induced by 1a,25-dihydroxyvitamin. Proc Natl Acad Sci USA 78: Baker AR, McDonnell DP, Hughes M, et al. (1988) Cloning and expression of full-length cdna encoding human vitamin D receptor. 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