ALEX J. BROWN, ADRIANA DUSSO, AND EDUARDO SLATOPOLSKY Renal Division, Washington University School of Medicine, St. Louis, Missouri 63110

invited review Vitamin D ALEX J. BROWN, ADRIANA DUSSO, AND EDUARDO SLATOPOLSKY Renal Division, Washington University School of Medicine, St. Louis, Missouri 63110 Brown, Alex J., Adriana Dusso, and Eduardo Slatopolsky. Vitamin D. Am. J. Physiol. 277 (Renal Physiol. 46): F157 F175, 1999. The vitamin D endocrine systems plays a critical role in calcium and phosphate homeostasis. The active form of vitamin D, 1,25-dihydroxyvitamin D 3 [1,25(OH) 2 D 3 ], binds with high affinity to a specific cellular receptor that acts as a ligand-activated transcription factor. The activated vitamin D receptor (VDR) dimerizes with another nuclear receptor, the retinoid X receptor (RXR), and the heterodimer binds to specific DNA motifs (vitamin D response elements, VDREs) in the promoter region of target genes. This heterodimer recruits nuclear coactivators and components of the transcriptional preinitiation complex to alter the rate of gene transcription. 1,25(OH) 2 D 3 also binds to a cell-surface receptor that mediates the activation of second messenger pathways, some of which may modulate the activity of the VDR. Recent studies with VDR-ablated mice confirm that the most critical role of 1,25(OH) 2 D 3 is the activation of genes that control intestinal calcium transport. However, 1,25(OH) 2 D 3 can control the expression of many genes involved in a plethora of biological actions. Many of these nonclassic responses have suggested a number of therapeutic applications for 1,25(OH) 2 D 3 and its analogs. gene expression; receptor; metabolism 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 D 2. 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 D 2 (ergocalciferol) (5) and vitamin D 3 (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 D 3 (146) permitted the identification of metabolites with greater antirachitic activity. 25-Hydroxyvitamin D 3 [25(OH)D 3 ] was found to be the major circulating metabolite of vitamin D 3 (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)D 3 in the nuclear fraction from intestine. This molecule, synthesized mainly in the kidney, was identified as 1,25-dihydroxyvitamin D 3 [1,25(OH) 2 D 3 ] (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) 2 D] (71). This 50- to 70-kDa protein facilitated association with nuclear chromatin, displayed saturable binding of 1,25(OH) 2 D 3, 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 ligandactivated transcription factor. The 1,25(OH) 2 D 3 -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) 2 D 3 with a diverse range of biological actions including immunomodulation, the control of other hormonal systems, 0363-6127/99 $5.00 Copyright 1999 the American Physiological Society F157

F158 inhibition of cell growth, and induction of cell differentiation. These actions have suggested a number of new therapeutic applications of 1,25(OH) 2 D 3 in immune dysfunction (autoimmune disease), endocrine disorders (hyperparathyroidism), and hyperproliferative disorders (leukemia, cancer, psoriasis). VITAMIN D METABOLISM 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. Sources of Vitamin D Vitamin D can be obtained from the diet and by the action of sunlight on the skin. Only a few food sources such as fish oils, egg yolks, and liver contain significant amounts of vitamins D 2 and D 3. However, many foods are now fortified with the vitamin, and minimum daily requirements are easily met. Vitamin D 3 is produced in the skin by an ultraviolet light-induced photolytic conversion of 7-dehydrocholesterol to previtamin D 3 (82, 154) followed by thermal isomerization to vitamin D 3 (67). 25-Hydroxylation of Vitamin D The first step in the metabolic activation of vitamin D is hydroxylation of carbon 25. This reaction occurs primarily in the liver, although other tissues including skin, intestine, and kidney have been reported to catalyze 25-hydroxylation of vitamin D. The contribution of the extrahepatic sources to the circulating levels of 25-hydroxyvitamin D [25(OH)D] is uncertain. The hepatic 25-hydroxylation involves cytochrome P-450 monooxygenase(s). At least two enzymes have been reported: one mitochondrial, the other microsomal. The microsomal enzyme present in rat liver has a higher affinity for vitamin D, but a microsomal P-450 (CYP2C11) capable of the reaction appears to be male specific in rats (73) and is apparently not present in human liver microsomes (163). The mitochondrial cytochrome P-450 (CYP27) can 25-hydroxylate vitamin D 3, is not sex specific, and can be found in all mammalian species. However, patients with cerebrotendinous xanthomatosis due to CYP27 mutations have normal levels of vitamin D metabolites, including 25(OH)D 3. Furthermore, the expressed recombinant CYP27 25-hydroxylates vitamin D 3 but not vitamin D 2 (64). Thus the identity of the cytochrome P-450s responsible for 25- hydroxylation remains to be determined. 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) 2 D The second and more important step in vitamin D bioactivation, the formation of 1,25(OH) 2 D from 25(OH)D occurs, under physiological conditions, mainly in the kidney (53). The renal enzyme responsible for producing 1,25(OH) 2 D, 25(OH)D-1 -hydroxylase, is located in the inner mitochondrial membrane and is a cytochrome P-450 monooxygenase requiring molecular oxygen and reduced ferredoxin (57). In recent years, many reports have demonstrated that the kidney is not unique in its ability to convert 25(OH)D to 1,25(OH) 2 D. Numerous cells and tissues express 1 -hydroxylase in vitro; however, in humans, these extrarenal sources of 1,25(OH) 2 D only contribute significantly to circulating 1,25(OH) 2 D levels during pregnancy, in chronic renal failure, and in pathological conditions such as sarcoidosis, tuberculosis, granulomatous disorders, and rheumatoid arthritis. 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)D 3. Further evidence for the identity of the human 1 -hydroxylase cdna came from chromosomal Fig. 1. Vitamin D metabolism: steps involved in production of vitamin D 3 in the skin and its subsequent activation to 1,25-dihydroxyvitamin D 3 [1,25(OH) 2 D 3 ] and catabolism to the water-soluble metabolite, calcitroic acid.

F159 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) 2 D 3 (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 The high potency of 1,25(OH) 2 D 3 in elevating serum calcium and phosphate levels requires its circulating levels to be tightly regulated. Control of serum 1,25(OH) 2 D 3 usually involves reciprocal changes in the rates of synthesis and degradation. Vitamin D compounds are catabolized primarily by oxidation of the side chain. The major catabolic enzyme is the vitamin D-24-hydroxylase, another mitochondrial cytochrome P-450 requiring molecular oxygen and reduced ferredoxin (27, 100). The oxidation of the side chain of 25(OH)D 3 and 1,25(OH) 2 D 3 is initiated at carbon C-24. This is followed by further oxidation of carbon C-24 to a ketone, oxidation of carbon C-23, and subsequent oxidative cleavage of the side chain (122, 161). Each oxidation step leads to progressive loss of biological activity. The final cleavage product of 1,25(OH) 2 D 3, calcitroic acid, is biologically inert. The 24-hydroxylase cdna (151) and gene (149) have been cloned. In contrast to the limited tissue distribution of the synthetic enzymes, the 24-hydroxylase is ubiquitously present in vitamin D target tissues. The 24-hydroxylase is highly inducible by 1,25(OH) 2 D 3, providing a mechanism for attenuating the response to the vitamin D hormone, and reducing 1,25(OH) 2 D 3 levels when they are abnormally high. In fact, mice lacking a functional 24- hydroxylase gene (169) have high serum 1,25(OH) 2 D 3 levels due to the decreased capacity to degrade it. Additional pathways for metabolism of 1,25(OH) 2 D 3 have been described. The vitamin D hormone can be converted to the 1,25(R)-(OH) 2 D 3-23(S),26-lactone following hydroxylations at the 23(S) and 26 positions (91). The lactone is a minor circulating metabolite of 1,25(OH) 2 D 3 in the blood (148). Interestingly, 1,25(R)- (OH) 2 D 3-23(S),26-lactone appears to have anti-vitamin D action in that it inhibits the 1,25(OH) 2 D 3 - 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) 2 D 3 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-D 3 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) 2 D 3 LEVELS The stringent control of serum 1,25(OH) 2 D 3 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) 2 D 3 levels are parathyroid hormone (PTH), calcium, phosphate, and 1,25(OH) 2 D 3 itself. There are many other factors that have been reported to influence 1,25(OH) 2 D 3 synthesis and/or degradation, but their physiological importance is not clear. Parathyroid Hormone Hypocalcemia increases serum 1,25(OH) 2 D 3 levels by stimulation of the kidney 1 -hydroxylase (155), whereas hypercalcemia depresses 1 -hydroxylase activity and increases 24-hydroxylase activity. Parathyroidectomy severely blunts the induction of the renal 1-hydroxylase by hypocalcemia (56), and administration of PTH to parathyroidectomized animals or intact animals increases the production of 1,25(OH) 2 D 3. Furthermore, patients with hypoparathyroidism have low calcitriol levels despite persistent hypocalcemia (69). 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) 2 D 3 (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 There is additional evidence for PTH-independent regulation of 1,25(OH) 2 D 3 by calcium. When rats that were parathyroidectomized and repleted with constantly infused PTH (to prevent the hyperphosphatemia which can decrease calcitriol), 1,25(OH) 2 D 3 levels decreased when serum calcium was raised by calcium chloride infusion, and levels increased when serum calcium was lowered by infusion with EGTA, a calcium chelator (129, 184). Thus, when constant PTH and phosphorus are maintained, an inverse correlation is seen between serum calcium and 1,25(OH) 2 D 3 levels. A recent report provided evidence for direct suppression of the 1 -hydroxylase activity and mrna by calcium in a human proximal tubule cell line (16). Acidosis Metabolic acidosis has also been shown to alter vitamin D metabolism through at least two mechanisms. Acidosis blunts the action of PTH on the 1 hydroxylase of the proximal convoluted tubule, an effect that can be overcome by camp (97). Since loss of PTH responsiveness with acidosis is not due to a loss of renal PTH receptors in the dog, the blunted camp production is attributed to altered coupling of these receptors with adenylate cyclase (9). Acidosis has also been shown to increase the activity of the renal 24- hydroxylase (110), which would enhance the degradation of 1,25(OH) 2 D 3. Another consequence of acidosis is an increase in ionized calcium due to the lower blood ph. It is possible that this increase in ionized calcium

F160 may be totally responsible for this altered vitamin D metabolism since EGTA, a calcium chelator, can block the effect of acidosis on calcitriol levels without changing blood ph (30). Thus acidosis appears to decrease calcitriol levels by raising serum ionized calcium and by decreasing the responsiveness of the kidney to PTH. Phosphate The importance of phosphate as a regulator of renal vitamin D metabolism is well established. Dietary phosphate restriction increases serum 1,25(OH) 2 D 3 levels (88) and renal 1 -hydroxylase activity (178). In humans, modulation of phosphate within the normal range can alter serum 1,25(OH) 2 D 3 levels (158). The mechanism by which phosphorus controls the production of 1,25(OH) 2 D 3 is still unclear, but it appears to be independent of PTH (88) and changes in serum calcium (29). It was originally proposed that hypophosphatemia lowered the renal cortical phosphate content leading to increased 1 -hydroxylase activity (88). Subsequent studies showed that during short periods of phosphate deprivation, decreased cortical phosphate levels were not found at a time when increases in circulating 1,25(OH) 2 D 3 and 1 -hydroxylase activity were maximal (62). A recent report demonstrated normal regulation of renal 1 -hydroxylase by phosphate in mice lacking the major phosphate transporter (NPT2), suggesting that phosphate flux in the proximal tubule cells is not involved in the regulation by phosphate. It is now known that the control of 1 -hydroxylase by phosphate occurs at the level of mrna (167). 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 An interesting aspect of the regulation of 1,25(OH) 2 D 3 by hypophosphatemia is the role of insulin and somatomedins. Hypophysectomy blocks the stimulatory action of dietary phosphate restriction on 1 -hydroxylase activity (59), but injection of growth hormone to hypophysectomized rats restores this response to low dietary phosphorus (61). Similarly, phosphate-deprived rats with streptozotocin-induced diabetes have an impaired stimulation of 1 -hydroxylase activity, but insulin administration restores this response (130). These results suggest a role for insulin and insulin-like growth factor-i (IGF-I) in the regulation of vitamin D metabolism by phosphate (60). Insulin and IGF-I have also been shown to enhance the actions of PTH on hydroxylase activities. PTH induction of the 1 hydroxylase is blunted in diabetic rats (187). In chick kidney cell cultures, insulin enhanced the PTH stimulation of 1,25(OH) 2 D 3 production, but this is not due to further increases in camp (74). In osteoblasts, insulin also enhanced the ability of PTH and 1,25(OH) 2 D 3 to induce 24-hydroxylase expression (3). 1,25(OH) 2 D 3 Feedback regulation by 1,25(OH) 2 D 3 on its own synthesis and catabolism provide an important feedback loop to minimize vitamin D intoxication. In vitamin D-deficient animals, 1 -hydroxylase activity is maximal and 24-hydroxylase is low or undetectable. Treatment with 1,25(OH) 2 D 3 reverses the expression of these two activities. However, at least some of this effect is mediated by changes in calcium, phosphate and PTH. These same effects on the hydroxylases can be observed in kidney cell cultures, indicating that the effects of 1,25(OH) 2 D 3 are direct (75, 179). The actions of 1,25(OH) 2 D 3 on the vitamin D hydroxylases in cell culture are evident only after several hours, require protein synthesis, and are reversed by removal of the hormone from the medium (76). Furthermore, both hydroxylases are now known to be regulated by 1,25(OH) 2 D 3 at the mrna level (134, 150, 167, 170, 177). The 24-hydroxylase gene contains at least two distinct vitamin D response elements that mediate the effects of 1,25(OH) 2 D 3 via its receptor on transcription (38, 152). It seems likely, then, that when the intake of calcium and phosphorus is adequate, this regulation of vitamin D metabolism is the primary mechanism for maintaining proper circulating calcitriol levels. Other factors have been reported to modulate vitamin D metabolism. However, many of these actions appear to be indirect and may be mediated by calcitriol, calcium, PTH, or phosphate. TRANSPORT OF VITAMIN D Vitamin D and its hydroxylated metabolites 25(OH)D, 24,25(OH) 2 D, and 1,25(OH) 2 D 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) 2 D 1,25(OH) 2 D vitamin D (43). The dissociation constants for 25(OH)D and 1,25(OH) 2 D differ by 10-fold. In mammals, vitamin D 2 and vitamin D 3 metabolites exhibit the same relative affinities for DBP, whereas avian DBP preferentially binds vitamin D 3 metabolites 100 times more avidly than their vitamin D 2 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

F161 great influence on vitamin D pharmacokinetics. DBPbound 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) 2 D 3 stimulation of 24-hydroxylase in keratinocytes and monocytes, as well as 1,25(OH) 2 D 3 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)D 3 and 1,25(OH) 2 D 3 (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)D 3 to 1,25(OH) 2 D 3 by the renal 1 -hydroxylase. In fact, megalin null mice elicit high urinary excretion of 25(OH)D 3 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)D 3 to 1,25(OH) 2 D 3 is blunted, thus preventing hypercalcemia and 1,25(OH) 2 D 3 toxicity. The contribution of megalin-mediated endocytosis to either the cellular uptake of 25(OH)D 3 from circulating 25(OH)D 3 -DBP complexes by extrarenal sources of 1,25(OH) 2 D 3 or in the delivery of DBP-bound 1,25(OH) 2 D 3 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) 2 D 3 (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) 2 D 3, 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 D 3 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) 2 D 3 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) 2 D 3 from plasma carriers into target cells expressing megalin and/or LDL receptor. This possibility remains to be examined. VITAMIN D RECEPTOR Most of the biological activities of 1,25(OH) 2 D 3 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) 2 D 3. 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) Fig. 2. Functional domains of the human vitamin D receptor (VDR).

F162 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. Ligand Binding The ligand-binding domain (LBD), located in the carboxy-terminal portion of the VDR molecule, is responsible for the high-affinity binding of 1,25(OH) 2 D 3 (K d 10 10 to 10 11 M). 25(OH)D 3 and 24,25(OH) 2 D 3 bind nearly 100 times less avidly (26, 133). Upon ligand binding, the cytoplasmic VDR rapidly translocates to the nucleus along microtubules. A critical role for VDR translocation on 1,25(OH) 2 D 3 transcriptional regulation was suggested by 1) studies in normal human monocytes, in which disruption of microtubular integrity abolished 1,25(OH) 2 D 3 induction of 24-hydroxylase mrna (95), and 2) the report of a phenotype of vitamin D-resistant rickets caused by a defective cytoplasmic-tonuclear translocation of an otherwise normal VDR (79). Two potential nuclear localization signals have been identified within the VDR molecule. One is a bipartite signal consisting of a cluster of basic residues at each end of the sequence between amino acids 79 and 105. The second is a basic sequence of seven amino acids (residues 49 55), unique to the VDR, identified between the two zinc fingers. Point mutations in both nuclear localization signals impair VDR translocation to the nucleus and cause vitamin D-resistant rickets (8, 165). Heterodimerization with RXR RXR dimerization surfaces in the VDR are found in the first zinc finger, in a region COOH-terminal of the second zinc finger, and in a structural motif designated as heptad repeats in the LBD. Asn 37 in the first zinc finger, Lys 91 and Glu 92 situated in the T-box COOHterminal of the second zinc finger, and two of the heptad repeats within the LBD are critical in determining selective association between the VDR and its protein partner, RXR. Heterodimerization of the ligand-activated VDR with RXR induces a VDR conformation that is essential for VDR transactivating function. Two natural mutations (I314S and R391C) in the LBD of the VDR suggest the potential interplay between hormone binding and heterodimerization domains. These mutations confer the phenotype of vitamin D resistance by significantly impairing both VDR-RXR heterodimerization and ligand retention (70). Binding of the VDR-RXR Heterodimer to DNA The DNA-binding domain of the VDR (DBD) is the most conserved region of various members of the superfamily of nuclear receptors for steroid and thyroid hormones that includes the VDR. There are nine cysteine residues within the DBD that are strictly conserved throughout the superfamily of receptor proteins. The first eight cysteines in the NH 2 terminus coordinate two zinc atoms to form the so-called zinc finger DNA-binding motifs that are responsible for high-affinity interaction with specific DNA sequences Fig. 3. Regulation vitamin D action. Major sites at which the genomic actions of 1,25(OH) 2 D 3 can be regulated are highlighted and discussed in detail in the text. RXR, retinoid X receptor; DBP, vitamin D binding protein.

F163 in the promoter region of 1,25(OH) 2 D 3 target genes. Most of the natural mutations found in the human VDR are located in the zinc finger region, resulting in defective DNA binding and the most severe clinical phenotypes of vitamin D resistance (72). 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) 2 D 3 - 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) 2 D 3 (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 The transactivation domains of the VDR serve as an adaptor surface for nuclear proteins necessary for VDR-mediated transcriptional regulation. One of these regions is the heterodimerization domain containing residue 246, which is highly conserved among nuclear receptors. It forms part of the binding interface with transcriptional coactivators, and its alteration severely compromises transactivation. The second region is a conserved -helix near the COOH terminus of steroid receptor LBD, which undergoes a dramatic conformational shift upon ligand binding. This region of the VDR is known as ligand-dependent activation function, or AF2. Removal of the AF2 domain eliminates 1,25(OH) 2 D 3 -VDR transcriptional activity with little effect on ligand binding or heterodimeric DNA binding. The AF2 region of the VDR directly interacts with components of the transcription initiation complex and nuclear transcriptional coactivators. One of the proteins, in the rapidly growing list of VDR-interacting proteins, is the general transcription factor TFIIB, a component of the basal transcription complex, with a critical role in ligand-dependent transcription (15, 121, 124). The VDR also associates with the steroid receptor coactivator SRC-1, which is required for full transcriptional activity of the steroid receptor superfamily (156). The VDR interacts with the 62-kDa nuclear coactivator (NCoA-62) in a ligand-independent manner. On the other hand, interaction of the VDR with coactivator p65 (48, 70), the TATA binding protein (TBP)-associated factor TAFII28, and TIF1 is ligand dependent. The CBP/p300 family of nuclear coactivators may constitute additional VDR-interacting proteins. The association of the VDR partners RXR and SRC-1 with CBP/ p300 potentiate the transcriptional activity of various members of the steroid receptor superfamily including retinoic acid and estrogen receptors (58, 68, 168). These findings suggest a potential for CBP/p300 to affect VDR-transactivating function. Both SRC-1 and the CBP/p300 family of nuclear coactivators possess histone acetyltransferase activity, which modifies nucleosome structure and exposes the DNA for transcription. In summary, interactions of the ligand-activated VDR- RXR complex with nuclear proteins facilitate the assembly of the transcription preinitiation complex and regulate the rate of transcription of the target gene by RNA-polymerase II (36, 58, 193). NONGENOMIC ACTIONS OF 1,25(OH) 2 D 3 ARE MEDIATED BY A CELL SURFACE RECEPTOR 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) 2 D 3 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) 2 D 3 in these cells (140). A candidate membrane receptor in rat osteoblastlike (ROS 24/1) cells has been identified as annexin-2 (7). This protein binds 1,25(OH) 2 D 3, 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) 2 D 3.

F164 The role of the nongenomic actions in most cells remains uncertain. In chick duodenum, 1,25(OH) 2 D 3 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) 2 D 3 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) 2 D 3. REGULATION OF 1,25(OH) 2 D 3 -VDR ACTIONS A number of factors can influence the VDR-mediated actions of 1,25(OH) 2 D 3, 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 The concentration of ligand in a target cell available for VDR binding is determined by the net balance between the rate of uptake of ligand into the cell and the rate of its metabolic inactivation within the cell. As mentioned earlier, the cellular uptake of vitamin D metabolites is greatly controlled by plasma DBP. However, the demonstration that megalin-mediated endocytosis is the major pathway for the uptake of DBP-bound 25(OH)D by renal proximal tubular cells (147) raises the possibility that 1,25(OH) 2 D 3, which circulates bound to DBP and lipoproteins, may also enter into target cells through cell-specific, receptor-mediated processes. Once inside the target cell, an important attenuator of the biological response to the vitamin D metabolites or analogs is the rate of catabolic inactivation of these molecules. In all vitamin D-responsive organs, the major route of degradation for vitamin D metabolites is the oxidation of the side chain of the molecule, catalyzed by the vitamin D-24-hydroxylase, an enzyme which is highly inducible by 1,25(OH) 2 D 3 and its analogs. Blocking 24-hydroxylase activity with the cytochrome P-450 inhibitor ketoconazole markedly enhances the potency of 1,25(OH) 2 D 3 and its analogs (197). Moreover, 24-hydroxylase null mice exhibit reduced 1,25(OH) 2 D 3 clearance and signs of vitamin D intoxication, clearly demonstrating the critical role of ligand inactivation in the in situ control of the response to vitamin D (169). VDR Content The intracellular levels of VDR in a target cell are regulated by VDR ligands (homologous regulation) (44, 45) and other hormones and growth factors that do not bind to the VDR (heterologous upregulation) (80, 109, 145, 157). Homologous and heterologous regulation of VDR abundance can involve the control of the rate of transcription of the VDR gene, stabilization of the VDR mrna, or alteration of the degradation rate of the VDR protein. There are profound species-, tissue-, and cellspecific variations in VDR regulation (39, 80, 159). For example, 1,25(OH) 2 D 3 upregulates VDR mrna in the parathyroid glands and the kidney but not in the intestine (25). The variability in controlling VDR content depends upon many factors including the stage of proliferation and differentiation of the target cell, differences in the intracellular signaling pathways activated in these cells by the regulator, and differential expression in the various target cells of downstream nuclear proteins involved in the regulation of gene transcription. VDR content can also be controlled at the level of synthesis and degradation. Although there is no evidence for translational control of the VDR, liganddependent 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) 2 D 3 -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 Several genetic polymorphisms within the human VDR have been identified (51, 52), and there have been suggestions of a relationship between the frequency of certain alleles and bone density (102, 137), the susceptibility to primary hyperparathyroidism (34), and the response to vitamin D therapy in psoriasis (101). However, most of these variations in the VDR gene are not located in areas that affect the structure of the protein (51, 52). An exception is the C-to-T transition in the translation-initiation site that results in a VDR that is three amino acids shorter. This truncated version of the VDR is an evolutionarily more recent polymorphism that exhibits higher transcriptional activity. Posttranslational Modifications of the VDR Ligand binding to the VDR promotes serine phosphorylation of the receptor. VDR phosphorylation occurs at several loci and is mediated by various kinases. Although phosphorylation by casein kinase II (at serine- 208) may enhance VDR activity (93), phosphorylation by PKC (at serine-51) appears to decrease VDR activity (85) (86). PKA phosphorylates the VDR between residues 103 and 201. Although activation of PKA resulted in enhanced 1,25(OH) 2 D 3 -mediated transcription in intact cell lines (104), possibly by increasing VDR content, direct phosphorylation of the human VDR by PKA in vitro resulted in decreased VDR-transcriptional activity (92). Ligand-dependent hyperphosphorylation

F165 of the VDR was shown to inhibit the interaction of VDR-RXR complexes with the osteocalcin VDRE and impede transactivation of the osteocalcin gene (49). These findings clearly suggested that the nuclear actions of the VDR could be modulated by other hormonal systems acting at the cell surface to activate protein kinases cascades. In fact, the ability of 1,25(OH) 2 D 3 itself to rapidly activate PKC and other kinases through interaction with cell membrane receptors indicates a potential cell-specific mechanism for modulation of the genomic actions of the vitamin D hormone. 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 The genomic actions of the hormonal form of vitamin D could also be influenced by changes in the nuclear levels, or in the availability, of other components of the transcriptional complex. Competition between the VDR and transcription factors for other hormonal systems for limiting amounts of common nuclear transcriptional modulators could also affect 1,25(OH) 2 D 3 -VDR regulation of gene expression. A great deal of work remains to be done to elucidate the molecular events involved in the interactions of the VDR with the transcriptional machinery mediating 1,25(OH) 2 D 3 modulation of gene transcription and to identify the genes responsible for the plethora of biological actions of this potent steroid hormone. BIOLOGICAL ACTIONS OF VITAMIN D 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) 2 D 3 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) 2 D 3. Classic Vitamin D-Responsive Tissues Intestine. The most critical role of 1,25(OH) 2 D 3 in mineral homeostasis is to enhance the efficiency of the small intestine to absorb dietary calcium and phosphate as demonstrated conclusively by recent studies in the VDR null mice (112). In the absence of VDR, normalization of circulating levels of calcium and phosphorus through dietary supplementation corrected most of the phenotypic features of vitamin D resistance including parathyroid gland growth, bone mineralization, and growth plate histology. These findings concur with prior clinical observations in patients with vitamin D-resistant rickets whose bone abnormalities were resolved by calcium infusions. 1,25(OH) 2 D 3 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) 2 D 3 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) 2 D 3 may not be the only mode of action by which the hormone stimulates calcium absorption by the enterocyte. Rapid effects of 1,25(OH) 2 D 3 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) 2 D 3 increases active phosphate transport. However, significant phosphate absorption also occurs in 1,25(OH) 2 D 3 -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) 2 D 3 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) 2 D 3 in regulating mineral homeostasis makes it the most likely vitamin D metabolite involved in bone mineralization. Although controversial, 24,25(OH) 2 D 3 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) 2 D 3 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

F166 Table 1. 1,25(OH) 2 D 3 actions in classic and nonclassic targets tissues Tissue Cell type Action Classic Intestine Epithelial Enhancement of calcium and phosphate absorption Bone Osteoblast Enhancement of bone matrix protein synthesis, bone mineralization, and synthesis of mediators of osteoclastogenis and osteoclastic activity Osteoclast Enhancement of bone resorption Kidney Epithelial (proximal and distal) Inhibition of 1,25(OH) 2 D 3 synthesis and induction of 24-hydroxylase Enhancement of calcium and phosphate reabsorption Parathyroid gland Chief Inhibition of cell growth and PTH synthesis Nonclassic Hematopoietic tissues Myeloid cell precursors Antiproliferative, prodifferentiating Colony forming units Prodifferentiating Immune system Monocyte/macrophages Enhancement of immune function to control viral and bacterial infections and tumor growth Lymphocyte Immunosuppression Skin Keratinocytes, fibroblasts, hair follicle, Langerghan Antiproliferative, prodifferentiating cells, melanocytes Muscle Smooth muscle cell, myoblast Antiproliferative, prodifferentiating Heart Cardiac muscle cell Antiproliferative, prodifferentiating Atrial myocytes Inhibition of antinatriuretic factor synthesis Pancreas cells Enhancement of insulin synthesis and secretion Cancer cells Melanoma, breast carcinoma, leukemia, osteosarcoma, Antiproliferative, prodifferentiating fibrosarcoma, pituitary medullary thyroid carcioma, adenoma, neuroblastoma, pancreatic adenocarcinoma, bladder, cervical, prostate and colon carcinomas Adrenal gland Medullary cells Control of catecolamine metabolism Brain Hippocampus/selected neurons Neuronal regeneration, enhancement of nerve growth factor and neurotrophin synthesis, control of sphyngomyelin cycle Cartilage Chondrocyte Antiproliferative, prodifferentiating Female reproductive Myometrial and endometrial cells Antiproliferative, control of foliculogenesis organs Liver Parenchymal cell (fetal, adult) Enhancement of liver regeneration, control of glycogen and transferrin synthesis Lung Fetal pneumocytes Enhancement of maturation, phospholipid synthesis and surfactant release Adult pneumocytes Cell growth Male reproductive organs Sertoli/semminiferus tubule Enhancement of Sertoli cell function and spermatogenesis Pituitary production Somatomammotroph Control of T 3 -induced growth hormone, prolactin and tyrotrophyn Thyroid Follicular cells (C cells) Inhibition of cell function and calcitonin synthesis 1,25(OH) 2 D 3, 1,25-dihydroxyvitamin D 3 ; PTH, parathyroid hormone; T 3, triiodothyronine. for 1,25(OH) 2 D 3 induction of osteoclastic bone resorption. An in vitro system for studying the osteoclastogenic activity of 1,25(OH) 2 D 3 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) 2 D 3 - dependent fashion in this model. However, other hormones, including PTH, can substitute for 1,25(OH) 2 D 3 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) 2 D 3 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) 2 D 3 plays a key role in the day-to-day maintenance of calcium balance. PTH stimulates the production of 1,25(OH) 2 D 3 by activating the renal 1 hydroxylase (21, 138), and 1,25(OH) 2 D 3 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) 2 D 3 suppression of PTH synthesis occurs through negative regulation of the rate of PTH gene transcription by the 1,25(OH) 2 D 3 -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) 2 D 3 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

F167 Fig. 4. Current model for the control of VDR-mediated actions of 1,25(OH) 2 D 3. The activated vitamin D hormone, 1,25(OH) 2 D 3, circulates in the blood bound to vitamin D binding protein (DBP). 1,25(OH) 2 D 3 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) 2 D 3 -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. phosphate, which affects renal filtered load, and on serum PTH levels. Comprehensive in vivo studies demonstrated, however, that 1,25(OH) 2 D 3 increases renal calcium reabsorption (192). 1,25(OH) 2 D 3 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) 2 D 3 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) 2 D 3 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. Nonclassic Vitamin D-Responsive Tissues Hematopoietic tissues. Anemia, decreased bone cellularity, extramedullary erythropoiesis, and a timedependent reduction in spleen colony-forming units have been reported in vitamin D deficiency and vitamin D-deficient rickets. Vitamin D administration rapidly improves the hematopoietic condition by unknown mechanisms (194). 1,25(OH) 2 D 3 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) 2 D 3 -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) 2 D 3 in other cell types.

F168 Fig. 5. Role of 1,25(OH) 2 D 3 in calcium and phosphate homeostasis. Integrated roles of 1,25(OH) 2 D 3 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. 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) 2 D 3 deficiency. In both conditions, the impaired immunity can be improved with 1,25(OH) 2 D 3 therapy. 1,25(OH) 2 D 3 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) 2 D 3 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) 2 D 3 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) 2 D 3 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) 2 D 3 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

F169 skin diseases reemerged as a result of the observation that psoriatic lesions dramatically improved in a patient receiving oral 1 -hydroxyvitamin D 3 to treat severe osteoporosis (136). The antiproliferative and prodifferentiating effects of 1,25(OH) 2 D 3 in keratinocytes, melanocytes, and fibroblasts and its immunosuppressive properties on Langerhan s cells, the antigenpresenting 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) 2 D 3 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)D 3. 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) 2 D 3 controls hypertrophy in cardiac myocytes (189) and the synthesis and release of atrial natriuretic factor (188). 1,25(OH) 2 D 3 and 25(OH)D 3 improve both the left ventricular function in patients with cardiomyopathies and the skeletal muscle weakness secondary to endstage 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) 2 D 3 therapy significantly increases serum insulin concentrations (160). It has been postulated that 1,25(OH) 2 D 3, 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) 2 D 3 action. In these tissues, 1,25(OH) 2 D 3 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) 2 D 3 have been exploited therapeutically to treat leukemia, cancer, and psoriasis (22). These properties of 1,25(OH) 2 D 3 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) 2 D 3, 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) 2 D 3 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) 2 D 3 (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) 2 D 3 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) 2 D 3 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) 2 D 3 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) 2 D 3 -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

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