Vitamin D: Metabolism, Molecular Mechanism of Action, and Pleiotropic Effects

Abstract

1,25-Dihydroxvitamin D3 [1,25(OH)2D3] is the hormonally active form of vitamin D. The genomic mechanism of 1,25(OH)2D3 action involves the direct binding of the 1,25(OH)2D3 activated vitamin D receptor/retinoic X receptor (VDR/RXR) heterodimeric complex to specific DNA sequences. Numerous VDR co-regulatory proteins have been identified, and genome-wide studies have shown that the actions of 1,25(OH)2D3 involve regulation of gene activity at a range of locations many kilobases from the transcription start site. The structure of the liganded VDR/RXR complex was recently characterized using cryoelectron microscopy, X-ray scattering, and hydrogen deuterium exchange. These recent technological advances will result in a more complete understanding of VDR coactivator interactions, thus facilitating cell and gene specific clinical applications. Although the identification of mechanisms mediating VDR-regulated transcription has been one focus of recent research in the field, other topics of fundamental importance include the identification and functional significance of proteins involved in the metabolism of vitamin D. CYP2R1 has been identified as the most important 25-hydroxylase, and a critical role for CYP24A1 in humans was noted in studies showing that inactivating mutations in CYP24A1 are a probable cause of idiopathic infantile hypercalcemia. In addition, studies using knockout and transgenic mice have provided new insight on the physiological role of vitamin D in classical target tissues as well as evidence of extraskeletal effects of 1,25(OH)2D3 including inhibition of cancer progression, effects on the cardiovascular system, and immunomodulatory effects in certain autoimmune diseases. Some of the mechanistic findings in mouse models have also been observed in humans. The identification of similar pathways in humans could lead to the development of new therapies to prevent and treat disease.

Figure 1.

The metabolic pathway for vitamin D. CYP2R1 has been identified as a key vitamin D 25-hydroxylase. PTH, FGF23/klotho, and 1,25(OH)₂D₃ play key roles in the regulation of optimal levels of 1,25(OH)₂D₃. Only products of 1,25(OH)₂D₃ are represented for the C23 lactone pathway. See text for 25(OH)D products of the C23 lactone pathway.

Figure 2.

Structure of the full human RXR/VDR nuclear receptor heterodimeric complex with its target DNA. The structure of the RXR/VDR complex was determined by single particle cryo-EM and 3D reconstruction. Representation of the cryo-EM map with the fitted crystal structure of the individual RXR and VDR LBDs and DBDs resulting in a molecular model of the full RXR/VDR/DNA complex (top view of the complex). It has been suggested that the carboxy-terminal extension (CTE) of the DBD of VDR extending into the hinge region has a critical role for VDR transcriptional activity. The LBD interface contact comprising helix 4, loop 8/9 of VDR, and helix H7 of RXR is marked with a star. [Adapted from Orlov et al. (335), with permission from John Wiley and Sons.]

Figure 3.

Effects of 1,25(OH)₂D₃ in the intestine. An important function of 1,25(OH)₂D₃ is the stimulation of transcellular intestinal calcium transport by increasing the expression of the apical membrane calcium channel TRPV6 and calcium binding protein calbindin-D_9k. The extrusion of calcium is across the basolateral membrane by PMCA1b. This process is especially enhanced when dietary calcium intake is low. Mouse genetic studies however suggest that other calcium transporters (X) are likely involved. When calcium intake is high, the paracellular calcium transport prevails, but studies suggest that this pathway may also be regulated by 1,25(OH)₂D₃.

Figure 4.

Transgenic (TG) expression of VDR specifically in ileum, cecum, and colon of VDR null mice prevents the abnormal calcium phenotype of VDR null mice. A: the full-length hVDR cDNA was introduced into the multiple cloning cassette under control of the 9.5-kb CDX2 promoter region (9.5 kb CDX2 from E. Fearon). Mice expressing VDR exclusively in the ileum, cecum, and colon were generated by breeding VDR null mice with TG mice expressing hVDR under the control of 9.5-kb CDX2. B, top panel: expression of hVDR was restricted to ileum, cecum, and colon. Mouse (m) VDR was present in WT but not in TG mice or VDR null (KO) mice. Levels of VDR in the distal intestine in TG mice were equivalent or 1.5 upregulated compared with WT. Bottom panel: Van Kossa staining of histolocial sections of tibia showing that the expression of hVDR in the distal intestine (KO/TG1 and KO/TG2) rescues the bone defects associated with systemic VDR deficiency. Serum PTH and serum calcium are normalized in KO/TG1 and KO/TG2 mice (not shown). [From Dhawan et al. (112).]

Figure 5.

Renal VDR actions. In the proximal tubule cells, CYP27B1 expression is suppressed by 1,25(OH)₂D₃ and FGF23. FGF23 also stimulates phosphate excretion by decreasing the expression of the phosphate transporters NPT2a/c in the apical membrane. FGF23 may signal by binding to the few FGFR1-Klotho complexes in the proximal tubules or by inducing a paracrine factor (factor X) in the distal tubules where abundant FGFR1-Klotho complexes are present. Renal calcium reabsorption in the distal tubule is stimulated by 1,25(OH)₂D₃. 1,25(OH)₂D₃ increases the expression of calbindin-D_9k and calbindin-D_28k and to a lesser extent of TRPV5. The extrusion of calcium at the basolateral side is mediated by PMCA1b and NCX1. Two models are proposed on how Klotho and FGF23 regulate TRPV5 expression: 1) secreted Klotho (sKlotho) is considered to hydrolyze sugar residues from the glycan chains on TRPV5 resulting in better entrapment of TRPV5 in the apical membrane; and 2) FGF23 binds to the basolateral FGFR1-Klotho complex, which stimulates intracellular transport of TRPV5 to the plasma membrane.

Figure 6.

Skeletal effects of 1,25(OH)₂D₃ signaling. During a negative calcium balance, when VDR action in the intestine is impaired or dietary calcium intake is low, intestinal calcium absorption is decreased. Normal serum calcium levels can however be maintained by increased 1,25(OH)₂D₃ and PTH levels, which will increase bone resorption and reduce bone matrix mineralization. During a normal or positive calcium balance, normal serum 1,25(OH)₂D₃ levels promote intestinal calcium absorption. This pathway will deliver sufficient calcium for adequate bone matrix mineralization. VDR signaling in osteoprogenitors increases RANKL expression and stimulates osteoclastogenesis, whereas VDR action in mature osteoblasts has anticatabolic actions, by decreasing RANKL expression, and anabolic effects by stimulating LRP-5 signaling.

Figure 7.

1,25(OH)₂D₃-induced signaling pathways involved in the regulation of cell proliferation, apoptosis, and inflammation in cancer. 1,25(OH)₂D₃ hampers the transition from the G₁ to the S phase of the cell cycle either directly, through upregulation of different cyclin-dependent kinase inhibitors, or indirectly through the induction of other growth factors (e.g., TGF-β, EGF). In addition, 1,25(OH)₂D₃ induces apoptosis through activation of the intrinsic apoptotic pathway or by interference with other signaling pathways such as TNF-α, EGF, β-catenin, and prostaglandins. 1,25(OH)₂D₃ has also an immunosuppressive activity, as indicated by the repression of NFκB-mediated gene transcription, which results in a suppressed production of inflammatory cytokines, such as IL-1, IL-6, IL-8, and TNF-α.

Figure 8.

Cooperative effects of dietary vitamin D and IFN-β treatments in attenuation of EAE. On day 0, EAE was induced by immunization with myelin oligodendrocyte glycoprotein (MOG) p35-55 in C57BL/6 mice and scored daily for degree of paralysis. On day 5, the diet was changed to 20 IU vitamin D/g diet or left at 1.5 IU vitamin D/g diet. On day 7, treatment with IFN-β (a first line treatment for multiple sclerosis) was initiated and continued every other day through day 16. The combination of high-dose vitamin D and IFN-β was more effective than high dietary vitamin D alone or IFN-β alone in diminishing paralysis in EAE (R. Axtell, L. Steinman, and S. Christakos, unpublished data).

Figure 9.

Chemical structure of 1,25(OH)₂D₃ and possible analog modifications.

Figure 10.

Overview of clinically approved vitamin D analogs.