Vitamin D and human health: lessons from vitamin D receptor null mice

Abstract

The vitamin D endocrine system is essential for calcium and bone homeostasis. The precise mode of action and the full spectrum of activities of the vitamin D hormone, 1,25-dihydroxyvitamin D [1,25-(OH)(2)D], can now be better evaluated by critical analysis of mice with engineered deletion of the vitamin D receptor (VDR). Absence of a functional VDR or the key activating enzyme, 25-OHD-1alpha-hydroxylase (CYP27B1), in mice creates a bone and growth plate phenotype that mimics humans with the same congenital disease or severe vitamin D deficiency. The intestine is the key target for the VDR because high calcium intake, or selective VDR rescue in the intestine, restores a normal bone and growth plate phenotype. The VDR is nearly ubiquitously expressed, and almost all cells respond to 1,25-(OH)(2)D exposure; about 3% of the mouse or human genome is regulated, directly and/or indirectly, by the vitamin D endocrine system, suggesting a more widespread function. VDR-deficient mice, but not vitamin D- or 1alpha-hydroxylase-deficient mice, and man develop total alopecia, indicating that the function of the VDR and its ligand is not fully overlapping. The immune system of VDR- or vitamin D-deficient mice is grossly normal but shows increased sensitivity to autoimmune diseases such as inflammatory bowel disease or type 1 diabetes after exposure to predisposing factors. VDR-deficient mice do not have a spontaneous increase in cancer but are more prone to oncogene- or chemocarcinogen-induced tumors. They also develop high renin hypertension, cardiac hypertrophy, and increased thrombogenicity. Vitamin D deficiency in humans is associated with increased prevalence of diseases, as predicted by the VDR null phenotype. Prospective vitamin D supplementation studies with multiple noncalcemic endpoints are needed to define the benefits of an optimal vitamin D status.

Figure 1

Structure and structural domains of VDR. A, Schematic representation of the functional domains in the human VDR. A/B, Aminoterminal region. B, Schematic representation of the regulation of gene transcription by ligand-activated VDR-RXR heterodimers. Transcriptional activation requires the action of many multisubunit coactivator complexes that are recruited in a parallel and/or sequential manner (98).

Figure 2

Model for transcellular intestinal calcium absorption. Uptake of calcium in the enterocyte is mediated by TRPV6 and TRPV5, followed by intracellular binding to CaBP-9k and energy-dependent, basolateral extrusion by PMCA_1b. This process is stimulated by 1,25-(OH)₂D/VDR signaling resulting in increased gene expression of TRPV6, TRPV5, and CaBP-9k.

Figure 3

Abnormal growth plate morphology and increased osteoid surface in VDR ablated mice. Goldner staining of tibiae showing abnormal growth plate and increased osteoid volume (dark pink) in VDR null mice compared with VDR WT mice.

Figure 4

Vitamin D endocrine system, phosphate homeostasis, and FGF23. Increased serum phosphate or decreased serum calcium levels (not shown) induce PTH secretion by the parathyroid gland, which stimulates renal 1,25-(OH)₂D synthesis. Increased 1,25-(OH)₂D levels induce FGF23 production by osteoblasts and osteocytes. Both increased FGF23 and PTH levels reduce Na/Pi-2a and Na/Pi-2b expression in kidney and intestine, respectively, resulting in decreased phosphate (re)absorption and lower serum phosphate levels. FGF23 also down-regulates renal 1α-hydroxylase and decreases PTH secretion, creating a multiloop feedback system.

Figure 5

Skin phenotype of VDR null mice. A, Phenotype of 8-month-old VDR null mice with keratinocyte-specific VDR transgene expression. A WT control littermate is on the left, followed by a transgene negative VDR null mouse (KO). The VDR null mouse with keratinocyte-specific expression of a VDR with the LBD mutation (LBDm) does not have a cutaneous phenotype, whereas that expressing the VDR with the AF-2 domain mutation (AF-2m) exhibits significant hair loss. The smaller size of the three KO mice is due to growth retardation associated with abnormal mineral ion homeostasis. [Adapted from K. Skorija et al.: Mol Endocrinol 19:855–862, 2005 (251) Copyright The Endocrine Society.] B, Hematoxylin and eosin staining of the skin from a 70 day old WT mouse (A) and VDR null mice with abnormal (B) and normal (C) mineral ion homeostasis. [Adapted from Y.C. Li et al.: Endocrinology 139:4391–4396, 1998 (165) Copyright The Endocrine Society.]

Figure 6

Skin phenotype, VDR, VDR protein partners, and signaling pathways. The unliganded VDR is part of a multiprotein complex with its heterodimerization partner RXR and the corepressor Hairless (Hr). Each and all together are needed for long-term functional survival of skin stem cells. Moreover they are all three needed for orientation of the stem cells toward a functional hair follicle. VDR physically interacts with wnt target genes, β-catenin, and Lef-1. Both VDR and Hr promote wnt signaling to maintain normal hair cycling.

Figure 7

Cell cycle: 1,25-(OH)₂D-induced signaling pathways involved in the regulation of cell proliferation and apoptosis. 1,25-(OH)₂D blocks the progression of cells from the G₁ to the S phase of the cell cycle either directly or through the induction of other growth factors (e.g., TGFβ). 1,25-(OH)₂D induces the expression of different cyclin-dependent kinase inhibitors (p18, p19, p21, and p27), which inhibit the activity of cyclin/cdk complexes. Reduced cyclin expression after treatment with 1,25-(OH)₂D also contributes to a reduced cyclin/cdk activity. As a result, the pocket proteins retinoblastoma (pRb), p107, and p130 remain in an underphosphorylated state and form complexes with the E2F family of transcription factors. Complexes between the repressor E2F family members E2F4 and E2F5 on the one hand and the pocket proteins p107 and p130 on the other hand are especially thought to associate with promoter regions of E2F target genes in cells that are treated with 1,25-(OH)₂D. In some cell types, 1,25-(OH)₂D is shown to affect cell proliferation through inhibition of EGF-induced Ras-signaling. 1,25-(OH)₂D not only retards cell cycle progression but also induces apoptotic cell death either directly by inhibition of the antiapoptotic protein bcl-2 and by induction of the proapoptotic protein bax or by interfering with other signaling pathways (e.g., EGF, β-catenin, prostaglandins). Inhibition of prostaglandin (PGE₂)-signaling, either by a reduction of prostaglandin synthesis or by a decrease in expression of prostaglandin receptors (EP1–4) after treatment with 1,25-(OH)₂D, also contributes to the growth-inhibitory and proapoptotic effects of 1,25-(OH)₂D. 1,25-(OH)₂D also interacts with β-catenin-signaling. β-catenin is required for cell-cell adhesion and for the regulation of gene expression in response to Wnt-signaling. 1,25-(OH)₂D is able to transrepress β-catenin/T cell factor (TCF) signaling through the rapid induction of VDR/β-catenin interaction and the subsequent expression of E-cadherin, which promotes the redistribution of β-catenin to the plasma membrane. On the other hand, high levels of β-catenin are shown to potentiate the ligand-dependent activation of VDR-regulated promoters.

Figure 8

Overview of vitamin D homeostasis in the immune system. Both 25-hydroxylase and 1α-hydroxylase are present in APCs such as DCs and macrophages resulting in the local production of active 1,25-(OH)₂D. In the steady state and the early phase of inflammation, 1α-hydroxylase is absent or low. In IFNγ- and TLR ligand (TLR-L)-activated macrophages, 1α-hydroxylase is induced and 1,25-(OH)₂D is generated. Under steady-state conditions, 1,25-(OH)₂D induces 24-hydroxylase expression through a VDR-dependent mechanism creating a self-regulatory feedback loop and enabling 1,25-(OH)₂D to fulfill its role in maintaining immune balance. In conditions of infection or inflammation, up-regulation of 24-hydroxylase is, however, hindered by IFNγ-induced STAT1α (signal transducers and activators of transcription-1α) activity resulting in continued high 1,25-(OH)₂D levels and thus sustained and protective antimicrobial activity. In parallel, VDR is up-regulated in the early stage of inflammation after TLR activation and down-regulated in APCs at later stages of activation. Solid line, Stimulatory pathway; dotted line, inhibitory pathway.

Figure 9

Overview of the pharmacological effects of 1,25-(OH)₂D in the immune system. 1,25-(OH)₂D inhibits the surface expression of MHC II-complexed antigen and of costimulatory molecules, as well as the production of the cytokine IL-12 in APCs (such as DC), thereby shifting the polarization of T cells from an (auto-)aggressive effector (Te) toward a protective or regulatory (Tr) phenotype. 1,25-(OH)₂D exerts its immunomodulatory effects also directly on the level of T cells. Together, these immunomodulatory effects of 1,25-(OH)₂D onto players of the adaptive immune system can lead to the protection of target tissues in autoimmune diseases and transplantation. In the innate immune system on the other hand, 1,25-(OH)₂D strengthens the antimicrobial function of monocytes and macrophages, for example through enhanced expression of the CAMP, eventually leading to better clearance of pathogenic microorganisms.

Figure 10

1,25-(OH)₂D-VDR effects on cardiovascular cells. The vitamin D endocrine system generates mostly positive direct (on cells of the vascular wall and cardiomyocytes) or indirect effects regulating the production of pro/antithrombotic/fibrinolytic factors. PAI-1, Plasminogen activator inhibitor 1; BNP,brain natriuretic peptide.

Figure 11

VDR-vitamin D endocrine system and the renin-angiotensin system. 1,25-(OH)₂D directly down-regulates the renal production of renin and thereby regulates the renin-angiotensin system, systemic blood pressure, and cardiac function.