- Email: [email protected]
Vitamin D
The International Journal of Biochemistry & Cell Biology 41 (2009) 982–985
Contents lists available at ScienceDirect
The International Journal of Biochemistry & Cell Biology journal homepage: www.elsevier.com/locate/biocel
Molecules in focus
Vitamin D Katie M. Dixon ∗ , Rebecca S. Mason Department of Physiology and Bosch Institute, University of Sydney, NSW 2006 Australia
a r t i c l e
i n f o
Article history: Received 3 June 2008 Received in revised form 10 June 2008 Accepted 10 June 2008 Available online 3 August 2008 Keywords: Vitamin D 1,25-Dihydroxyvitamin D3 Cancer
a b s t r a c t The primary source of vitamin D is the skin, following exposure to ultraviolet radiation. Vitamin D is well known for its effects on stimulating calcium absorption and is thus essential for maintenance of normal bone. It is also important for muscle function and has more recently been implicated in protection against several diseases including diabetes. Different pathways of action have been described for vitamin D compounds and various analogs specific to these pathways have demonstrated potential for therapeutic use. Recent studies suggest a novel role for vitamin D compounds in protection against cancer, a proposal supported by substantial epidemiological evidence. © 2008 Elsevier Ltd. All rights reserved.
1. Introduction
2. Structure
Following the discovery of vitamin D by Mellanby in 1920, its physiological significance has been progressively realized. It was originally classified as a “vitamin” essential for normal skeletal development and maintenance of calcium homeostasis. However, it has since been revealed that vitamin D is not strictly a vitamin since it can be synthesized in the body as a result of UVR exposure. Vitamin D is rather a steroid, more specifically, a secosteroid, indicating that one of the rings of the cyclopentanoperhydrophenanthrene ring structure (in the case of vitamin D, the 9–10 carbon–carbon bond of ring B) is broken. Vitamin D can be obtained from dietary sources or can be synthesized in the body. Dietary vitamin D is available in two forms: vitamin D2 (ergocalciferol) from plant sources and vitamin D3 (cholecalciferol) from animal sources, both of which are collectively termed vitamin D. However, dietary sources generally account for a very small amount of the total vitamin D in the body. Vitamin D in the form of vitamin D3 can be made from 7-dehydrocholesterol (7DHC) in the skin by exposure to ultraviolet light, mainly from light in the UVB spectrum (Holick, 2004). Vitamin D is a prohormone, converted ultimately to the active form 1␣,25-dihydroxyvitamin D3 (1,25(OH)2 D3 ).
The molecular structure of vitamin D is closely allied to that of classical steroid hormones. Vitamin D and all its metabolites, including the steroid hormone 1,25(OH)2 D3 are, in comparison to other steroid hormones, unusually conformationally flexible. This arises from the flexible properties of the three major regions of the molecule (Fig. 1A). The side chain has a 360◦ rotation around each of the five carbon–carbon single bonds; the A-ring undergoes a cyclohexane-like chair–chair interconversion, which changes the orientation of the 1␣-hydroxyl and 3-hydroxyl groups; and the broken B-ring has 360◦ rotation around the 6–7 single carbon bond. This flexibility results in rapid conformational changes, occurring millions of times per second. Since vitamin D can undergo rotation about the 6–7 carbon–carbon bond, a wide variety of potential ligand shapes can be generated, extending from the 6-s-cis steroidlike conformation to the 6-s-trans extended steroid conformation (Fig. 1B) (Norman et al., 2001).
∗ Corresponding author. Tel.: +61 2 9351 2561; fax: +61 2 9351 2510. E-mail address: [email protected] (K.M. Dixon). 1357-2725/$ – see front matter © 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.biocel.2008.06.016
3. Expression, activation and turnover Upon exposure of the skin to ultraviolet light, the 9–10 carbon–carbon bond of 7-DHC is cleaved, forming pre-vitamin D3 , which at body temperature thermally isomerizes over several hours to days to vitamin D3 (Holick, 2004). However, the vitamin D molecule itself has no demonstrated intrinsic biological activity. Vitamin D3 binds to the vitamin D-binding protein, which selectively transports vitamin D in the bloodstream to target cells of the vitamin D endocrine system for metabolism. It then undergoes two sequential biochemical alterations involving the addition of two hydroxyl groups by cytochrome P450 enzymes. The first of
K.M. Dixon, R.S. Mason / The International Journal of Biochemistry & Cell Biology 41 (2009) 982–985
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4. Biological function
Fig. 1. Structure and conformational flexibility of 1,25(OH)2 D3 . (A) Structure and sites of flexibility. (B) 6-s-cis steroid-like conformation and 6-s-trans extended steroid conformation.
these hydroxylation reactions occurs in the liver, where vitamin D is hydroxylated by the enzyme 25-hydroxylase at the carbon-25 position to form 25-hydroxyvitamin D (25(OH)D). This compound is further hydroxylated in the kidney by 25-hydroxyvitamin D-1␣hydroxylase (1␣-hydroxylase) to the biologically active metabolite 1,25(OH)2 D (Holick, 2004), which can then generate biological responses in over 30 target tissues by acting as a ligand for the receptors that mediate pathways for vitamin D action (Norman et al., 2001). Prolonged sunlight exposure cannot result in excessive production of vitamin D3 . During extended sun exposure, both previtamin D3 and the thermal isomerization product, vitamin D3 , absorb solar UVR and are converted to several “overirradiation products” (Holick, 2004). The two vitamin D activation steps have been shown to occur in several other tissues. Activity of 25-hydroxylase has also been observed in the intestine, adrenal gland, lung, kidney and bone, whilst 1␣-hydroxylation of 25(OH)D3 to form 1,25(OH)2 D3 also occurs in many tissues, including colon, breast, prostate, lung, activated macrophages, and parathyroid cells (Holick, 2004). Recent studies in vitamin D metabolism have revealed that the complete pathway of vitamin D synthesis, from 7-DHC to 1,25(OH)2 D3 , can also occur in epidermal cells (Lehmann et al., 2001; Bikle et al., 1986). This may be of significance in the photoprotection of skin cells by 1,25(OH)2 D3 .
Vitamin D compounds are best known for their role in stimulating calcium absorption and thus contributing to optimal bone mineralization and reduced fracture risk. The role of vitamin D in calcium homeostasis is highlighted in genetic mouse models of targeted deletion of either the 1,25(OH)2 D3 -synthesizing enzyme, 25 hydroxyvitamin D-1␣-hydroxylase [1␣(OH)ase or CYP27B1], or of the nuclear receptor for 1,25(OH)2 D3 , the vitamin D receptor (VDR). Even with heroic calcium supplementation, which almost abolishes most of the phenotype, these mice have reduced osteoblast numbers and bone volume (Goltzman et al., 2004). Furthermore, vitamin D compounds play an important role in muscle function, and more recently, adequate vitamin D status has been implicated in protection against a number of diseases including diabetes, infection and a number of cancers (Peterlik and Cross, 2005). The major hormonally active product of the vitamin D endocrine system is 1,25(OH)2 D3 . The various biological responses produced by 1,25(OH)2 D3 depend upon possibly two classes of vitamin D receptors which, with their shape-sensitive and stereo-selective ligand binding domains, determine the signal transduction pathways that become activated (Norman et al., 2001). It is well established that 1,25(OH)2 D3 can produce genomic biological responses via signal transduction pathways that utilize a nuclear receptor (VDR) for 1,25(OH)2 D3 to regulate gene transcription. The genomic effects depend upon the interaction between 1,25(OH)2 D3 and the VDR followed by interaction of the activated steroid receptor complex in the nucleus with vitamin D response elements (VDREs) present in the promoter regions of the regulated genes which are either to be activated or repressed (Norman et al., 2001). The biological responses that occur as a result of the genomic pathway are not immediate since they involve time-consuming processes such as gene transcription. A rapid-acting, non-genomic pathway for 1,25(OH)2 D3 action has been proposed to generate a variety of biological responses within seconds to minutes. The receptor involved in this pathway, however, has not yet been definitively identified. The first report of a rapid response-related membrane binding protein/receptor for 1,25(OH)2 D3 was in the basal lateral membrane of chick epithelial cells and was linked to the rapid calcium absorption from the duodenum, a process known as transcaltachia (Nemere et al., 1984). One group has presented evidence of a membrane-associated rapid response steroid-binding protein (1,25D-MARRS), identical to ERp57, as the receptor involved in rapid responses by 1,25(OH)2 D3 (Nemere et al., 2004). Recent reports implicate an alternate pocket in the ligand binding domain of the classical vitamin D receptor (VDR) in the rapid response (Mizwicki et al., 2004). The binding of 1,25(OH)2 D3 to its proposed rapid-response receptor can stimulate various signaling pathways including protein kinase C, cAMP, intracellular calcium and MAP kinase (Norman et al., 2001). The conformational flexibility of 1,25(OH)2 D3 enables it to generate a wide variety of ligand shapes for available receptor(s) in the vitamin D endocrine system. Studies of the X-ray structure of the VDR have shown that the preferred ligand shape for this receptor, when 1,25(OH)2 D3 is present as a ligand, is that represented by a twisted 6-s-trans bowl. Structure-function studies involving a variety of analogs of 1,25(OH)2 D3 locked in a particular conformational shape suggest that the preferred shape for the receptor linked to rapid responses is the 6-s-cis conformation (Norman et al., 2001). Conformationally flexible analogs can, in principle, initiate either genomic or rapid responses. The known agonists and antagonists have been used to dissect signal pathways in various systems. Fig. 2 summarizes the pathways of action for vitamin D and its analogs.
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Fig. 2. Pathways of action for 1,25(OH)2 D3 and analogs. In principle, 1,25(OH)2 D3 and conformationally flexible analogs can activate either the genomic pathway or rapid response pathway. Genomic responses are initiated following interaction between the vitamin D compound and the VDR followed by interaction of the activated steroid receptor complex in the nucleus with vitamin D response elements in the promoter regions of the genes which are either to be activated or repressed. Analogs locked in the 6-s-cis conformation such as 1,25-dihydroxylumisterol3 (JN) are full agonists for the rapid pathway and can only weakly bind the VDR. A membrane-associated rapid response steroid-binding protein (1,25D-MARRS) has been proposed as the receptor for the rapid response pathway though recent reports implicate an alternate pocket in the ligand-binding domain of the classical VDR in rapid responses. Binding of vitamin D compounds to the receptor for rapid responses triggers activation of other signal transduction pathways including MAP kinase.
5. Possible medical applications Recent studies suggest a role for vitamin D compounds in the prevention of skin cancer. The active metabolite 1,25(OH)2 D3 has been shown to inhibit ultraviolet radiation (UVR)-induced cell loss when added to cultured human skin cells prior to and/or immediately after UVR (Wong et al., 2004). The improved cell survival in 1,25(OH)2 D3 -treated cells does not appear to come at the expense of increased DNA damage in remaining cells. It has also been shown that 1,25(OH)2 D3 has a dose-dependent inhibitory effect on cyclobutane pyrimidine dimers (CPD) following UVR (Wong et al., 2004; De Haes et al., 2005). The protective effects of 1,25(OH)2 D3 against UVR-induced skin damage have also been observed in vivo. Topical application or intraperitoneal injection of 1,25(OH)2 D3 prior to UVR (Hanada et al., 1995), or topical application immediately after UVR only (Gupta et al., 2007) reduced apoptotic sunburn cells in mouse skin. Furthermore, a reduction in skin CPD has been noted in mice treated topically with 1,25(OH)2 D3 (Dixon et al., 2005).
While high concentrations of vitamin D have been shown to be immunosuppressive, vitamin D deficiency also causes immunosuppression (Yang et al., 1993). In mice, topical 1,25(OH)2 D3 protected against UVR-induced systemic immunosuppression (Dixon et al., 2005). Furthermore, it has been shown that 1,25(OH)2 D3 dose-dependently suppresses epidermal interleukin6 which usually increases after UVR exposure (De Haes et al., 2003). Several studies support a role for the rapid response pathway in protection against UVR-induced skin damage by 1,25(OH)2 D3 . Two 6-s-cis locked low calcemic rapid-acting agonists, 1,25-dihydroxylumisterol3 (JN) and 1␣,25-dihydroxy-7dehydrocholesterol (JM), entirely mimicked the photoprotective effects of 1,25(OH)2 D3 in human skin cells (Wong et al., 2004). Furthermore, these effects were completely abolished by a rapidacting antagonist whilst a genomic antagonist had no effect. Of significance, the low calcemic compound JN was shown to be effective in an in vivo model, reducing UVR-induced sunburn cells, CPD and immunosuppression (Dixon et al., 2005).
K.M. Dixon, R.S. Mason / The International Journal of Biochemistry & Cell Biology 41 (2009) 982–985
These studies raise the possibility of the use of low calcemic vitamin D compounds in skin cancer prevention. For example, 1␣-hydroxymethyl-16-ene-24,24-difluoro-25-hydroxy-26,27bis-homovitamin D3 or QW-1624F2-2 (QW) is a transcriptionally active analog that is approximately 80–100 times less calciuric than 1,25(OH)2 D3 , and does not cause cachexia (Posner et al., 2004). The agent QW has high anti-proliferative and pro-differentiating activity and has been shown to inhibit chemical-induced skin tumorigenesis and tumour latency (Kensler et al., 2000). QW has been fast tracked by the United States Food and Drug Administration (USFDA) for approval for clinical use in humans. A randomized trial in postmenopausal women receiving vitamin D and/or calcium supplementation showed a decreased all-cancer risk with improved vitamin D status (Lappe et al., 2007). Furthermore, VDR−/− mice were shown to be more susceptible to chemical-induced skin carcinogenesis than their wild-type counterparts (Zinser et al., 2002). There is substantial epidemiological evidence correlating decreased sunlight exposure and/or dietary vitamin D intake with increased incidence of breast, prostate and colon cancers in humans (Holick, 2004; Peterlik and Cross, 2005). A cohort study of skin and non-skin cancer patients was consistent with a role for vitamin D in decreasing the risk of several solid cancers, particularly stomach, colorectal, liver and gallbladder, pancreas, lung, breast, prostate, bladder and kidney. Incidence of second solid primary cancers after skin melanoma was significantly lower for countries with higher sunlight exposure than for those with lower sunlight exposure. The difference was more obvious after non-melanoma skin cancers, which is consistent with earlier reports that non-melanoma skin cancers reflect cumulative sun exposure, and thus effective vitamin D production. Conversely, melanoma is more related to early intermittent exposure and sunburn (Tuohimaa et al., 2007). The studies outlined support the use of vitamin D compounds in both therapeutic and preventative approaches to cancer. Vitamin D synthesis and metabolism in skin may contribute to endogenous photoprotection. Synthesis of vitamin D analogs with less calcemic side effects is already resulting in the development of a novel class of anticancer agents. Finally, the epidemiological data highlight the importance of vitamin D status in protection from cancers. Acknowledgements This work was supported by the National Health and Medical Research Council of Australia and the Cancer Council of New South Wales. K.M. Dixon was the recipient of a Cancer Institute N.S.W. Research Scholar Award. The authors apologize for the omission of references due to space limitations. References Bikle DD, Nemanic MK, Gee E, Elias P. 1,25-Dihydroxyvitamin D3 production by human keratinocytes. Kinetics and regulation. Journal of Clinical Investigation 1986;78:557–66.
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De Haes P, Garmyn M, Degreef H, Vantieghem K, Bouillon R, Segaert S. 1,25Dihydroxyvitamin D3 inhibits ultraviolet B-induced apoptosis, Jun kinase activation, and interleukin-6 production in primary human keratinocytes. Journal of Cellular Biochemistry 2003;89:663–73. De Haes P, Garmyn M, Verstuyf A, De Clercq P, Vandewalle M, Degreef H, et al. 1,25-Dihydroxyvitamin D3 and analogues protect primary human keratinocytes against UVB-induced DNA damage. Journal of Photochemistry & Photobiology B Biology 2005;78:141–8. Dixon KM, Deo SS, Wong G, Slater M, Norman AW, Bishop JE, et al. Skin cancer prevention: a possible role of 1,25dihydroxyvitamin D3 and its analogs. Journal of Steroid Biochemistry & Molecular Biology 2005;97:137–43. Goltzman D, Miao D, Panda DK, Hendy GN. Effects of calcium and of the Vitamin D system on skeletal and calcium homeostasis: lessons from genetic models. Journal of Steroid Biochemistry & Molecular Biology 2004;89/90:485–9. Gupta R, Dixon KM, Deo SS, J. Holliday C, Slater M, Halliday GM, et al. Photoprotection by 1,25 dihydroxyvitamin D3 is associated with an increase in p53 and a decrease in nitric oxide products. Journal of Investigative Dermatology 2007;127: 707–15. Hanada K, Sawamura D, Nakano H, Hashimoto I. Possible role of 1,25dihydroxyvitamin D3 -induced metallothionein in photoprotection against UVB injury in mouse skin and cultured rat keratinocytes. Journal of Dermatological Science 1995;9:203–8. Holick MF. Vitamin D: importance in the prevention of cancers, type 1 diabetes, heart disease, and osteoporosis. American Journal of Clinical Nutrition 2004;79:362–71 [erratum appears in Am J Clin Nutr 2004;79(May (5)):890]. Kensler TW, Dolan PM, Gange SJ, Lee JK, Wang Q, Posner GH. Conceptually new deltanoids (vitamin D analogs) inhibit multistage skin tumorigenesis. Carcinogenesis 2000;21:1341–5. Lappe JM, Travers-Gustafson D, Davies KM, Recker RR, Heaney RP. Vitamin D and calcium supplementation reduces cancer risk: results of a randomized trial. American Journal of Clinical Nutrition 2007;85:1586–91. Lehmann B, Genehr T, Knuschke P, Pietzsch J, Meurer M. UVB-induced conversion of 7-dehydrocholesterol to 1alpha,25-dihydroxyvitamin D3 in an in vitro human skin equivalent model. Journal of Investigative Dermatology 2001;117:1179–85. Mizwicki MT, Keidel D, Bula CM, Bishop JE, Zanello LP, Wurtz JM, et al. Identification of an alternative ligand-binding pocket in the nuclear vitamin D receptor and its functional importance in 1alpha, 25(OH)2 -vitamin D3 signaling. Proceedings of the National Academy of Sciences of the United States of America 2004;101:12876–81. Nemere I, Yoshimoto Y, Norman AW, Nemere I, Yoshimoto Y, Norman AW. Calcium transport in perfused duodena from normal chicks: enhancement within fourteen minutes of exposure to 1,25-dihydroxyvitamin D3 . Endocrinology 1984;115:1476–83. Nemere I, Farach-Carson MC, Rohe B, Sterling TM, Norman AW, Boyan BD, et al. Ribozyme knockdown functionally links a 1,25(OH)2 D3 membrane binding protein (1,25D3 -MARRS) and phosphate uptake in intestinal cells. Proceedings of the National Academy of Sciences of the United States of America 2004;101:7392–7. Norman AW, Henry HL, Bishop JE, Song XD, Bula C, Okamura WH. Different shapes of the steroid hormone 1alpha, 25(OH)(2)-vitamin D(3) act as agonists for two different receptors in the vitamin D endocrine system to mediate genomic and rapid responses. Steroids 2001;66:147–58. Peterlik M, Cross HS. Vitamin D and calcium deficits predispose for multiple chronic diseases. European Journal of Clinical Investigation 2005;35:290–304. Posner GH, Jeon HB, Sarjeant A, Riccio ES, Doppalapudi RS, Kapetanovic IM, et al. Low-calcemic, efficacious, 1␣,25-dihydroxyvitamin D3 analog QW-1624F2 2: calcemic dose–response determination, preclinical genotoxicity testing, and revision of A-ring stereochemistry. Steroids 2004;69:757–62. Tuohimaa P, Pukkala E, Scelo G, Olsen JH, Brewster DH, Hemminki K, et al. Does solar exposure, as indicated by the non-melanoma skin cancers, protect from solid cancers: vitamin D as a possible explanation. European Journal of Cancer 2007;43:1701–12. Wong G, Gupta R, Dixon KM, Deo SS, Choong SM, Halliday GM, et al. 1,25Dihydroxyvitamin D and three low-calcemic analogs decrease UV-induced DNA damage via the rapid response pathway. Journal of Steroid Biochemistry & Molecular Biology 2004;89/90:567–70. Yang S, Smith C, Prahl JM, Luo X, DeLuca HF. Vitamin D deficiency suppresses cell-mediated immunity in vivo. Archives of Biochemistry & Biophysics 1993;303:98–106. Zinser GM, Sundberg JP, Welsh J. Vitamin D(3) receptor ablation sensitizes skin to chemically induced tumorigenesis. Carcinogenesis 2002;23:2103–9.
Contents lists available at ScienceDirect
The International Journal of Biochemistry & Cell Biology journal homepage: www.elsevier.com/locate/biocel
Molecules in focus
Vitamin D Katie M. Dixon ∗ , Rebecca S. Mason Department of Physiology and Bosch Institute, University of Sydney, NSW 2006 Australia
a r t i c l e
i n f o
Article history: Received 3 June 2008 Received in revised form 10 June 2008 Accepted 10 June 2008 Available online 3 August 2008 Keywords: Vitamin D 1,25-Dihydroxyvitamin D3 Cancer
a b s t r a c t The primary source of vitamin D is the skin, following exposure to ultraviolet radiation. Vitamin D is well known for its effects on stimulating calcium absorption and is thus essential for maintenance of normal bone. It is also important for muscle function and has more recently been implicated in protection against several diseases including diabetes. Different pathways of action have been described for vitamin D compounds and various analogs specific to these pathways have demonstrated potential for therapeutic use. Recent studies suggest a novel role for vitamin D compounds in protection against cancer, a proposal supported by substantial epidemiological evidence. © 2008 Elsevier Ltd. All rights reserved.
1. Introduction
2. Structure
Following the discovery of vitamin D by Mellanby in 1920, its physiological significance has been progressively realized. It was originally classified as a “vitamin” essential for normal skeletal development and maintenance of calcium homeostasis. However, it has since been revealed that vitamin D is not strictly a vitamin since it can be synthesized in the body as a result of UVR exposure. Vitamin D is rather a steroid, more specifically, a secosteroid, indicating that one of the rings of the cyclopentanoperhydrophenanthrene ring structure (in the case of vitamin D, the 9–10 carbon–carbon bond of ring B) is broken. Vitamin D can be obtained from dietary sources or can be synthesized in the body. Dietary vitamin D is available in two forms: vitamin D2 (ergocalciferol) from plant sources and vitamin D3 (cholecalciferol) from animal sources, both of which are collectively termed vitamin D. However, dietary sources generally account for a very small amount of the total vitamin D in the body. Vitamin D in the form of vitamin D3 can be made from 7-dehydrocholesterol (7DHC) in the skin by exposure to ultraviolet light, mainly from light in the UVB spectrum (Holick, 2004). Vitamin D is a prohormone, converted ultimately to the active form 1␣,25-dihydroxyvitamin D3 (1,25(OH)2 D3 ).
The molecular structure of vitamin D is closely allied to that of classical steroid hormones. Vitamin D and all its metabolites, including the steroid hormone 1,25(OH)2 D3 are, in comparison to other steroid hormones, unusually conformationally flexible. This arises from the flexible properties of the three major regions of the molecule (Fig. 1A). The side chain has a 360◦ rotation around each of the five carbon–carbon single bonds; the A-ring undergoes a cyclohexane-like chair–chair interconversion, which changes the orientation of the 1␣-hydroxyl and 3-hydroxyl groups; and the broken B-ring has 360◦ rotation around the 6–7 single carbon bond. This flexibility results in rapid conformational changes, occurring millions of times per second. Since vitamin D can undergo rotation about the 6–7 carbon–carbon bond, a wide variety of potential ligand shapes can be generated, extending from the 6-s-cis steroidlike conformation to the 6-s-trans extended steroid conformation (Fig. 1B) (Norman et al., 2001).
∗ Corresponding author. Tel.: +61 2 9351 2561; fax: +61 2 9351 2510. E-mail address: [email protected] (K.M. Dixon). 1357-2725/$ – see front matter © 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.biocel.2008.06.016
3. Expression, activation and turnover Upon exposure of the skin to ultraviolet light, the 9–10 carbon–carbon bond of 7-DHC is cleaved, forming pre-vitamin D3 , which at body temperature thermally isomerizes over several hours to days to vitamin D3 (Holick, 2004). However, the vitamin D molecule itself has no demonstrated intrinsic biological activity. Vitamin D3 binds to the vitamin D-binding protein, which selectively transports vitamin D in the bloodstream to target cells of the vitamin D endocrine system for metabolism. It then undergoes two sequential biochemical alterations involving the addition of two hydroxyl groups by cytochrome P450 enzymes. The first of
K.M. Dixon, R.S. Mason / The International Journal of Biochemistry & Cell Biology 41 (2009) 982–985
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4. Biological function
Fig. 1. Structure and conformational flexibility of 1,25(OH)2 D3 . (A) Structure and sites of flexibility. (B) 6-s-cis steroid-like conformation and 6-s-trans extended steroid conformation.
these hydroxylation reactions occurs in the liver, where vitamin D is hydroxylated by the enzyme 25-hydroxylase at the carbon-25 position to form 25-hydroxyvitamin D (25(OH)D). This compound is further hydroxylated in the kidney by 25-hydroxyvitamin D-1␣hydroxylase (1␣-hydroxylase) to the biologically active metabolite 1,25(OH)2 D (Holick, 2004), which can then generate biological responses in over 30 target tissues by acting as a ligand for the receptors that mediate pathways for vitamin D action (Norman et al., 2001). Prolonged sunlight exposure cannot result in excessive production of vitamin D3 . During extended sun exposure, both previtamin D3 and the thermal isomerization product, vitamin D3 , absorb solar UVR and are converted to several “overirradiation products” (Holick, 2004). The two vitamin D activation steps have been shown to occur in several other tissues. Activity of 25-hydroxylase has also been observed in the intestine, adrenal gland, lung, kidney and bone, whilst 1␣-hydroxylation of 25(OH)D3 to form 1,25(OH)2 D3 also occurs in many tissues, including colon, breast, prostate, lung, activated macrophages, and parathyroid cells (Holick, 2004). Recent studies in vitamin D metabolism have revealed that the complete pathway of vitamin D synthesis, from 7-DHC to 1,25(OH)2 D3 , can also occur in epidermal cells (Lehmann et al., 2001; Bikle et al., 1986). This may be of significance in the photoprotection of skin cells by 1,25(OH)2 D3 .
Vitamin D compounds are best known for their role in stimulating calcium absorption and thus contributing to optimal bone mineralization and reduced fracture risk. The role of vitamin D in calcium homeostasis is highlighted in genetic mouse models of targeted deletion of either the 1,25(OH)2 D3 -synthesizing enzyme, 25 hydroxyvitamin D-1␣-hydroxylase [1␣(OH)ase or CYP27B1], or of the nuclear receptor for 1,25(OH)2 D3 , the vitamin D receptor (VDR). Even with heroic calcium supplementation, which almost abolishes most of the phenotype, these mice have reduced osteoblast numbers and bone volume (Goltzman et al., 2004). Furthermore, vitamin D compounds play an important role in muscle function, and more recently, adequate vitamin D status has been implicated in protection against a number of diseases including diabetes, infection and a number of cancers (Peterlik and Cross, 2005). The major hormonally active product of the vitamin D endocrine system is 1,25(OH)2 D3 . The various biological responses produced by 1,25(OH)2 D3 depend upon possibly two classes of vitamin D receptors which, with their shape-sensitive and stereo-selective ligand binding domains, determine the signal transduction pathways that become activated (Norman et al., 2001). It is well established that 1,25(OH)2 D3 can produce genomic biological responses via signal transduction pathways that utilize a nuclear receptor (VDR) for 1,25(OH)2 D3 to regulate gene transcription. The genomic effects depend upon the interaction between 1,25(OH)2 D3 and the VDR followed by interaction of the activated steroid receptor complex in the nucleus with vitamin D response elements (VDREs) present in the promoter regions of the regulated genes which are either to be activated or repressed (Norman et al., 2001). The biological responses that occur as a result of the genomic pathway are not immediate since they involve time-consuming processes such as gene transcription. A rapid-acting, non-genomic pathway for 1,25(OH)2 D3 action has been proposed to generate a variety of biological responses within seconds to minutes. The receptor involved in this pathway, however, has not yet been definitively identified. The first report of a rapid response-related membrane binding protein/receptor for 1,25(OH)2 D3 was in the basal lateral membrane of chick epithelial cells and was linked to the rapid calcium absorption from the duodenum, a process known as transcaltachia (Nemere et al., 1984). One group has presented evidence of a membrane-associated rapid response steroid-binding protein (1,25D-MARRS), identical to ERp57, as the receptor involved in rapid responses by 1,25(OH)2 D3 (Nemere et al., 2004). Recent reports implicate an alternate pocket in the ligand binding domain of the classical vitamin D receptor (VDR) in the rapid response (Mizwicki et al., 2004). The binding of 1,25(OH)2 D3 to its proposed rapid-response receptor can stimulate various signaling pathways including protein kinase C, cAMP, intracellular calcium and MAP kinase (Norman et al., 2001). The conformational flexibility of 1,25(OH)2 D3 enables it to generate a wide variety of ligand shapes for available receptor(s) in the vitamin D endocrine system. Studies of the X-ray structure of the VDR have shown that the preferred ligand shape for this receptor, when 1,25(OH)2 D3 is present as a ligand, is that represented by a twisted 6-s-trans bowl. Structure-function studies involving a variety of analogs of 1,25(OH)2 D3 locked in a particular conformational shape suggest that the preferred shape for the receptor linked to rapid responses is the 6-s-cis conformation (Norman et al., 2001). Conformationally flexible analogs can, in principle, initiate either genomic or rapid responses. The known agonists and antagonists have been used to dissect signal pathways in various systems. Fig. 2 summarizes the pathways of action for vitamin D and its analogs.
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K.M. Dixon, R.S. Mason / The International Journal of Biochemistry & Cell Biology 41 (2009) 982–985
Fig. 2. Pathways of action for 1,25(OH)2 D3 and analogs. In principle, 1,25(OH)2 D3 and conformationally flexible analogs can activate either the genomic pathway or rapid response pathway. Genomic responses are initiated following interaction between the vitamin D compound and the VDR followed by interaction of the activated steroid receptor complex in the nucleus with vitamin D response elements in the promoter regions of the genes which are either to be activated or repressed. Analogs locked in the 6-s-cis conformation such as 1,25-dihydroxylumisterol3 (JN) are full agonists for the rapid pathway and can only weakly bind the VDR. A membrane-associated rapid response steroid-binding protein (1,25D-MARRS) has been proposed as the receptor for the rapid response pathway though recent reports implicate an alternate pocket in the ligand-binding domain of the classical VDR in rapid responses. Binding of vitamin D compounds to the receptor for rapid responses triggers activation of other signal transduction pathways including MAP kinase.
5. Possible medical applications Recent studies suggest a role for vitamin D compounds in the prevention of skin cancer. The active metabolite 1,25(OH)2 D3 has been shown to inhibit ultraviolet radiation (UVR)-induced cell loss when added to cultured human skin cells prior to and/or immediately after UVR (Wong et al., 2004). The improved cell survival in 1,25(OH)2 D3 -treated cells does not appear to come at the expense of increased DNA damage in remaining cells. It has also been shown that 1,25(OH)2 D3 has a dose-dependent inhibitory effect on cyclobutane pyrimidine dimers (CPD) following UVR (Wong et al., 2004; De Haes et al., 2005). The protective effects of 1,25(OH)2 D3 against UVR-induced skin damage have also been observed in vivo. Topical application or intraperitoneal injection of 1,25(OH)2 D3 prior to UVR (Hanada et al., 1995), or topical application immediately after UVR only (Gupta et al., 2007) reduced apoptotic sunburn cells in mouse skin. Furthermore, a reduction in skin CPD has been noted in mice treated topically with 1,25(OH)2 D3 (Dixon et al., 2005).
While high concentrations of vitamin D have been shown to be immunosuppressive, vitamin D deficiency also causes immunosuppression (Yang et al., 1993). In mice, topical 1,25(OH)2 D3 protected against UVR-induced systemic immunosuppression (Dixon et al., 2005). Furthermore, it has been shown that 1,25(OH)2 D3 dose-dependently suppresses epidermal interleukin6 which usually increases after UVR exposure (De Haes et al., 2003). Several studies support a role for the rapid response pathway in protection against UVR-induced skin damage by 1,25(OH)2 D3 . Two 6-s-cis locked low calcemic rapid-acting agonists, 1,25-dihydroxylumisterol3 (JN) and 1␣,25-dihydroxy-7dehydrocholesterol (JM), entirely mimicked the photoprotective effects of 1,25(OH)2 D3 in human skin cells (Wong et al., 2004). Furthermore, these effects were completely abolished by a rapidacting antagonist whilst a genomic antagonist had no effect. Of significance, the low calcemic compound JN was shown to be effective in an in vivo model, reducing UVR-induced sunburn cells, CPD and immunosuppression (Dixon et al., 2005).
K.M. Dixon, R.S. Mason / The International Journal of Biochemistry & Cell Biology 41 (2009) 982–985
These studies raise the possibility of the use of low calcemic vitamin D compounds in skin cancer prevention. For example, 1␣-hydroxymethyl-16-ene-24,24-difluoro-25-hydroxy-26,27bis-homovitamin D3 or QW-1624F2-2 (QW) is a transcriptionally active analog that is approximately 80–100 times less calciuric than 1,25(OH)2 D3 , and does not cause cachexia (Posner et al., 2004). The agent QW has high anti-proliferative and pro-differentiating activity and has been shown to inhibit chemical-induced skin tumorigenesis and tumour latency (Kensler et al., 2000). QW has been fast tracked by the United States Food and Drug Administration (USFDA) for approval for clinical use in humans. A randomized trial in postmenopausal women receiving vitamin D and/or calcium supplementation showed a decreased all-cancer risk with improved vitamin D status (Lappe et al., 2007). Furthermore, VDR−/− mice were shown to be more susceptible to chemical-induced skin carcinogenesis than their wild-type counterparts (Zinser et al., 2002). There is substantial epidemiological evidence correlating decreased sunlight exposure and/or dietary vitamin D intake with increased incidence of breast, prostate and colon cancers in humans (Holick, 2004; Peterlik and Cross, 2005). A cohort study of skin and non-skin cancer patients was consistent with a role for vitamin D in decreasing the risk of several solid cancers, particularly stomach, colorectal, liver and gallbladder, pancreas, lung, breast, prostate, bladder and kidney. Incidence of second solid primary cancers after skin melanoma was significantly lower for countries with higher sunlight exposure than for those with lower sunlight exposure. The difference was more obvious after non-melanoma skin cancers, which is consistent with earlier reports that non-melanoma skin cancers reflect cumulative sun exposure, and thus effective vitamin D production. Conversely, melanoma is more related to early intermittent exposure and sunburn (Tuohimaa et al., 2007). The studies outlined support the use of vitamin D compounds in both therapeutic and preventative approaches to cancer. Vitamin D synthesis and metabolism in skin may contribute to endogenous photoprotection. Synthesis of vitamin D analogs with less calcemic side effects is already resulting in the development of a novel class of anticancer agents. Finally, the epidemiological data highlight the importance of vitamin D status in protection from cancers. Acknowledgements This work was supported by the National Health and Medical Research Council of Australia and the Cancer Council of New South Wales. K.M. Dixon was the recipient of a Cancer Institute N.S.W. Research Scholar Award. The authors apologize for the omission of references due to space limitations. References Bikle DD, Nemanic MK, Gee E, Elias P. 1,25-Dihydroxyvitamin D3 production by human keratinocytes. Kinetics and regulation. Journal of Clinical Investigation 1986;78:557–66.
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De Haes P, Garmyn M, Degreef H, Vantieghem K, Bouillon R, Segaert S. 1,25Dihydroxyvitamin D3 inhibits ultraviolet B-induced apoptosis, Jun kinase activation, and interleukin-6 production in primary human keratinocytes. Journal of Cellular Biochemistry 2003;89:663–73. De Haes P, Garmyn M, Verstuyf A, De Clercq P, Vandewalle M, Degreef H, et al. 1,25-Dihydroxyvitamin D3 and analogues protect primary human keratinocytes against UVB-induced DNA damage. Journal of Photochemistry & Photobiology B Biology 2005;78:141–8. Dixon KM, Deo SS, Wong G, Slater M, Norman AW, Bishop JE, et al. Skin cancer prevention: a possible role of 1,25dihydroxyvitamin D3 and its analogs. Journal of Steroid Biochemistry & Molecular Biology 2005;97:137–43. Goltzman D, Miao D, Panda DK, Hendy GN. Effects of calcium and of the Vitamin D system on skeletal and calcium homeostasis: lessons from genetic models. Journal of Steroid Biochemistry & Molecular Biology 2004;89/90:485–9. Gupta R, Dixon KM, Deo SS, J. Holliday C, Slater M, Halliday GM, et al. Photoprotection by 1,25 dihydroxyvitamin D3 is associated with an increase in p53 and a decrease in nitric oxide products. Journal of Investigative Dermatology 2007;127: 707–15. Hanada K, Sawamura D, Nakano H, Hashimoto I. Possible role of 1,25dihydroxyvitamin D3 -induced metallothionein in photoprotection against UVB injury in mouse skin and cultured rat keratinocytes. Journal of Dermatological Science 1995;9:203–8. Holick MF. Vitamin D: importance in the prevention of cancers, type 1 diabetes, heart disease, and osteoporosis. American Journal of Clinical Nutrition 2004;79:362–71 [erratum appears in Am J Clin Nutr 2004;79(May (5)):890]. Kensler TW, Dolan PM, Gange SJ, Lee JK, Wang Q, Posner GH. Conceptually new deltanoids (vitamin D analogs) inhibit multistage skin tumorigenesis. Carcinogenesis 2000;21:1341–5. Lappe JM, Travers-Gustafson D, Davies KM, Recker RR, Heaney RP. Vitamin D and calcium supplementation reduces cancer risk: results of a randomized trial. American Journal of Clinical Nutrition 2007;85:1586–91. Lehmann B, Genehr T, Knuschke P, Pietzsch J, Meurer M. UVB-induced conversion of 7-dehydrocholesterol to 1alpha,25-dihydroxyvitamin D3 in an in vitro human skin equivalent model. Journal of Investigative Dermatology 2001;117:1179–85. Mizwicki MT, Keidel D, Bula CM, Bishop JE, Zanello LP, Wurtz JM, et al. Identification of an alternative ligand-binding pocket in the nuclear vitamin D receptor and its functional importance in 1alpha, 25(OH)2 -vitamin D3 signaling. Proceedings of the National Academy of Sciences of the United States of America 2004;101:12876–81. Nemere I, Yoshimoto Y, Norman AW, Nemere I, Yoshimoto Y, Norman AW. Calcium transport in perfused duodena from normal chicks: enhancement within fourteen minutes of exposure to 1,25-dihydroxyvitamin D3 . Endocrinology 1984;115:1476–83. Nemere I, Farach-Carson MC, Rohe B, Sterling TM, Norman AW, Boyan BD, et al. Ribozyme knockdown functionally links a 1,25(OH)2 D3 membrane binding protein (1,25D3 -MARRS) and phosphate uptake in intestinal cells. Proceedings of the National Academy of Sciences of the United States of America 2004;101:7392–7. Norman AW, Henry HL, Bishop JE, Song XD, Bula C, Okamura WH. Different shapes of the steroid hormone 1alpha, 25(OH)(2)-vitamin D(3) act as agonists for two different receptors in the vitamin D endocrine system to mediate genomic and rapid responses. Steroids 2001;66:147–58. Peterlik M, Cross HS. Vitamin D and calcium deficits predispose for multiple chronic diseases. European Journal of Clinical Investigation 2005;35:290–304. Posner GH, Jeon HB, Sarjeant A, Riccio ES, Doppalapudi RS, Kapetanovic IM, et al. Low-calcemic, efficacious, 1␣,25-dihydroxyvitamin D3 analog QW-1624F2 2: calcemic dose–response determination, preclinical genotoxicity testing, and revision of A-ring stereochemistry. Steroids 2004;69:757–62. Tuohimaa P, Pukkala E, Scelo G, Olsen JH, Brewster DH, Hemminki K, et al. Does solar exposure, as indicated by the non-melanoma skin cancers, protect from solid cancers: vitamin D as a possible explanation. European Journal of Cancer 2007;43:1701–12. Wong G, Gupta R, Dixon KM, Deo SS, Choong SM, Halliday GM, et al. 1,25Dihydroxyvitamin D and three low-calcemic analogs decrease UV-induced DNA damage via the rapid response pathway. Journal of Steroid Biochemistry & Molecular Biology 2004;89/90:567–70. Yang S, Smith C, Prahl JM, Luo X, DeLuca HF. Vitamin D deficiency suppresses cell-mediated immunity in vivo. Archives of Biochemistry & Biophysics 1993;303:98–106. Zinser GM, Sundberg JP, Welsh J. Vitamin D(3) receptor ablation sensitizes skin to chemically induced tumorigenesis. Carcinogenesis 2002;23:2103–9.