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Review
Is there more to learn about functional vitamin D metabolism? Hector F. DeLuca * Department of Biochemistry, University of Wisconsin-Madison, 433 Babcock Drive, Madison, WI 53706-1544, USA
A R T I C L E I N F O
A B S T R A C T
Article history: Received 8 July 2014 Received in revised form 26 August 2014 Accepted 30 August 2014 Available online xxx
The state of information on the enzymes responsible for the conversion of vitamin D3 to 1a, 25-dhydroxyvitamin D3 (1,25-(OH)2D3), the metabolic active form responsible for the well-known function of vitamin D on calcium metabolism and bone mineralization has been briefly reviewed. There remains an unidentified enzyme responsible for 25% of the 25-hydroxylation of vitamin D3, while 75% of serum 25-hydroxyvitamin D3 (25-OH-D3) arises from CYP2R1. The well-established suppression of multiple sclerosis (MS) by sunlight has been confirmed using the mouse model, experimental autoimmune encephalomyelitis (EAE). This suppression results from a narrow band of ultraviolet light (300–315 nm) that does not increase serum 25-OH-D3. Thus, UV light suppresses EAE by a mechanism not involving vitamin D. Vitamin D deficiency unexpectedly suppresses the development of EAE. Further, vitamin D receptor knockout in susceptible mice also prevents the development of EAE. On the other hand, deletion of CYP2R1 and the 1a-hydroxylase, CYP27B1, does not impair the development of EAE. Thus, either vitamin D itself or a heretofore-unknown metabolite is needed for the development of a component of the immune system necessary for development of EAE. This article is part of a Special Issue entitled ‘17th Vitamin D Workshop’. ã 2014 Elsevier Ltd. All rights reserved.
Keywords: Functional vitamin D metabolism Narrow band UV light Vitamin D and immunology
1. Introduction Kodicek concluded, on the basis of early work with either unlabeled vitamin D or low-specific activity radioactive vitamin D, that vitamin D itself carried out its functions [1,2]. However, Lund et al., using high specific activity radiolabeled vitamin D3, discovered a more polar metabolite of vitamin D that acted earlier and appeared more potent than vitamin D3 itself in stimulating intestinal calcium transport and in healing rickets [3–5]. This led to the isolation, identification, and determination of the biological activity of the first active metabolite of vitamin D, 25-hydroxyvitamin D3 (25-OH-D3) in 1968 [6]. This compound proved to be more potent and acted more rapidly in inducing intestinal calcium transport and in the healing of rachitic lesions than vitamin D3 itself [7]. When this compound was chemically synthesized that allowed the introduction of tritium in its molecule, it was quickly learned that it is rapidly metabolized to an even more polar metabolite [8]. This metabolite was successfully isolated in 1970–1971 by Holick et al. [9,10] and its structure unequivocally identified as 1a,25-dihydroxyvitamin D3 (1,25-(OH)2D3). This compound has proved to be the metabolically
* Tel.: +1 608 262 1620. E-mail address: [email protected] (H.F. DeLuca).
active form of vitamin D in initiation of intestinal calcium transport, bone calcium mobilization, and the mineralization of the skeleton [11]. Because of the finding of Fraser and Kodicek [12] that the kidney is the primary site of synthesis of this compound, it was possible with anephric animals to prove that 1,25-(OH)2D3 and not its precursor or the vitamin itself is the active form in calcium metabolism [13,14]. Thus, the idea of functional metabolism of vitamin D was clearly established. This was followed by an intense investigation in the regulation of the production of 1,25-(OH)2D3 giving rise to the discovery of the vitamin D endocrine system which was largely defined by 1974 [11,15]. In that same year, Brumbaugh and Haussler first demonstrated the existence of a nuclear protein that proved to be the vitamin D receptor (VDR) [16]. The confirmation of that finding [17] then led to an attempt to isolate it and determine its structure [18,19]. This was not successful until the advent of molecular cloning in which two groups successfully cloned the VDR [20,21]. The basic elements then of the vitamin D endocrine system were clearly defined. In the meantime, many additional metabolites of vitamin D were isolated and identified predominantly in the Wisconsin laboratory [22]. One of the most prominent metabolites proved to be 24, 25-dihydroxyvitamin D3 (24,25-(OH)2D3) [23] Repeated attempts to demonstrate that 24,25-(OH)2D3 is a metabolically active form of vitamin D has proved to be unsuccessful [24]. This metabolite has been postulated to be a hormone required for hatching in birds
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Please cite this article in press as: H.F. DeLuca, Is there more to learn about functional vitamin D metabolism?, J. Steroid Biochem. Mol. Biol. (2014), http://dx.doi.org/10.1016/j.jsbmb.2014.08.020
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[25], a hormone effective on bone [26], and there still remains a persistent feeling that it may retain some direct metabolic activity. Thus, far, this has not proved to be the case [24]. The multiple pathways of vitamin D metabolism discovered up to now have largely yielded pathways relating to degradation and elimination of vitamin D compounds. The question now is do we have evidence that there may be other functional metabolism of vitamin D besides 25- and 1a-hydroxylation. 2. Defining enzymes in the functional metabolism pathway In the absence of the discovery of additional functional pathways of vitamin D metabolism, a great deal of energy has been focused on defining the enzymes responsible for the two-step activation pathway for function. The CYP27B1 has been cloned and clearly demonstrated to be the enzyme responsible for 1a-hydroxylation, largely in the kidney but perhaps in other tissues in an autocrine or paracrine fashion [27–29]. In addition to knocking out this enzyme in mice, experiments of nature have long proved that this activation pathway is essential for the vitamin D’s well-known action in healing rickets and osteomalacia [30,31]. These experiments leave no doubt that the CYP27B1 is a key enzyme in the function of vitamin D on calcium metabolism, phosphorus metabolism, and the skeleton [32]. Unfortunately we have yet to totally define the initial step in the activation pathway, namely 25-hydroxylation. A very major step forward in defining the enzyme for this initial step was brought about by the work of Cheng et al. in the cloning of the CYP2R1 enzyme [33]. Recently, a knockout of the CYP2R1 was achieved in mice by Zhu et al. [34], and this knockout demonstrated very clearly that the CYP2R1 is the major enzyme involved in 25-hydroxylation of vitamin D (Fig. 1). But it also demonstrates that it is not the exclusive enzyme since the knockout only eliminated 75% of the circulating 25-OH-D. Thus, an additional 25-hydroxylase must exist. The several 25-hydroxylases that have been discussed in the literature have not eliminated nor confirmed that they function in this initial pathway. In fact, of those that have been suggested in the past, there are reasons to doubt that they account for the 25-hydroxylation that is carried out by an enzyme other than the CYP2R1. Those arguments have been put forth [34]. It appears that there remains another unidentified enzyme responsible for 25-hydroxylation. 3. VDR: its essentiality for function and its location It is well established that the VDR is located not only in the expected target tissues responsible for calcium homeostasis, the
Fig. 1. Serum 25-hydroxyvitamin 25-hydroxyvitamin D3 in CYP2R1 / methods.
D3 levels in CYP2R1 / mice. Serum mice measured using the DiaSorin liaison
healing of rickets and osteomalacia, but are found in tissues that have no role in this process [35–37]. It is believed that the vitamin D system functions beyond skeletal health and growth. A great deal of information has been published on the location of the VDR. In fact, the literature is far too massive to be reviewed here. However, in recent years it has been possible to define very specific anti-receptor antibodies that are very selective and can yield unquestionable information on tissue location [38]. It is also possible with these antibodies to exclude some tissues that previously were reported to have VDR. Among them are skeletal and heart muscle that are devoid of VDR [39]. However, beyond intestine, osteoblasts, and renal tissue, the VDR can be found in such important cells as keratinocytes of skin, in induced lymphocytes, in macrophages, and several other tissues suggesting that vitamin D plays a role beyond skeletal health. Of great importance is whether the VDR is required for all of the known functions of vitamin D. In fact, there have been many in vitro experiments tailored to the idea that vitamin D may have non-genomic functions [40]. So far, none of these experiments is convincing and the question of whether there is any non-receptor, hence non-genomic-related functions in vivo remain open. All of the proven functions of vitamin D require the presence of VDR [41]. Further, it is well established that there is a single VDR and there have been attempts to define other forms of the VDR but so far none have reached confirmation. 4. The role of vitamin D in the immune system Despite numerous publications on the VDR and metabolites in immune cells, there is no universal agreement as to how vitamin D functions in the immune system. That vitamin D does function in the immune system was clearly demonstrated early with the delayed hypersensitivity studies in which vitamin D deficiency largely eliminated delayed hypersensitivity reaction to dinitrofluorobenzene [42]. Furthermore, an excess of vitamin D hormone also suppressed the delayed hypersensitivity response [43]. Thus both vitamin D deficiency and vitamin D excess appeared to play a role in immunosuppression. The possible relationship between vitamin D and multiple sclerosis (MS) was first suggested by Goldberg based on the observation that the incidence of MS is inversely related to sunlight exposure as determined by geographic location [44]. Clearly it is well known that ultraviolet light (UV) between 280 and 310 nm converts 7-dehydrocholesterol to previtamin D that then spontaneously isomerizes to vitamin D3 [45]. Thus the question: Is the vitamin D produced by the UV light responsible for the suppression of MS or is it merely a correlation and UV light suppresses MS by a mechanism not involving vitamin D? Many scientists and physicians have assumed that vitamin D mediates the suppression of MS by UV light. This has resulted in both clinical and experimental attempts to suppress MS with vitamin D compounds. Early experiments by Lemire reported suppression of experimental autoimmune encephalomyelitis (EAE) by the active form of vitamin D [46]. Cantorna et al. convincingly demonstrated that 1,25-(OH)2D3 can completely suppress EAE [47]. However, the suppression was accompanied by hypercalcemia. Low doses of 1,25-(OH)2D3 that do not produce hypercalcemia do not suppress EAE [48]. Certainly hypercalcemia has not been found in subjects exposed to intense sunlight or UV light [49]. Hypercalcemia is an unwanted side effect that eliminated the possibility of use of active vitamin D compounds to suppress MS and other autoimmune diseases. Furthermore, Cantorna et al. [48] showed that, when animals were given a very low calcium diet, 1,25-(OH)2D3 lost its ability to suppress EAE until doses that caused hypercalcemia by mobilization of the skeleton were given. In females, Meehan et al. was able to show that
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Fig. 2. Ultraviolet light suppression of EAE. Mice (BL/6) were immunized with Moog protein as previously described (52) and then irradiated for 1 h/day with broadband UVB (285–375 nm) or narrow band UVB (305–315 nm). Clinical score: 1. limp tail; 2. impaired gait; 3. leg paralysis; 4. all limbs paralyzed.
hypercalcemia itself can suppress EAE [50]. The idea that the vitamin D hormone could act directly to suppress EAE and hence MS did not seem feasible. We then returned to the question of what is responsible for sunlight exposure’s suppression of MS. Certainly UV light clearly suppresses EAE (Fig. 2), and also increases serum 25-OH-D3 levels but does not increase serum calcium concentrations [51]. Of great interest is that a narrow wave band of light (305–315 nm) accounts for the suppression of EAE [51] without increasing serum 25-OH-D3 [51] (Fig. 2). Thus, UV light suppresses EAE independent of vitamin D. Our research group then returned to the original observation of Yang et al. with delayed hypersensitivity in which vitamin D deficiency markedly reduced if not prevented the development of delayed hypersensitivity [42]. In our experiments with EAE, we could clearly demonstrate that vitamin D deficiency prevents or markedly reduces the development of EAE [53]. An identical observation was produced by Fernandes de Abreu et al. [54]. Therefore two laboratories clearly demonstrated that vitamin D deficiency, rather than increasing the incidence of EAE, markedly suppresses incidence and severity. The absence of the VDR also markedly suppressed if not prevented the appearance of EAE in mice [52] (Fig. 3). Thus, the development of this autoimmune disease requires the presence of vitamin D and the VDR. An obvious question is: Does it involve the vitamin D hormone and its precursor, 25-OH-D3? Experiments in which the CYP2R1 was knocked out, the incidence of EAE and its severity was not affected by the absence of CYP2R1. This argued that perhaps 25-OH-D might not be involved, although this cannot be concluded since CYP2R1 does not eliminate circulating 25-OH-D completely.
Fig. 3. The vitamin D receptor (VDR) is required for the development of EAE. EAE was induced as in Fig. 2 in BL/6 mice (52).
3
Fig. 4. The 25-hydroxyvitamin D-1a-hydroxylase (CYP27B1) is not required for the development of EAE. EAE was induced as described in Fig. 3.
However, the knockout of CYP27B1, the 1a-hydroxlyase also did not prevent the development of EAE (Fig. 4). We have before us the fact that vitamin D3 is required for the development of EAE as is the VDR. However, 1,25-(OH)2D3 does not appear to be required. This suggests that the development of the immune cells responsible for EAE require either vitamin D3 itself or some form that has not yet been identified. The work also suggests that the form of vitamin D requires its interaction with the receptor to allow the development of EAE responsible immune cells. The conclusion, therefore, is that vitamin D3 or an unknown metabolite is responsible for the development of a component(s) of the immune system that plays a role in the development of EAE and presumably other autoimmune diseases. Because vitamin D and the VDR are both required for the development of EAE and vitamin D itself is a poor ligand for VDR, it appears that a metabolite of vitamin D is likely required. Since the 1a-hydroxylase / mutant develops EAE, 1,25-(OH) 2D3 is not that metabolite. Thus, we may not yet know all there is to know about functional metabolism of vitamin D. Acknowledgment This work was supported by the Wisconsin Alumni Research Foundation. References [1] E. Kodicek, Metabolic studies on vitamin D, in: G.W.E. Wolstenholme, C.M. O’Connor (Eds.), Ciba Foundation Symposium on Bone Structure and Metabolism, Little, Brown and Co., Boston, MA, 1956, pp. 161–174. [2] E. Kodicek, The metabolism of vitamin D, in: W. Umbreit, M. Molitor (Eds.), Proceedings of the Fourth International Congress of Biochemistry, vol. XI, Pergamon Press Ltd., London, 1960, pp. 198–208. [3] J. Lund, H.F. DeLuca, Biologically active metabolite of vitamin D3 from bone, liver, and blood serum, J. Lipid Res. 7 (1966) 739–744. [4] A.W. Norman, J. Lund, H.F. DeLuca, Biologically active forms of vitamin D3 in kidney and intestine, Arch. Biochem. Biophys. 108 (1964) 12–21. [5] H. Morii, J. Lund, P.F. Neville, H.F. DeLuca, Biological activity of a vitamin D metabolite, Arch. Biochem. Biophys. 120 (1967) 508–512. [6] J.W. Blunt, H.F. DeLuca, H.K. Schnoes, 25-Hydroxycholecalciferol. A biologically active metabolite of vitamin D3, Biochemistry 7 (1968) 3317–3322. [7] J.W. Blunt, Y. Tanaka, H.F. DeLuca, The biological activity of 25-hydroxycholecalciferol: a metabolite of vitamin D3, Proc. Natl. Acad. Sci. U. S. A. 61 (1968) 1503–1506. [8] R.J. Cousins, H.F. DeLuca, R.W. Gray, Metabolism of 25-hydroxycholecalciferol in target and nontarget tissues, Biochemistry 9 (1970) 3649–3652. [9] M.F. Holick, H.K. Schnoes, H.F. DeLuca, Identification of 1,25-dihydroxycholecalciferol, a form of vitamin D3 metabolically active in the intestine, Proc. Natl. Acad. Sci. U. S. A. 68 (1971) 803–804. [10] M.F. Holick, H.F. Schnoes, T. Suda, R.J. Cousins, Isolation and identification of 1,25-dihydroxycholecalciferol. A metabolite of vitamin D active in intestine, Biochemistry 10 (1971) 2799–2804. [11] H.F. DeLuca, Vitamin D. The vitamin and the hormone, Fed. Proc. 33 (1974) 2211–2219.
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Journal of Steroid Biochemistry & Molecular Biology xxx (2014) xxx–xxx
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Review
Is there more to learn about functional vitamin D metabolism? Hector F. DeLuca * Department of Biochemistry, University of Wisconsin-Madison, 433 Babcock Drive, Madison, WI 53706-1544, USA
A R T I C L E I N F O
A B S T R A C T
Article history: Received 8 July 2014 Received in revised form 26 August 2014 Accepted 30 August 2014 Available online xxx
The state of information on the enzymes responsible for the conversion of vitamin D3 to 1a, 25-dhydroxyvitamin D3 (1,25-(OH)2D3), the metabolic active form responsible for the well-known function of vitamin D on calcium metabolism and bone mineralization has been briefly reviewed. There remains an unidentified enzyme responsible for 25% of the 25-hydroxylation of vitamin D3, while 75% of serum 25-hydroxyvitamin D3 (25-OH-D3) arises from CYP2R1. The well-established suppression of multiple sclerosis (MS) by sunlight has been confirmed using the mouse model, experimental autoimmune encephalomyelitis (EAE). This suppression results from a narrow band of ultraviolet light (300–315 nm) that does not increase serum 25-OH-D3. Thus, UV light suppresses EAE by a mechanism not involving vitamin D. Vitamin D deficiency unexpectedly suppresses the development of EAE. Further, vitamin D receptor knockout in susceptible mice also prevents the development of EAE. On the other hand, deletion of CYP2R1 and the 1a-hydroxylase, CYP27B1, does not impair the development of EAE. Thus, either vitamin D itself or a heretofore-unknown metabolite is needed for the development of a component of the immune system necessary for development of EAE. This article is part of a Special Issue entitled ‘17th Vitamin D Workshop’. ã 2014 Elsevier Ltd. All rights reserved.
Keywords: Functional vitamin D metabolism Narrow band UV light Vitamin D and immunology
1. Introduction Kodicek concluded, on the basis of early work with either unlabeled vitamin D or low-specific activity radioactive vitamin D, that vitamin D itself carried out its functions [1,2]. However, Lund et al., using high specific activity radiolabeled vitamin D3, discovered a more polar metabolite of vitamin D that acted earlier and appeared more potent than vitamin D3 itself in stimulating intestinal calcium transport and in healing rickets [3–5]. This led to the isolation, identification, and determination of the biological activity of the first active metabolite of vitamin D, 25-hydroxyvitamin D3 (25-OH-D3) in 1968 [6]. This compound proved to be more potent and acted more rapidly in inducing intestinal calcium transport and in the healing of rachitic lesions than vitamin D3 itself [7]. When this compound was chemically synthesized that allowed the introduction of tritium in its molecule, it was quickly learned that it is rapidly metabolized to an even more polar metabolite [8]. This metabolite was successfully isolated in 1970–1971 by Holick et al. [9,10] and its structure unequivocally identified as 1a,25-dihydroxyvitamin D3 (1,25-(OH)2D3). This compound has proved to be the metabolically
* Tel.: +1 608 262 1620. E-mail address: [email protected] (H.F. DeLuca).
active form of vitamin D in initiation of intestinal calcium transport, bone calcium mobilization, and the mineralization of the skeleton [11]. Because of the finding of Fraser and Kodicek [12] that the kidney is the primary site of synthesis of this compound, it was possible with anephric animals to prove that 1,25-(OH)2D3 and not its precursor or the vitamin itself is the active form in calcium metabolism [13,14]. Thus, the idea of functional metabolism of vitamin D was clearly established. This was followed by an intense investigation in the regulation of the production of 1,25-(OH)2D3 giving rise to the discovery of the vitamin D endocrine system which was largely defined by 1974 [11,15]. In that same year, Brumbaugh and Haussler first demonstrated the existence of a nuclear protein that proved to be the vitamin D receptor (VDR) [16]. The confirmation of that finding [17] then led to an attempt to isolate it and determine its structure [18,19]. This was not successful until the advent of molecular cloning in which two groups successfully cloned the VDR [20,21]. The basic elements then of the vitamin D endocrine system were clearly defined. In the meantime, many additional metabolites of vitamin D were isolated and identified predominantly in the Wisconsin laboratory [22]. One of the most prominent metabolites proved to be 24, 25-dihydroxyvitamin D3 (24,25-(OH)2D3) [23] Repeated attempts to demonstrate that 24,25-(OH)2D3 is a metabolically active form of vitamin D has proved to be unsuccessful [24]. This metabolite has been postulated to be a hormone required for hatching in birds
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[25], a hormone effective on bone [26], and there still remains a persistent feeling that it may retain some direct metabolic activity. Thus, far, this has not proved to be the case [24]. The multiple pathways of vitamin D metabolism discovered up to now have largely yielded pathways relating to degradation and elimination of vitamin D compounds. The question now is do we have evidence that there may be other functional metabolism of vitamin D besides 25- and 1a-hydroxylation. 2. Defining enzymes in the functional metabolism pathway In the absence of the discovery of additional functional pathways of vitamin D metabolism, a great deal of energy has been focused on defining the enzymes responsible for the two-step activation pathway for function. The CYP27B1 has been cloned and clearly demonstrated to be the enzyme responsible for 1a-hydroxylation, largely in the kidney but perhaps in other tissues in an autocrine or paracrine fashion [27–29]. In addition to knocking out this enzyme in mice, experiments of nature have long proved that this activation pathway is essential for the vitamin D’s well-known action in healing rickets and osteomalacia [30,31]. These experiments leave no doubt that the CYP27B1 is a key enzyme in the function of vitamin D on calcium metabolism, phosphorus metabolism, and the skeleton [32]. Unfortunately we have yet to totally define the initial step in the activation pathway, namely 25-hydroxylation. A very major step forward in defining the enzyme for this initial step was brought about by the work of Cheng et al. in the cloning of the CYP2R1 enzyme [33]. Recently, a knockout of the CYP2R1 was achieved in mice by Zhu et al. [34], and this knockout demonstrated very clearly that the CYP2R1 is the major enzyme involved in 25-hydroxylation of vitamin D (Fig. 1). But it also demonstrates that it is not the exclusive enzyme since the knockout only eliminated 75% of the circulating 25-OH-D. Thus, an additional 25-hydroxylase must exist. The several 25-hydroxylases that have been discussed in the literature have not eliminated nor confirmed that they function in this initial pathway. In fact, of those that have been suggested in the past, there are reasons to doubt that they account for the 25-hydroxylation that is carried out by an enzyme other than the CYP2R1. Those arguments have been put forth [34]. It appears that there remains another unidentified enzyme responsible for 25-hydroxylation. 3. VDR: its essentiality for function and its location It is well established that the VDR is located not only in the expected target tissues responsible for calcium homeostasis, the
Fig. 1. Serum 25-hydroxyvitamin 25-hydroxyvitamin D3 in CYP2R1 / methods.
D3 levels in CYP2R1 / mice. Serum mice measured using the DiaSorin liaison
healing of rickets and osteomalacia, but are found in tissues that have no role in this process [35–37]. It is believed that the vitamin D system functions beyond skeletal health and growth. A great deal of information has been published on the location of the VDR. In fact, the literature is far too massive to be reviewed here. However, in recent years it has been possible to define very specific anti-receptor antibodies that are very selective and can yield unquestionable information on tissue location [38]. It is also possible with these antibodies to exclude some tissues that previously were reported to have VDR. Among them are skeletal and heart muscle that are devoid of VDR [39]. However, beyond intestine, osteoblasts, and renal tissue, the VDR can be found in such important cells as keratinocytes of skin, in induced lymphocytes, in macrophages, and several other tissues suggesting that vitamin D plays a role beyond skeletal health. Of great importance is whether the VDR is required for all of the known functions of vitamin D. In fact, there have been many in vitro experiments tailored to the idea that vitamin D may have non-genomic functions [40]. So far, none of these experiments is convincing and the question of whether there is any non-receptor, hence non-genomic-related functions in vivo remain open. All of the proven functions of vitamin D require the presence of VDR [41]. Further, it is well established that there is a single VDR and there have been attempts to define other forms of the VDR but so far none have reached confirmation. 4. The role of vitamin D in the immune system Despite numerous publications on the VDR and metabolites in immune cells, there is no universal agreement as to how vitamin D functions in the immune system. That vitamin D does function in the immune system was clearly demonstrated early with the delayed hypersensitivity studies in which vitamin D deficiency largely eliminated delayed hypersensitivity reaction to dinitrofluorobenzene [42]. Furthermore, an excess of vitamin D hormone also suppressed the delayed hypersensitivity response [43]. Thus both vitamin D deficiency and vitamin D excess appeared to play a role in immunosuppression. The possible relationship between vitamin D and multiple sclerosis (MS) was first suggested by Goldberg based on the observation that the incidence of MS is inversely related to sunlight exposure as determined by geographic location [44]. Clearly it is well known that ultraviolet light (UV) between 280 and 310 nm converts 7-dehydrocholesterol to previtamin D that then spontaneously isomerizes to vitamin D3 [45]. Thus the question: Is the vitamin D produced by the UV light responsible for the suppression of MS or is it merely a correlation and UV light suppresses MS by a mechanism not involving vitamin D? Many scientists and physicians have assumed that vitamin D mediates the suppression of MS by UV light. This has resulted in both clinical and experimental attempts to suppress MS with vitamin D compounds. Early experiments by Lemire reported suppression of experimental autoimmune encephalomyelitis (EAE) by the active form of vitamin D [46]. Cantorna et al. convincingly demonstrated that 1,25-(OH)2D3 can completely suppress EAE [47]. However, the suppression was accompanied by hypercalcemia. Low doses of 1,25-(OH)2D3 that do not produce hypercalcemia do not suppress EAE [48]. Certainly hypercalcemia has not been found in subjects exposed to intense sunlight or UV light [49]. Hypercalcemia is an unwanted side effect that eliminated the possibility of use of active vitamin D compounds to suppress MS and other autoimmune diseases. Furthermore, Cantorna et al. [48] showed that, when animals were given a very low calcium diet, 1,25-(OH)2D3 lost its ability to suppress EAE until doses that caused hypercalcemia by mobilization of the skeleton were given. In females, Meehan et al. was able to show that
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Fig. 2. Ultraviolet light suppression of EAE. Mice (BL/6) were immunized with Moog protein as previously described (52) and then irradiated for 1 h/day with broadband UVB (285–375 nm) or narrow band UVB (305–315 nm). Clinical score: 1. limp tail; 2. impaired gait; 3. leg paralysis; 4. all limbs paralyzed.
hypercalcemia itself can suppress EAE [50]. The idea that the vitamin D hormone could act directly to suppress EAE and hence MS did not seem feasible. We then returned to the question of what is responsible for sunlight exposure’s suppression of MS. Certainly UV light clearly suppresses EAE (Fig. 2), and also increases serum 25-OH-D3 levels but does not increase serum calcium concentrations [51]. Of great interest is that a narrow wave band of light (305–315 nm) accounts for the suppression of EAE [51] without increasing serum 25-OH-D3 [51] (Fig. 2). Thus, UV light suppresses EAE independent of vitamin D. Our research group then returned to the original observation of Yang et al. with delayed hypersensitivity in which vitamin D deficiency markedly reduced if not prevented the development of delayed hypersensitivity [42]. In our experiments with EAE, we could clearly demonstrate that vitamin D deficiency prevents or markedly reduces the development of EAE [53]. An identical observation was produced by Fernandes de Abreu et al. [54]. Therefore two laboratories clearly demonstrated that vitamin D deficiency, rather than increasing the incidence of EAE, markedly suppresses incidence and severity. The absence of the VDR also markedly suppressed if not prevented the appearance of EAE in mice [52] (Fig. 3). Thus, the development of this autoimmune disease requires the presence of vitamin D and the VDR. An obvious question is: Does it involve the vitamin D hormone and its precursor, 25-OH-D3? Experiments in which the CYP2R1 was knocked out, the incidence of EAE and its severity was not affected by the absence of CYP2R1. This argued that perhaps 25-OH-D might not be involved, although this cannot be concluded since CYP2R1 does not eliminate circulating 25-OH-D completely.
Fig. 3. The vitamin D receptor (VDR) is required for the development of EAE. EAE was induced as in Fig. 2 in BL/6 mice (52).
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Fig. 4. The 25-hydroxyvitamin D-1a-hydroxylase (CYP27B1) is not required for the development of EAE. EAE was induced as described in Fig. 3.
However, the knockout of CYP27B1, the 1a-hydroxlyase also did not prevent the development of EAE (Fig. 4). We have before us the fact that vitamin D3 is required for the development of EAE as is the VDR. However, 1,25-(OH)2D3 does not appear to be required. This suggests that the development of the immune cells responsible for EAE require either vitamin D3 itself or some form that has not yet been identified. The work also suggests that the form of vitamin D requires its interaction with the receptor to allow the development of EAE responsible immune cells. The conclusion, therefore, is that vitamin D3 or an unknown metabolite is responsible for the development of a component(s) of the immune system that plays a role in the development of EAE and presumably other autoimmune diseases. Because vitamin D and the VDR are both required for the development of EAE and vitamin D itself is a poor ligand for VDR, it appears that a metabolite of vitamin D is likely required. Since the 1a-hydroxylase / mutant develops EAE, 1,25-(OH) 2D3 is not that metabolite. Thus, we may not yet know all there is to know about functional metabolism of vitamin D. Acknowledgment This work was supported by the Wisconsin Alumni Research Foundation. References [1] E. Kodicek, Metabolic studies on vitamin D, in: G.W.E. Wolstenholme, C.M. O’Connor (Eds.), Ciba Foundation Symposium on Bone Structure and Metabolism, Little, Brown and Co., Boston, MA, 1956, pp. 161–174. [2] E. Kodicek, The metabolism of vitamin D, in: W. Umbreit, M. Molitor (Eds.), Proceedings of the Fourth International Congress of Biochemistry, vol. XI, Pergamon Press Ltd., London, 1960, pp. 198–208. [3] J. Lund, H.F. DeLuca, Biologically active metabolite of vitamin D3 from bone, liver, and blood serum, J. Lipid Res. 7 (1966) 739–744. [4] A.W. Norman, J. Lund, H.F. DeLuca, Biologically active forms of vitamin D3 in kidney and intestine, Arch. Biochem. Biophys. 108 (1964) 12–21. [5] H. Morii, J. Lund, P.F. Neville, H.F. DeLuca, Biological activity of a vitamin D metabolite, Arch. Biochem. Biophys. 120 (1967) 508–512. [6] J.W. Blunt, H.F. DeLuca, H.K. Schnoes, 25-Hydroxycholecalciferol. A biologically active metabolite of vitamin D3, Biochemistry 7 (1968) 3317–3322. [7] J.W. Blunt, Y. Tanaka, H.F. DeLuca, The biological activity of 25-hydroxycholecalciferol: a metabolite of vitamin D3, Proc. Natl. Acad. Sci. U. S. A. 61 (1968) 1503–1506. [8] R.J. Cousins, H.F. DeLuca, R.W. Gray, Metabolism of 25-hydroxycholecalciferol in target and nontarget tissues, Biochemistry 9 (1970) 3649–3652. [9] M.F. Holick, H.K. Schnoes, H.F. DeLuca, Identification of 1,25-dihydroxycholecalciferol, a form of vitamin D3 metabolically active in the intestine, Proc. Natl. Acad. Sci. U. S. A. 68 (1971) 803–804. [10] M.F. Holick, H.F. Schnoes, T. Suda, R.J. Cousins, Isolation and identification of 1,25-dihydroxycholecalciferol. A metabolite of vitamin D active in intestine, Biochemistry 10 (1971) 2799–2804. [11] H.F. DeLuca, Vitamin D. The vitamin and the hormone, Fed. Proc. 33 (1974) 2211–2219.
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