INTRODUCTION

TOP ABSTRACT INTRODUCTION VITAMIN D METABOLISM REGULATION OF 1,25(OH)2D3... TRANSPORT OF VITAMIN D VITAMIN D RECEPTOR NONGENOMIC ACTIONS OF 1,25(OH)2... REGULATION OF 1,25(OH)2D3-VDR... BIOLOGICAL ACTIONS OF VITAMIN... REFERENCES

VITAMIN D plays a central role in calcium and phosphate homeostasis and is essential for the proper development and maintenance of bone. The recognition by Sir Edward Mellanby in 1919 (132) that rickets could be caused by a nutritional deficiency led to the isolation of a fat-soluble antirachitic substance in fish liver oil and other foods that was identified as vitamin D₂. At the same time, Huldschinsky (89) and Hess and Unger (78) discovered that children with rickets could be cured by exposing them to ultraviolet light. Antirachitic activity could also be induced in various foods by ultraviolet irradiation. Continuing studies of these antirachitic substances led to the structural identification of vitamin D₂ (ergocalciferol) (5) and vitamin D₃ (cholecalciferol) (186) as secosterols, derived from the photolytic cleavage of the B rings of ergosterol and 7-dehydrocholesterol, respectively.

These two compounds were considered the biologically active forms of vitamin D until the mid-1960s, when the availability of radiolabeled vitamin D₃ (146) permitted the identification of metabolites with greater antirachitic activity. 25-Hydroxyvitamin D₃ [25(OH)D₃] was found to be the major circulating metabolite of vitamin D₃ (17) and subsequently was shown to be produced primarily in the liver. In 1969, Haussler et al. (71) found a metabolite more polar than 25(OH)D₃ in the nuclear fraction from intestine. This molecule, synthesized mainly in the kidney, was identified as 1,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃] (53, 83, 108) and is now known to be the most active metabolite of vitamin D.

The next major breakthrough in vitamin D research was the discovery of a high-affinity receptor for 1,25-dihydroxyvitamin D [1,25(OH)₂D] (71). This 50- to 70-kDa protein facilitated association with nuclear chromatin, displayed saturable binding of 1,25(OH)₂D₃, and had a specificity for other vitamin D metabolites that precisely matched their in vivo biopotency.

The development of monoclonal antibodies directed against the vitamin D receptor (VDR) allowed the isolation of cDNAs coding for the avian, human, mouse, and rat VDRs (131),(6, 28), (94). The sequence of the VDR revealed considerable similarity to other members of the steroid receptor superfamily including the characteristic two zinc finger motifs in a DNA-binding domain. This suggested that VDR was also a ligand-activated transcription factor. The 1,25(OH)₂D₃-activated VDR interacts with specific DNA sequences within vitamin D-responsive genes and regulates their rates of transcription.

The VDR was found originally in the classic vitamin D target organs involved in mineral homeostasis: the intestine, bone, kidney, and the parathyroid glands. More recently, the VDR has been detected in many other tissues and cells types as well. These nonclassic vitamin D target organs respond to 1,25(OH)₂D₃ with a diverse range of biological actions including immunomodulation, the control of other hormonal systems, inhibition of cell growth, and induction of cell differentiation. These actions have suggested a number of new therapeutic applications of 1,25(OH)₂D₃ in immune dysfunction (autoimmune disease), endocrine disorders (hyperparathyroidism), and hyperproliferative disorders (leukemia, cancer, psoriasis).

	VITAMIN D METABOLISM

TOP ABSTRACT INTRODUCTION VITAMIN D METABOLISM REGULATION OF 1,25(OH)2D3... TRANSPORT OF VITAMIN D VITAMIN D RECEPTOR NONGENOMIC ACTIONS OF 1,25(OH)2... REGULATION OF 1,25(OH)2D3-VDR... BIOLOGICAL ACTIONS OF VITAMIN... REFERENCES

Vitamin D, derived from the diet or by bioactivation of 7-dehydrocholesterol, is inert and must be activated to exert its biological activity. The steps involved are illustrated in Fig. 1 and are discussed below.

View larger version (15K): [in this window] [in a new window]   Fig. 1.   Vitamin D metabolism: steps involved in production of vitamin D3 in the skin and its subsequent activation to 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] and catabolism to the water-soluble metabolite, calcitroic acid.

Sources of Vitamin D

25-Hydroxylation of Vitamin D

The 25-hydroxylation of vitamin D is poorly regulated. The levels of 25(OH)D increase in proportion to vitamin D intake, and for this reason, plasma 25(OH)D levels are commonly used as an indicator of vitamin D status (81).

Formation of 1,25(OH)₂D

The 1-hydroxylase has been cloned from mouse kidney (177), rat kidney (167, 170), human kidney (134), and keratinocytes (55). Expression of the protein in cultured cells promotes 1-hydroxylation of 25(OH)D₃. Further evidence for the identity of the human 1-hydroxylase cDNA came from chromosomal mapping and mutational analysis. The cDNA hybridizes solely to chromosomal locus 12q13.1-q13.3, the site to which the defect in patients unable to produce 1,25(OH)₂D₃ (vitamin D-dependent rickets type I) has been mapped (107). Moreover, mutations in the coding regions of the 1-hydroxylase gene have been identified in patients with the disease (55, 99, 195).

Vitamin D Catabolism

Additional pathways for metabolism of 1,25(OH)₂D₃ have been described. The vitamin D hormone can be converted to the 1,25(R)-(OH)₂D₃-23(S),26-lactone following hydroxylations at the 23(S) and 26 positions (91). The lactone is a minor circulating metabolite of 1,25(OH)₂D₃ in the blood (148). Interestingly, 1,25(R)-(OH)₂D₃-23(S),26-lactone appears to have anti-vitamin D action in that it inhibits the 1,25(OH)₂D₃-stimulated fusion of mouse bone marrow mononuclear cells (90). More recent studies by Reddy and coworkers (14, 24) demonstrated that the 3-hydroxyl group of 1,25(OH)₂D₃ can be epimerized to the 3 position. The activity is cell specific and has been detected in intestinal cells, parathyroid cells, keratinocytes, and osteoblast-like cells, but not in myeloid leukemia cells or perfused kidney. The 1,25(OH)₂-3-epi-D₃ appears to be catabolized more slowly than the parent hormone and retains significant biological activity. The role of this pathway is under investigation.

	REGULATION OF 1,25(OH)2D3 LEVELS

TOP ABSTRACT INTRODUCTION VITAMIN D METABOLISM REGULATION OF 1,25(OH)2D3... TRANSPORT OF VITAMIN D VITAMIN D RECEPTOR NONGENOMIC ACTIONS OF 1,25(OH)2... REGULATION OF 1,25(OH)2D3-VDR... BIOLOGICAL ACTIONS OF VITAMIN... REFERENCES

The stringent control of serum 1,25(OH)₂D₃ levels is dictated by the calcium and phosphorus needs of the animal, and exerted through the coordinated action of classic mineral-regulating target organs, the kidney, intestine, bone, and the parathyroid glands. The major regulators of 1,25(OH)₂D₃ levels are parathyroid hormone (PTH), calcium, phosphate, and 1,25(OH)₂D₃ itself. There are many other factors that have been reported to influence 1,25(OH)₂D₃ synthesis and/or degradation, but their physiological importance is not clear.

Parathyroid Hormone

These effects of PTH are exerted directly on the renal proximal tubular cells (77) and are mediated by cAMP (162). In addition, PTH decreases 24-hydroxylase activity and slows degradation of 1,25(OH)₂D₃ (74). Recent studies have shown that PTH treatment alters the levels of 1-hydroxylase mRNA (167, 170) through effects on 1-hydroxylase gene transcription (21, 138).

Calcium

Acidosis

Phosphate

The 24-hydroxylase is regulated by phosphate in the opposite manner (178). Recent evidence indicates that the control of the 1-hydroxylase and 24-hydroxylase by phosphate is through changes in mRNA levels (190). In vitro effects of phosphate on the vitamin D hydroxylases in isolated kidney mitochondria, tubules, and cultured cells are inconclusive.

Insulin and IGF-I

1,25(OH)₂D₃

	TRANSPORT OF VITAMIN D

TOP ABSTRACT INTRODUCTION VITAMIN D METABOLISM REGULATION OF 1,25(OH)2D3... TRANSPORT OF VITAMIN D VITAMIN D RECEPTOR NONGENOMIC ACTIONS OF 1,25(OH)2... REGULATION OF 1,25(OH)2D3-VDR... BIOLOGICAL ACTIONS OF VITAMIN... REFERENCES

Vitamin D and its hydroxylated metabolites 25(OH)D, 24,25(OH)₂D, and 1,25(OH)₂D are lipophilic molecules. Because of their low solubility in the aqueous media of plasma, vitamin D compounds are transported in the circulation bound to plasma proteins. The most important of these carrier proteins is the vitamin D-binding protein (DBP). The relative affinities of vitamin D metabolites for DBP are 25(OH)D = 24,25(OH)₂D > 1,25(OH)₂D > vitamin D (43). The dissociation constants for 25(OH)D and 1,25(OH)₂D differ by ~10-fold. In mammals, vitamin D₂ and vitamin D₃ metabolites exhibit the same relative affinities for DBP, whereas avian DBP preferentially binds vitamin D₃ metabolites 100 times more avidly than their vitamin D₂ counterparts. DBP is synthesized in the liver and circulates in plasma at concentrations 20 times higher than the total amount of vitamin D metabolites. The role of the large molar excess of circulating DBP, which differs from carrier proteins for other steroid hormones, is uncertain. Since DBP has a single sterol binding site, only 5% of the total DBP of normal human plasma is occupied with vitamin D compounds. Therefore, under normal physiological conditions, nearly all circulating vitamin D compounds are protein bound, which has a great influence on vitamin D pharmacokinetics. DBP-bound vitamin D metabolites have limited access to target cells (43) and are, therefore, less susceptible to hepatic metabolism and subsequent biliary excretion, which prolongs their half-life in circulation. In fact, recent studies in the DBP null mice confirm the critical role of DBP in prolonging the half-life of vitamin D and its metabolites in circulation (164). However, several studies demonstrated that it is the free, DBP-unbound form of vitamin D metabolites that exhibits greater accessibility to target cells and therefore a higher biological response both in vitro and in vivo (11, 23, 50). For example, 1,25(OH)₂D₃ stimulation of 24-hydroxylase in keratinocytes and monocytes, as well as 1,25(OH)₂D₃ inhibition of lymphocyte proliferation correlate with free rather than total concentration of the hormone. Moreover, the DBP null mice fed a vitamin D-replete diet lack the abnormalities in serum PTH and bone morphology associated with vitamin D deficiency despite low "total" serum levels of 25(OH)D₃ and 1,25(OH)₂D₃ (164), thus supporting the "free hormone hypothesis" of simple diffusion of the unbound sterol through the plasma membrane into the target cell. Therefore, by buffering the levels of free vitamin D compounds, DBP plays an important role in guarding against vitamin D intoxication (19). The validity of this assertion has recently been challenged by the unexpected finding that, after a vitamin D overload, the DBP null mouse was less susceptible than its normal counterpart to hypercalcemia and its toxic effects (164). An explanation for this apparent contradiction came from the unexpected finding that DBP and DBP-bound vitamin D metabolites are filtered through the glomerulus and reabsorbed by the endocytic receptor megalin into the proximal tubular cell (147). Megalin-mediated endocytosis of DBP-bound 25(OH)D appears to be the major pathway to preserve circulating levels of 25(OH)D and for the metabolic activation of 25(OH)D₃ to 1,25(OH)₂D₃ by the renal 1-hydroxylase. In fact, megalin null mice elicit high urinary excretion of 25(OH)D₃ and DBP, severe vitamin D deficiency, and bone disease (147). Therefore, in the absence of DBP, despite vitamin D overload, the major pathway for renal uptake and activation of 25(OH)D₃ to 1,25(OH)₂D₃ is blunted, thus preventing hypercalcemia and 1,25(OH)₂D₃ toxicity.

The contribution of megalin-mediated endocytosis to either the cellular uptake of 25(OH)D₃ from circulating 25(OH)D₃-DBP complexes by extrarenal sources of 1,25(OH)₂D₃ or in the delivery of DBP-bound 1,25(OH)₂D₃ from the circulation to target cells is unclear at present.

The plasma concentrations of DBP are not regulated by vitamin D, but megalin levels are regulated by 1,25(OH)₂D₃ (119). DBP levels decrease under pathological conditions such as liver disease, nephrotic syndrome, and malnutrition, and increase during pregnancy and estrogen therapy. The concentration of free 1,25(OH)₂D₃, however, remains constant when DBP levels change, an example of the tight self-regulation of vitamin D metabolism.

Albumin and lipoproteins are also important plasma carrier proteins with lower affinities for vitamin D metabolites than DBP. Vitamin D administered parenterally binds to both lipoproteins and DBP. However, lipoproteins are more efficient than DBP to deliver the vitamin D₃ synthesized in the skin to the hepatocyte for 25-hydroxylation, whereas lymph chylomicrons mediate the intestinal absorption and hepatic uptake of the vitamin D ingested in the diet (65). Since a fraction of serum 1,25(OH)₂D₃ circulates bound to lipoproteins (153) and there are striking similarities between megalin- and LDL receptor-mediated endocytosis, it is possible that a cell-specific, receptor-mediated process also contributes to the delivery of 1,25(OH)₂D₃ from plasma carriers into target cells expressing megalin and/or LDL receptor. This possibility remains to be examined.

	VITAMIN D RECEPTOR

TOP ABSTRACT INTRODUCTION VITAMIN D METABOLISM REGULATION OF 1,25(OH)2D3... TRANSPORT OF VITAMIN D VITAMIN D RECEPTOR NONGENOMIC ACTIONS OF 1,25(OH)2... REGULATION OF 1,25(OH)2D3-VDR... BIOLOGICAL ACTIONS OF VITAMIN... REFERENCES

Most of the biological activities of 1,25(OH)₂D₃ are mediated by a high-affinity receptor that acts as a ligand-activated transcription factor. The VDR has been cloned from chicken, human, mouse, and rat (131, 28, 6, 94). Expression of the VDR cDNA in a variety of systems has allowed the structure-function analysis of the VDR protein (Fig. 2) and provided new insights into the mechanisms mediating the actions of 1,25(OH)₂D₃. The majors steps involved in the control of gene transcription by the VDR include 1) ligand binding, 2) heterodimerization with retinoid X receptor (RXR), 3) binding of the heterodimer to vitamin D response elements (VDREs), and 4) recruitment of other nuclear proteins into the transcriptional preinitiation complex. These steps are illustrated in Fig. 3 and are discussed in detail below.

View larger version (20K): [in this window] [in a new window]   Fig. 2.   Functional domains of the human vitamin D receptor (VDR).

View larger version (33K): [in this window] [in a new window]   Fig. 3.   Regulation vitamin D action. Major sites at which the genomic actions of 1,25(OH)2D3 can be regulated are highlighted and discussed in detail in the text. RXR, retinoid X receptor; DBP, vitamin D binding protein.

Ligand Binding

10

11

Heterodimerization with RXR

Binding of the VDR-RXR Heterodimer to DNA

Several VDREs have been characterized in the promoter region of vitamin D-regulated genes. Although there is considerable variation between natural VDREs, a consensus positive VDRE can be defined as a direct repeat (DR) of two six-base half elements of the sequence AGGTCA, separated by a spacer of three nucleotides (DR-3). This sequence directs the VDR-RXR heterodimer to the promoter region of 1,25(OH)₂D₃-regulated genes, with the RXR binding the 5' half site and the VDR occupying the 3' half site (72). The VDREs of genes that are negatively regulated by vitamin D have been characterized. These genes include avian and human PTH, mouse osteocalcin, rat PTH-related peptide, rat bone sialoprotein, protein kinase A (PKA) inhibitor, and interleukin-2 genes. The VDREs of the human PTH and mouse osteocalcin genes are similar to the DR-3 sequence found in genes in which transcription is induced by vitamin D. This finding raises important questions regarding the mechanisms determining whether gene transcription will be induced or suppressed by 1,25(OH)₂D₃ (47). Interestingly, by changing the two 3'-terminal bases GT of the avian PTH VDRE to the consensus CA, the VDRE reversed from a negative to a positive regulator of transcription (103). Similarly, rat and mouse osteocalcin VDREs differ only in the two bases at position 4 and 5 in the 5' half element. It is possible that, as for RAR-RXR heterodimers, the polarity of the VDR-RXR changes so that for negative regulation of gene transcription, the VDR occupies the 5'-half element (72). VDR-RXR binding to the DNA causes a bend of ~55 degrees from the horizontal. The impact of DNA bending on transactivation by the VDR is still unclear (172).

VDR interactions with Nuclear Transcriptional Components and Coactivators

	NONGENOMIC ACTIONS OF 1,25(OH)2D3 ARE MEDIATED BY A CELL SURFACE RECEPTOR

TOP ABSTRACT INTRODUCTION VITAMIN D METABOLISM REGULATION OF 1,25(OH)2D3... TRANSPORT OF VITAMIN D VITAMIN D RECEPTOR NONGENOMIC ACTIONS OF 1,25(OH)2... REGULATION OF 1,25(OH)2D3-VDR... BIOLOGICAL ACTIONS OF VITAMIN... REFERENCES

Steroid hormones can also elicit responses that are too rapid to involve changes in gene expression and appear to be mediated by cell surface receptors. Nongenomic effects of 1,25(OH)₂D₃ include rapid changes in phosphoinositide metabolism (20, 116, 135), increases in intracellular calcium levels (115, 173) (84, 120, 135), stimulation of intestinal calcium transport (141) and phosphate fluxes (96), elevation in cGMP levels (63, 180), and activation of protein kinase C (PKC) (174).

The receptor that mediates the rapid actions has been partially characterized. It is clear that the nongenomic responses are carried out by a distinct receptor (7, 113, 139). The ligand specificity for the rapid actions is different from that for the genomic response (139), and these rapid responses still occur in osteoblasts from VDR knockout mice (113). A plasma membrane protein from chick duodenum has been isolated recently that binds vitamin D analogs with affinities that correlate with their activation of the rapid responses (139). Subsequent studies utilizing an antibody raised against this protein demonstrated the same 66-kDa protein in rat chondrocytes. The antibody blocks the rapid stimulation of PKC by 1,25(OH)₂D₃ in these cells (140). A candidate membrane receptor in rat osteoblast-like (ROS 24/1) cells has been identified as annexin-2 (7). This protein binds 1,25(OH)₂D₃, and antibodies to annexin-2 inhibit the nongenomic actions of the hormone. However, annexin-2 is significantly smaller (36 kDa) than the membrane receptor in chick intestine and rat chondrocytes. These initial findings indicate that there may be multiple membrane receptors that mediate the rapid actions of 1,25(OH)₂D₃.

The role of the nongenomic actions in most cells remains uncertain. In chick duodenum, 1,25(OH)₂D₃ stimulates calcium movement from the lumen to the basolateral surface within minutes. In other cells, the role is less clear. However, it is tempting to speculate that activation of second messenger pathways may influence the activity of the nuclear VDR. For example, one rapid action of 1,25(OH)₂D₃ is to increase PKC activity. Phosphorylation of the VDR by PKC has been shown to decrease its transcriptional activity. Thus the rapid actions may modulate the genomic response to 1,25(OH)₂D₃.

	REGULATION OF 1,25(OH)2D3-VDR ACTIONS

TOP ABSTRACT INTRODUCTION VITAMIN D METABOLISM REGULATION OF 1,25(OH)2D3... TRANSPORT OF VITAMIN D VITAMIN D RECEPTOR NONGENOMIC ACTIONS OF 1,25(OH)2... REGULATION OF 1,25(OH)2D3-VDR... BIOLOGICAL ACTIONS OF VITAMIN... REFERENCES

A number of factors can influence the VDR-mediated actions of 1,25(OH)₂D₃, including ligand accessibility to the VDR, the cellular content of the VDR, posttranslational modifications to the receptor, and the availability of nuclear coactivators. These regulatory factors are highlighted in Fig. 3.

Ligand Availability

VDR Content

VDR content can also be controlled at the level of synthesis and degradation. Although there is no evidence for translational control of the VDR, ligand-dependent stabilization of the VDR protein appears to occur in virtually all cell types (2). The mechanisms implicated in VDR degradation have only recently been addressed. Masuyama et al. (123) documented a direct interaction of liganded VDR with SUG1, a component of the proteasome complex, known to target many cellular proteins for ubiquitination and subsequent proteolysis. The use of proteasome inhibitors confirmed the degradation of the VDR by this pathway. The role of SUG1 binding to the VDR in the termination of the signal for 1,25(OH)₂D₃-VDR transcriptional activity is currently under investigation, since mSUG1 was also shown to act as a coactivator more specific for the VDR than SRC-1 (181).

VDR Polymorphisms

Posttranslational Modifications of the VDR

Induction of posttranslational modifications of the VDR by substances from uremic plasma ultrafiltrate also disrupt VDR-RXR binding to DNA, presumably by covalently modifying the VDR at or near the DBD (87). This may partially account for the resistance to vitamin D commonly associated with chronic uremia.

Nuclear Levels of Transcriptional Cofactors

	BIOLOGICAL ACTIONS OF VITAMIN D

TOP ABSTRACT INTRODUCTION VITAMIN D METABOLISM REGULATION OF 1,25(OH)2D3... TRANSPORT OF VITAMIN D VITAMIN D RECEPTOR NONGENOMIC ACTIONS OF 1,25(OH)2... REGULATION OF 1,25(OH)2D3-VDR... BIOLOGICAL ACTIONS OF VITAMIN... REFERENCES

The genomic and nongenomic actions of vitamin D combine to produce a multitude of responses in an ever-increasing list of target cells. The VDR has been found in the classic vitamin D target organs, namely, the intestine, bone, kidney, and the parathyroid glands, as well as a host of target tissues not involved in calcium homeostasis, such as skin, muscle, pancreas, reproductive organs, and the hematopoietic, immune, and nervous systems (10, 42, 71). This section and Table 1 summarize the VDR distribution and the biological actions of 1,25(OH)₂D₃ in these target tissues. In addition, Fig. 4 illustrates the basic mechanism of action and lists some of the genes regulated by 1,25(OH)₂D₃.

View larger version (23K): [in this window] [in a new window]   Fig. 4.   Current model for the control of VDR-mediated actions of 1,25(OH)2D3. The activated vitamin D hormone, 1,25(OH)2D3, circulates in the blood bound to vitamin D binding protein (DBP). 1,25(OH)2D3 taken up by target cells is either degraded by the mitochondrial 24-hydroxylase or bound to the vitamin D receptor (VDR). The 1,25(OH)2D3-VDR complex heterodimerizes with retinoid X receptor (RXR), and the VDR/RXR heterodimer binds specific sequences in the promoter regions of target genes. The DNA-bound heterodimer attracts components of the RNA polymerase II (Pol II) preinitiation complex and nuclear transcriptional coactivators, thereby altering the rate of gene transcription. The mRNA is processed and released to the cytoplasm where it is translated into proteins. A list of gene products known to be up- () or downregulated () at the transcriptional level is shown. PTH, parathyroid hormone; PTH-RP, PTH-related peptide; IL-2, interleukin-2.

Classic Vitamin D-Responsive Tissues

1,25(OH)₂D₃ increases the entry of calcium through the plasma membrane into the enterocyte, the movement of calcium through the cytoplasm, and the transfer of calcium across the basolateral membrane into the circulation. 1,25(OH)₂D₃ is the only hormone known to stimulate intestinal calcium transport directly. Other vitamin D metabolites can stimulate calcium transport, but only at higher doses, consistent with their lower affinity for the VDR. The mechanism for stimulation of transcellular calcium transport is not entirely clear, but induction of a cytosolic calcium-binding protein (calbindin D) and the basolateral calcium pump undoubtedly are important components (183).

Increasing evidence suggests that the VDR-mediated effects of 1,25(OH)₂D₃ may not be the only mode of action by which the hormone stimulates calcium absorption by the enterocyte. Rapid effects of 1,25(OH)₂D₃ appear to mediate an increase in both the vesicular and paracellular pathways for intestinal calcium absorption. The actual contribution of these nongenomic pathways to intestinal calcium absorption in vivo is unclear.

In addition to its effects on calcium absorption, 1,25(OH)₂D₃ increases active phosphate transport. However, significant phosphate absorption also occurs in 1,25(OH)₂D₃-deficient states (185). The sterol directly stimulates the expression of the Na-P_i cotransporter (191) and affects the composition of the enterocyte plasma membrane, increasing fluidity and phosphate uptake. Sodium-independent entry of phosphate occurs independently of vitamin D status (46). Little is known, however, concerning the molecular mechanisms involved in the extrusion of phosphate across the basolateral membrane into the circulation.

Skeleton. Vitamin D is essential for the development and maintenance of a mineralized skeleton. Vitamin D deficiency results in rickets in young growing animals and osteomalacia in adults. 1,25(OH)₂D₃ induces bone formation by inducing the synthesis of bone matrix proteins and mineral apposition. However, several studies, including the most recent findings from the VDR null mice, have demonstrated that vitamin D is not absolutely essential for the ossification process. It is apparent, therefore, that vitamin D induces bone mineralization by increasing serum levels of calcium and phosphate. The higher potency of 1,25(OH)₂D₃ in regulating mineral homeostasis makes it the most likely vitamin D metabolite involved in bone mineralization. Although controversial, 24,25(OH)₂D₃ may be required in bone and cartilage formation, as suggested by the bone abnormalities found in the 24-hydroxylase knockout mice (169).

1,25(OH)₂D₃ also maintains normal serum calcium and phosphate by inducing bone resorption through enhancement of osteoclastogenesis and osteoclastic activity. Strong evidence suggests, however, that osteoblasts and osteoblast-derived substances are required for 1,25(OH)₂D₃ induction of osteoclastic bone resorption. An in vitro system for studying the osteoclastogenic activity of 1,25(OH)₂D₃ has been established in which osteoblast/stromal cells are cocultured with osteoclast precursor cells (e.g., spleen cells). Mature, multinucleated osteoclasts are produced in a 1,25(OH)₂D₃-dependent fashion in this model. However, other hormones, including PTH, can substitute for 1,25(OH)₂D₃ in this system and may explain the normal bone development in the calcium-supplemented VDR-ablated mice (112).

Parathyroid glands. PTH and 1,25(OH)₂D₃ directly affect calcium homeostasis, and each exerts important regulatory effects on the other. Whereas PTH is the principal hormone involved in the minute-to-minute regulation of ionized calcium levels in the extracellular fluid, 1,25(OH)₂D₃ plays a key role in the day-to-day maintenance of calcium balance. PTH stimulates the production of 1,25(OH)₂D₃ by activating the renal 1-hydroxylase (21, 138), and 1,25(OH)₂D₃ in turn suppresses the synthesis and secretion of PTH (32, 37) and controls parathyroid cell growth (175). Vitamin D deficiency, therefore, causes parathyroid hyperplasia and secondary hyperparathyroidism. 1,25(OH)₂D₃ suppression of PTH synthesis occurs through negative regulation of the rate of PTH gene transcription by the 1,25(OH)₂D₃-VDR/RXR complex (118). The correction of parathyroid gland growth and circulating levels of PTH in the VDR null mice (176), through dietary supplementation, suggests that the VDR is not essential but cooperative with calcium and phosphate in the control of PTH synthesis and parathyroid cell proliferation (112).

Kidney. The most important effects of 1,25(OH)₂D₃ in the kidney are the suppression of 1-hydroxylase activity and the stimulation of 24-hydroxylase activity. Both effects of the sterol are VDR mediated. The role of vitamin D in the renal handling of calcium and phosphate continues to be controversial due to the simultaneous effects of the sterol on intestinal calcium and phosphate, which affects renal filtered load, and on serum PTH levels. Comprehensive in vivo studies demonstrated, however, that 1,25(OH)₂D₃ increases renal calcium reabsorption (192). 1,25(OH)₂D₃ enhances calcium reabsorption (12) and calbindin expression (13), and it accelerates PTH-dependent calcium transport in the distal tubule (54), the site with the highest VDR content (98) and where active calcium transport is known to occur. The effect of 1,25(OH)₂D₃ enhancing renal absorption of phosphate only in the presence of PTH suggests that this may not be a direct action of the sterol on the kidney but rather the result of 1,25(OH)₂D₃ suppression of PTH.

Control of mineral homeostasis. The role of the vitamin D endocrine system in calcium and phosphate homeostasis is illustrated in Fig. 5. The responses to increases or decreases in serum calcium and phosphate involve coordinated actions of the parathyroid glands, kidney, intestine, and bone. The complexity of these interactions ensure the availability of the minerals for a host of biological functions as well as skeletal mineralization.

View larger version (66K): [in this window] [in a new window]   Fig. 5.   Role of 1,25(OH)2D3 in calcium and phosphate homeostasis. Integrated roles of 1,25(OH)2D3 and PTH in the tissues (kidney, intestine, and bone) involved in calcium and phosphate homeostasis. A: response to hypocalcemia. B: response to hypercalcemia. C: response to hypophosphatemia. D: response to hyperphosphatemia.

Nonclassic Vitamin D-Responsive Tissues

1,25(OH)₂D₃ also inhibits clonal proliferation in a variety of human leukemia cell lines and promotes the differentiation of normal and leukemic myeloid precursors toward more mature, less aggressive phenotypes, thus rendering the sterol potentially useful in the treatment of leukemias and other myeloproliferative disorders (1). The 1,25(OH)₂D₃-VDR directly induces the expression of the cyclin-dependent kinase inhibitor p21 (117). Increases of p21 appear to be sufficient to arrest growth and promote differentiation in cells of the monocyte-macrophage lineage and may mediate the antiproliferative, prodifferentiating activities of 1,25(OH)₂D₃ in other cell types.

The immune system. Several clinical observations suggested a role for vitamin D in immunology prior to the finding of the VDR in cells of the immune system. Recurrent infections are commonly associated with vitamin D-deficient rickets (171), and an impaired defense mechanism often accompanies chronic renal failure (4), a state of prolonged 1,25(OH)₂D₃ deficiency. In both conditions, the impaired immunity can be improved with 1,25(OH)₂D₃ therapy.

1,25(OH)₂D₃ interacts with mature monocytes and macrophages, enhancing their immune function and improving host defense against both bacterial infection and tumor cell growth. In addition, 1,25(OH)₂D₃ promotes macrophage survival and function at the increased temperatures associated with tissue inflammation by inducing heat shock protein synthesis.

In contrast to the stimulatory effects of the hormone on monocytes and macrophages, the principal action of 1,25(OH)₂D₃ in lymphocytes is to act as an immunosuppressive agent (111). It does so by decreasing both the rate of proliferation and the activity of T cells and B cells, and by inducing the availability of suppressor T cells, which further contributes to limiting lymphocyte activity. An important aspect of these immunosuppressive actions of 1,25(OH)₂D₃ is the therapeutic application of the sterol in the control of autoimmune diseases such as systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, juvenile (type I) diabetes, experimental autoimmune encephalomyelitis in mice, and transplant rejection. The production of 1,25(OH)₂D₃ by activated macrophages at sites of active inflammation may represent an autocrine-paracrine system that inhibits further T cell activation and lymphokine production, thus preventing a potentially self-destructive immune response.

Skin. Vitamin D was used to treat a variety of skin diseases including psoriasis in the 1930s. It was in the mid 1980s, however, when its therapeutical potential in skin diseases reemerged as a result of the observation that psoriatic lesions dramatically improved in a patient receiving oral 1-hydroxyvitamin D₃ to treat severe osteoporosis (136). The antiproliferative and prodifferentiating effects of 1,25(OH)₂D₃ in keratinocytes, melanocytes, and fibroblasts and its immunosuppressive properties on Langerhan's cells, the antigen-presenting cells of the skin, have been exploited in the treatment of psoriasis, melanoma, and scleroderma. The alopecia in patients with hereditary vitamin D-resistant rickets and the high expression of VDR in the hair follicle suggest a role for 1,25(OH)₂D₃ or the VDR in the hair cycle. Interestingly, unlike the other phenotypic features of vitamin D-resistant in mice lacking VDR, normalization of serum calcium and phosphate fails to correct the alopecia, dilated hair follicles, and dermal cysts (112). These findings demonstrate an essential role of the VDR in hair and skin development.

Muscle. Skeletal muscle weakness and atrophy, with electrophysiological abnormalities in muscle contraction and relaxation, often occur in vitamin D deficiency, in calcitriol deficiency due to chronic renal failure, and with the prolonged use of anticonvulsant agents that decrease serum 25(OH)D₃. These effects were originally attributed to low serum calcium levels; however, various studies showed direct effects of vitamin D on skeletal muscle function (18). In the heart, vitamin D deficiency results in cardiomegaly, whereas 1,25(OH)₂D₃ controls hypertrophy in cardiac myocytes (189) and the synthesis and release of atrial natriuretic factor (188). 1,25(OH)₂D₃ and 25(OH)D₃ improve both the left ventricular function in patients with cardiomyopathies and the skeletal muscle weakness secondary to end-stage renal disease. Although the mechanisms involved are still unclear, the selectivity of vitamin D analogs in modulating muscle cell calcium metabolism and growth suggest their therapeutic potential to treat vitamin D-dependent myopathies (166).

Pancreas. Vitamin D deficiency results in impaired glucose-mediated insulin secretion that can be reversed by vitamin D repletion (40). In uremic patients, 1,25(OH)₂D₃ therapy significantly increases serum insulin concentrations (160). It has been postulated that 1,25(OH)₂D₃, through a VDR-mediated modulation of calbindin expression, controls intracellular calcium flux in the islet cells, which in turn affects insulin release (41). The immunomodulatory properties of the sterol have extended its therapeutic application to reducing the incidence of insulinitis and diabetes in animal models for type I diabetes (125, 128) and to preventing the recurrence of autoimmune diabetes after islet transplantation (126, 127).

Additional nonclassic target tissues. Table 1 lists additional nonclassic target tissues for 1,25(OH)₂D₃ action. In these tissues, 1,25(OH)₂D₃ exerts a diverse range of biological actions including the control of growth and differentiation of numerous normal and cancerous cell types, modulation of hormone secretion by several endocrine glands (182), regulation of reproductive function (105, 106), and protection of specific neurons from degenerative processes (35).

The antiproliferative, prodifferentiating properties of 1,25(OH)₂D₃ have been exploited therapeutically to treat leukemia, cancer, and psoriasis (22). These properties of 1,25(OH)₂D₃ suggested an important role for the sterol during embryonic development. However, the lack of a functional VDR both in patients and in the VDR null mice produces significant phenotype only after weaning, suggesting that the VDR is not essential in the development of major organ systems during embryogenesis (114). An exception is the essential role of the VDR in skin and hair development discussed earlier in this section.

A role for vitamin D in reproduction was suggested by the demonstration of reduced female fertility in vitamin D-deficient rats (66) and the uterine hypoplasia of the VDR null mice (196). Female fertility could be corrected by 1,25(OH)₂D₃, but not by simply raising serum calcium (105). In contrast, the reduced fertility of vitamin D-deficient males can be restored by raising serum calcium, suggesting that the VDR may not be essential for spermatogenesis and male reproduction (106).

The role of vitamin D in the nervous system has been addressed only recently, despite the early demonstration of VDR expression in the brain and several regions of the central and peripheral nervous system (35). In vivo, 1,25(OH)₂D₃ administration to rat or mice prevents or halts the progression of encephalomyelitis (33), which suggests that the nervous system is a target for the immunosuppressive actions of the sterol. In fact, cultures of newborn brain microglia, cells of the monocyte-macrophage lineage, synthesize 1,25(OH)₂D₃ (144). The sterol, in turn, promotes phagocytic activity of adult retinal glia. Ex vivo studies in isolated avian nerves suggest a role for vitamin D in conductance velocity in motor neurons (31).

The potential of 1,25(OH)₂D₃ to prevent the loss of injured neurons was suggested by the antiproliferative, prodifferentiating effects of the sterol in a neuroblastoma cell line. In addition, 1,25(OH)₂D₃ was shown to induce the expression of VDR, as well as neurotrophic factors such as nerve growth factor and neurotrophyns T3 and T4/5 in primary cultures of brain glia (142, 143), and to enhance monoamineoxidase activity, an enzyme involved in polyamine metabolism and cell growth, in certain brain nuclei (35). 1,25(OH)₂D₃ induces nerve growth factor production in the cortex, hippocampus, and basal forebrain, the most affected sites in Alzheimer's disease. The ability of nerve growth factor and 1,25(OH)₂D₃-induced neurotrophyns to prevent the loss of injured neurons suggested a potential therapeutical application of the sterol for treatment of neurodegenerative disorders such as Alzheimer's disease.

It is important to emphasize, however, that no obvious hematopoietic, immune, or neurological abnormalities were demonstrated in the VDR null mice, raising numerous questions on the direct involvement of the intracellular VDR that we know today. A great deal of work, remains to be done to identify the precise molecular mechanisms as well as the target genes mediating the plethora of biological actions of this potent steroid hormone.