1. Chapter 13 Chapter 13 Vitamin D: Biology, Actions, and Clinical Implications Copyright © 2013 Elsevier Inc. All rights reserved.

2. FIGURE 13.1 Pathway of native hormone or 1α,25-dihydroxyvitamin D 3 production. The major source of vitamin D 3 is through ultraviolet irradiation of 7-dehydrocholesterol in the skin. The liver 25-hydroxylase enzyme (encoded by CYP2R1) then converts vitamin D 3 to 25-hydroxyvitamin D 3 (25-OH-D 3 ), the major circulating form of the vitamin. Generation of 1α,25-dihydroxyvitamin D3 occurs primarily in the kidney by the 25-OH-D-1α- hydroxylase enzyme (encoded by CYP27B1). See text for more details. Source: Plum and DeLuca (2010). Nat Rev Drug Discov 9(12):941–55. 2

3. Copyright © 2013 Elsevier Inc. All rights reserved. FIGURE 13.2 Overview of the vitamin D metabolic pathway. 3

4. Copyright © 2013 Elsevier Inc. All rights reserved. FIGURE 13.3 Regulation of 1α-hydroxylase in the kidney. Low levels of calcium stimulate the secretion of parathyroid hormone (PTH) from parathyroid glands as shown in the top of the figure. PTH then binds to its receptor (PTHR) and stimulates CYP27B1 in the kidney leading to synthesis of 1α,25(OH) 2 D 3. 1α,25(OH) 2 D 3 inhibits its own production by: (i) inhibiting CYP27B1 in the kidney; (ii) inhibiting PTH secretion; and (iii) stimulating FGF23 transcription in the bone. FGF23 binds to Klotho and the FGF receptor (FGFR) to inhibit CYP27B1 providing a third negative feedback loop in the synthesis of 1α,25(OH) 2 D 3. See text for more detailed explanations. Broken lines with arrows indicate stimulation and solid lines indicate inhibition. 4

5. Copyright © 2013 Elsevier Inc. All rights reserved. FIGURE 13.4 Structure–function relationships and proposed mechanisms of gene induction and repression by vitamin D receptor (VDR). (A) A schematic view of the functional domains in human VDR. (B) Allosteric model of VDR–retinoid X receptor (RXR) activation after binding 1α,25(OH) 2 D 3 and coactivator, phosphorylation and docking on a high-affinity positive vitamin D response element (VDRE) (mouse osteopontin). See text for explanation. (C) Allosteric model for VDR–RXR inactivation after binding 1α,25(OH) 2 D 3 and corepressor, dephosphorylation and docking in reverse polarity on a high affinity negative VDRE(chicken PTH). See text for explanation. Source: reproduced with permission from Haussler et al. (2011) [3]. 5

6. Copyright © 2013 Elsevier Inc. All rights reserved. FIGURE 13.5 The vitamin D receptor (VDR). (A) Organization of the VDR chromosomal gene. The human VDR gene is located on chromosome 12q13-14 and spans approximately 60 kilobases of DNA. The gene is composed of at least 5 noncoding exons and 8 coding exons. Alternative splicing results in at least 14 types of transcripts. The translation start site (ATG) and termination (TGA) signals are shown in chapter 73. (B) Domains A–Eare shown below the protein model. The DNA-binding domain consists of two zinc finger modules located at the amino terminal portion of the receptor. The ligand-binding domain contains 12α-helices shown as open boxes and 3β-turns shown as a filled box. The E1 and AF-2 subregions of the receptor are important in transactivation. (C) A molecular model of the full retinoid X receptor (RXR)/VDR/deoxyribonucleic acid (DNA) complex. A stereo representation of the cryo-electron microscopic (cryo-EM) map of the complex is fitted with the crystal structures of individual RXR and VDR ligand-binding domains (LBDs) and DNA binding domain (DBDs), resulting in a molecular model of the whole complex. The cryo-EM map is shown in magenta. The DNA is shown in blue with the 5 half-site of the response element in green and the 3 half-site in red. The VDR DBD and LBD are shown in orange and the RXR DBD and LBD shown in cyan. The ligands for VDR and RXR are shown in yellow and blue van der Waals spheres. Helix 12 in the VDR LBD is shown in red. The VDR and RXR LBDs are oriented perpendicular to the DNA and are anchored through the 5 end of the response element. The hinges of both VDR and RXR hold the complex in an open conformation to which coregulators can bind. Source: reproduced with permission from Orlov et al. (2012) [6]. 6

7. Copyright © 2013 Elsevier Inc. All rights reserved. FIGURE 13.6 Mutations in the vitamin D receptor (VDR) causing hereditary vitamin D resistant rickets (HVDRR). A) depicts the two zinc finger modules and the amino acid composition of the deoxyribonucleic acid binding domain (DBD). Conserved amino acids are depicted as shaded circles. Natural mutations are indicated by large arrows. The location of the intron separating exon 2 and exon 3 which encode the separate zinc finger modules is indicated by an arrow labeled intron. Numbers specify amino acid number. B) depicts the location of the α- helices (H1-H12) of the VDR LBD. The α-helices are depicted as filled boxes and the region containing the β- turns is drawn as a cross-hatched box. The E1 and AF-2 regions are shown below the α-helices. The locations of the mutations are indicated. Fs refers to a frameshift mutation. 7

8. Copyright © 2013 Elsevier Inc. All rights reserved. FIGURE 13.7 Mechanism of epithelial calcium transport. Paracellular calcium transport through tight junctions is represented by the paracellular arrow. Transcellular calcium transport is carried out in three steps: (i) following entry through the calcium channels TRPV5 and TRPV6, (ii) calcium will diffuse across the cell bound to calbindin, and (iii) be extruded at the basolateral membrane via an adenosine triphosphate (ATP)-dependent Ca 2+ -ATP-ase (PMCA1b) and Na + /Ca 2+ (NCX1) exchanger mechanism. 1,25(OH) 2 D increases the expression of calcium channels, calbindins, and the extrusion systems (arrows). Source: reproduced with permission from Bindels (2005) [302]. 8

9. Copyright © 2013 Elsevier Inc. All rights reserved. FIGURE 13.8 Immune modulatory effects of vitamin D 3. Vitamin D 3 is acquired via diet or synthesized in the skin upon ultraviolet (UV) exposure. Cells of the immune system possess enzymes (CYP27A1, CYP27B1) to perform the two hydroxylation steps needed to generate bioactive 1,25(OH) 2 D 3. Next, 1,25(OH) 2 D 3 binds to the VDR to induce a range of immune modulatory effects. Source: reproduced with permission from Van Belle et al. (2011) [398]. 9

10. Copyright © 2013 Elsevier Inc. All rights reserved. FIGURE 13.9 The pathophysiologic pathways from vitamin D deficiency to osteoporosis, osteomalacia, falls, and fracture. Source: reproduced with permission from Lips and van Schoor (2011) [409]. 10