B. Overview of Metabolism
Vitamin D, in the form of vitamin D3 , is made from 7-dehydrocholesterol in the skin by exposure to ultraviolet light (270-300 nm range). Alternatively, vitamin D, in the form of either vitamin D2 or vitamin D3 , can be derived from dietary sources (Fig. 1A). Both vitamin D3 and vitamin D2 undergo the same activation process, involving first 25-hydroxylation in the liver, followed by 1
-hydroxylation in the kidney, to make the biologically active compounds 1,25-(OH)2D3 and 1,25-(OH)2D2 , respectively (Fig. 1B). There is little evidence that these two active forms differ in their mode of action, and because most is known about the synthesis and action of 1,25-(OH)2D3 , this review focuses on the natural D3 compound. The metabolic activations of vitamin D3 are carried out by specific cytochrome P-450-containing enzymes, the vitamin D3-25-hydroxylase (CYP27) and possibly another P-450 in the hepatocyte and the 25-hydroxyvitamin D-1-hydroxylase (CYP1) in the renal proximal tubular cell. Both of the known hydroxylases are located in the inner mitochondrial membrane of these cells (Fig. 2). The synthesis of 25-hydroxyvitamin D3 (25-OH-D3) by the liver appears to be only loosely regulated, whereas the synthesis of 1,25-(OH)2D3 by the renal 1-hydroxylase is tightly regulated by the levels of plasma 1,25-(OH)2D3 and calcium. The renal enzyme is strongly upregulated by the hormone parathyroid hormone (PTH), a point that is discussed further in section II. A third vitamin D-related mitochondrial cytochrome P-450-containing enzyme, the 25-hydroxyvitamin D-24-hydroxylase (CYP24), was originally believed to be exclusively located in the kidney and to be involved only in the metabolism of 25-OH-D3 to 24,25-dihydroxyvitamin D3 [24,25-(OH)2D3]. The 24-hydroxylation of 1,25-(OH)2D3 was first realized with the isolation of 1,24,25-(OH)3D3 and subsequently shown to occur in all vitamin D target tissues including enterocytes, osteoblasts, keratinocytes, and parathyroid cells. Thus it is now known that CYP24 will use 1,25-(OH)2D3 as substrate also. Because CYP24 is widely distributed around the body, is strongly induced in target cells by 1,25-(OH)2D3 , and prefers 1,25-(OH)2D3 as substrate to 25-OH-D3 , its role appears to be catabolic. The enzyme CYP24 catalyzes several steps of 1,25-(OH)2D3 degradation, collectively known as the C-24 oxidation pathway which starts with 24-hydroxylation and culminates in the formation of the biliary excretory form, calcitroic acid (Fig. 3). Thus our current view is that both the synthesis and degradation of 1,25-(OH)2D3 are tightly regulated events, attesting to the fact that the concentration of this potent hormone requires fine control at the cellular level, and hence a set of highly specific and finely tuned cytochrome P-450 exist for the purpose.
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| FIG. 2.
Electron transport chain for mitochondrial steroid hydroxylases. Concept for 3-dimensional arrangement of components of mitochondrial cytochrome P-450-containing hydroxylases is shown.
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C. Hepatic 25-Hydroxylation
Vitamin D does not circulate for long in the bloodstream but, instead, is immediately taken up by adipose tissue for storage or liver for further metabolism. In humans, tissue storage of vitamin D can last for months or even years. Ultimately, vitamin D3 undergoes its first step of activation, namely, 25-hydroxylation, in the liver (28) (Fig. 1B). Early data suggested that the liver is the only significant site of 25-hydroxylation in vivo, although there were occasional reports of intestinal and renal extracts containing this activity (346). Research, therefore, focused on purification of the major hepatic enzyme activity. Over the years, there has been some controversy over whether 25-hydroxylation is carried out by one enzyme or two and whether this cytochrome P-450-based enzyme is found in the mitochondrial or microsomal fractions of liver. Madhok and DeLuca (207) reported that a rat liver microsomal system requiring NADPH, molecular oxygen, a flavoprotein, and a cytochrome P-450 was capable of 25-hydroxylation of vitamin D3 , but the cytochrome P-450 responsible has never been cloned. There was some speculation that the microsomal enzyme might be CYP2C11, but this cytochrome is male specific (129) and other data have also been presented that indicate that human microsomes do not possess 25-hydroxylase activity (285). Recently, Axen et al. (9) have purified a pig liver microsomal 25-hydroxylase with an NH2-terminal sequence different from that of CYP2C11 and that is capable of the 25-hydroxylation of both vitamins D2 and D3 . Currently, only the mitochondrial 25-hydroxylase has been purified to homogeneity and subsequently cloned (7, 44, 351). The cytochrome P-450 involved is known as CYP27 or P-450c27 because it is a bifunctional cytochrome P-450 which in addition to 25-hydroxylating vitamin D3 also carries out side-chain hydroxylation, including 27-hydroxylation of cholesterol-derived intermediates involved in bile acid biosynthesis (from which it derives its name) (252).1 The primary amino acid sequences of three species of CYP27 are depicted in Figure 4. Even though 25-hydroxylation of a variety of vitamin D compounds, including vitamin D3 , has been clearly demonstrated in cells transfected with CYP27 (119), there is still some skepticism in the vitamin D field that a single cytochrome P-450 can explain all the metabolic findings observed over the past two decades of research. The many unexplained observations suggesting that other cytochrome P-450 might perform 25-hydroxylation of vitamin D at nanomolar concentrations of substrate that exist in vivo include the following. 1) Perfused rat liver studies by Fukushima et al. (104) demonstrate kinetics consistent with two 25-hydroxylase enzyme activities: a high-affinity, low-capacity form (presumably microsomal) and a low-affinity, high-capacity form (presumably mitochondrial; CYP27).
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| FIG. 4.
Amino acid alignments of all published vitamin D-related cytochromes P-450 from various species: CYP1 (25-OH-D3 1-hydroxylase); CYP27 (mitochondrial vitamin D3 25-hydroxylase); CYP24 (25-OH-D3 24-hydroxylase). Note high degree of sequence similarity between all family members, particularly toward COOH terminus of each isoform. Conserved cysteine residue in block CMGRRLAELEL in extreme COOH terminus is where heme group is covalently bonded to protein. Slightly more NH2 terminal is putative ferredoxin-binding site involving LPLLKAVVKEVLRL. Another highly conserved site is putative oxygen-binding site ELLLAGVDTVSNTL.
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2) Dietary studies show regulation, albeit weak, of the liver 25-hydroxylase in animals given normal intakes of vitamin D after a period of vitamin D deficiency (20), which is not explained by a transcriptional mechanism, since the gene promoter of CYP27 lacks a vitamin D-responsive element (VDRE) (118) or demonstrable responsiveness to 1,25-(OH)2D3 whereas it is regulated by bile acids (354).
3) Clinical studies show no obvious 25-OH-D3 or 1,25-(OH)2D3 deficiency occurs in patients suffering from the genetically inherited disease cerebrotendinous xanthomatosis, in which CYP27 is mutated. [Although a subset of these patients suffer from osteoporosis, this is more likely because of biliary defects leading to altered enterohepatic circulation of 25-OH-D3 (18).]
4) Substrate specificity studies using transfected recombinant human CYP27 show that the enzyme does not 25-hydroxylate vitamin D2 (119); it 24-hydroxylates vitamin D2 instead, evoking the question: Which cytochrome P-450 synthesizes 25-OH-D2?
Observations that are explained by the existence of CYP27 include 1) the occasional reports of extrahepatic 25-hydroxylation of vitamin D3 mentioned above (346), which are consistent with the detection of CYP27 mRNA in a number of extrahepatic tissues including kidney and bone (osteoblast) (10, 147), and 2) the abundance of 24-hydroxylated metabolites [e.g., 24-OH-D2 , 1,24-(OH)2D2 , and 24,26-(OH)2D2] in the blood of vitamin D2-intoxicated animals (143, 161, 178, 320). It is worth noting from perusal of Figure 4 that the recently cloned CYP1 is more closely related to CYP27 than it is to CYP24, a surprising fact given that CYP1 and CYP24 are both renal cytochromes P-450 and appear to be reciprocally regulated, thereby implying CYP27 might have evolved to metabolize vitamin D after all. Thus, although CYP27 remains the best-characterized cytochrome P-450 capable of 25-hydroxylation, it may not be the only 25-hydroxylase, and its full physiological importance remains to be established.
The product of the 25-hydroxylation step, 25-OH-D3 , is the major circulating form of vitamin D3 and in humans is present in plasma at concentrations in the range 10-40 ng/ml (25-125 nM) (140). The main reason for the stability of this metabolite is its strong affinity for the vitamin D-binding (globulin) protein of blood (DBP) (70). The metabolic fate of 25-OH-D3 is dependent on the calcium requirements of the animal. An urgent need for calcium results in renal 1-hydroxylation, whereas an abundance of calcium results in 24-hydroxylation (see sect. II). These two alternative pathways are discussed in turn below.
D. Renal 1-Hydroxylation
The enzyme 25-hydroxyvitamin D3-1-hydroxylase is responsible for the tightly regulated step that involves the introduction of a 1-hydroxyl group into the A ring of 25-OH-D3 , thereby creating the hormone 1,25-(OH)2D3 . The specific location of this enzyme in the kidney became apparent (100) even before the unequivocal identification of 1,25-(OH)2D3 (139). Experiments involving nephrectomized animals have confirmed that the kidney as the major source of the circulating pool of 1,25-(OH)2D3 . The renal 1-hydroxylase enzyme comprises a cytochrome P-450, a ferredoxin, and a ferredoxin reductase (112) (see Fig. 2). The cytochrome P-450 for the 1-hydroxylase enzyme, CYP1, was recently cloned from rat, mouse, and human (228a, 298, 316, 332), and the amino acid sequences of these are compared with the other known vitamin D-related cytochromes P-450 in Figure 4. Because of the close resemblance with CYP27, it has been suggested that CYP1 (P-4501) be termed CYP27B1 (298). Both 1-hydroxylases have short mitochondrial targeting sequences but share many regions of similarity with other members of the family including the classical heme-, ferredoxin-, and oxygen-binding sites indicated in Figure 4. The 1-hydroxylase is induced by PTH through a cAMP/phosphatidylinositol 4,5-bisphosphate (PIP2)-mediated signal transduction mechanism that is still to be defined at the molecular level (132). The enzyme appears to be downregulated by vitamin D status, possibly through a VDR-mediated transcriptional mechanism involving the hormonal product 1,25-(OH)2D3 , although there were early claims that 1,25-(OH)2D3 might act directly on its own synthesis through an allosteric mechanism (117). Work using the perfused vitamin D-deficient rat kidney (281) elegantly shows that the downregulation of 1-hydroxylation takes 2-4 h after exposure to 1,25-(OH)2D3 and is blocked by inhibitors of protein synthesis and transcription. In the same model, the disappearance of the 1-hydroxylase is mirrored by the reciprocal appearance of the renal 25-OH-D3-24-hydroxylase, in effect a complete "switchover" from 1- to 24-hydroxylating activity in the isolated organ over the 4-h period. The exact reciprocal regulation of the two enzymes, first demonstrated in vivo by Tanaka et al. (336) two decades ago, led some workers to postulate that the 1- and 24-hydroxylases might share a single cytochrome P-450 polypeptide chain, its catalytic properties modified by NH2-terminal truncation (111) or regulated by the phosphorylation state of the ferredoxin component of the enzyme (300). The cloning of two distinct cytochromes P-450, representing 1-hydroxylation and 24-hydroxylation, suggests the first hypothesis to be incorrect, the switchover process probably being accomplished by de novo protein synthesis of the required cytochrome P-450. The second hypothesis involving regulation of enzyme activity through ferredoxin phosphorylation remains a possibility for fine-tuning of enzyme activity, although the details of this remain obscure at this point. The cloning of CYP1 has already led to mapping of the human gene to chromosome 12q13.1-q13.3 (316), the same locus as the gene defect for vitamin D dependency rickets type I (192), a disease cured by small doses of exogenous 1,25-(OH)2D3 and which had been previously postulated to be because of a mutated version of the 1-hydroxylase (99). The next decade will see a clarification of the molecular and clinical aspects surrounding this key regulatory step of calcium homeostasis.
There have been indications that there are extrarenal versions of the 1-hydroxylase existing in cells of monocyte/macrophage, placental, and keratinocyte lineages (4, 22, 83). There is strong evidence that the extrarenal enzyme located in macrophages plays a major role in certain granulomatous conditions (e.g., sarcoidosis), causing uncontrolled elevations of blood 1,25-(OH)2D3 levels, which subsequently result in troublesome hypercalcemia and hypercalciuria (3). The normal function of this and other extrarenal 1-hydroxylases remains obscure at this time, although some have postulated a paracrine or autocrine role for locally produced 1,25-(OH)2D3 . Once again, it is safe to predict that the molecular basis for various reports of extrarenal activity in cultured cells in vitro (4, 22, 83) and in certain human disease states will be resolved shortly with the cloning of the renal enzyme (298, 316, 332), as will its mechanisms of regulation.
E. 24-Hydroxylation
The discovery of 24,25-(OH)2D3 (138, 327) predated even the identification of 1,25-(OH)2D3 and the recognition of 24-hydroxylation as a metabolic step allowed for the search for the enzyme activity. The relative ease with which 24,25-(OH)2D3 was generated in such large amounts was a clue that the metabolic step was upregulated rather than downregulated by vitamin D administration. The earliest report of the 25-OH-D3-24-hydroxylase was a subcellular localization study (174) using vitamin D-replete chicken kidney tissue which established that the 24-hydroxylase is a mitochondrial cytochrome P-450-containing enzyme, and this was followed by a reconstitution experiment involving partially purified enzyme components (248). Evidence was also emerging both in vivo and in vitro that the 24-hydroxylase was not confined to the kidney but could be found in classical vitamin D target tissues including the intestine and bone (185, 347). In the late 1970s, it became evident that 24-hydroxylation was probably only the first step in an inactivation process. First, it was shown to be induced by 1,25-(OH)2D3 itself (172, 187, 335, 337, 340), and the product 1,24,25-(OH)3D3 was 10 times less biologically active than 1,25-(OH)2D3 (56, 335). Second, 24,25-(OH)2D3 and its 1-hydroxylated analog, 1,24,25-(OH)3D3 , could be converted to further metabolic products containing a 24-oxo and/or 23-hydroxy groups as well (237, 246, 330). The perfused rat kidney was helpful in establishing the production of these catabolites (160) but revealed two further pieces to the puzzle of the metabolic role of 24-hydroxylation. First, the perfused rat kidney allowed for a clarification of the temporal relationship of these catabolites, in effect, suggesting the existence of a pathway from 1,25-(OH)2D3 and/or 25-OH-D3 (160), and second, the perfused kidney generated two side chain-cleaved molecules: a 23-alcohol (159) and a 23-acid (208, 276), not observed previously in vitro. The 1-hydroxylated 23-acid, calcitroic acid, is observed in vivo and has been shown to be the principal biliary excretory form of 1,25-(OH)2D3 (94). These discoveries led to the rationalization of many findings from a number of laboratories into a hypothesis that 24-hydroxylation is the first step of a target cell C-24 oxidation pathway (Fig. 3) whose major function is to convert 1,25-(OH)2D3 to calcitroic acid (208, 276). The demonstration of vitamin D-inducible, calcitroic acid production in bone (UMR106) and kidney (208) was followed by demonstration of vitamin D-inducible C-24 oxidation pathway activity in a number of vitamin D target cells including intestine (Caco-2), keratinocyte (HPKIA-ras), and breast (T47D and MCF-7) (215, 278, 344).
In the early 1990s, Okuda's group succeeded in cloning the cytochrome P-450, CYP24 or P-450cc24, representing the 24-hydroxylase (62, 148, 247). The amino acid sequences of three species of CYP24 are shown in Figure 4. It belongs to the same subfamily as the other two known vitamin D-related cytochromes. The structure of the gene for CYP24 has been described for several species (rat, mouse, and human), and in each case shown to possess two VDRE in its proximal promoter (249, 367, 368). These VDRE allow for the 1,25-(OH)2D3 VDR-mediated upregulation of CYP24 from undetectable to strongly detectable expression at the mRNA level within 4 h (297). This is consistent with the enzyme activity pattern first observed in the kidney but subsequently reported for a variety of vitamin D target cells after initial exposure to hormone (127, 202, 209). Metabolic studies with the recombinant CYP24 protein produced in bacteria or insect cells have been equally enlightening. Akiyoshi-Shibata et al. (5) and Beckman et al. (15) have shown that CYP24 is a multicatalytic enzyme capable of several if not all of the steps illustrated in Figure 3. It is likely that CYP24 is able to catalyze three successive oxidations, two at C-24 and one at C-23, to give an intermediate that is subsequently cleaved by an unknown mechanism. All prevailing evidence suggests that C-24 oxidation is a highly efficient process giving rise to molecules of lower biological activity (e.g., calcitroic acid) such that if C-24 hydroxylation is blocked by a general cytochrome P-450 inhibitor such as ketoconazole, 1,25-(OH)2D3 hormone action is extended (265). This argues that the main role of the C-24 oxidation pathway is attenuation of the biological signal inside target cells.
Recently, this hypothesis was tested when St. Arnaud et al. (315) engineered a CYP24-null mouse. At this time, the results have been reported only in abstract form, but it is clear that the defect is not lethal during embryonic development. Instead, at least one-half the mice exhibit hypercalcemia/hypercalciuria in early neonatal life and quickly die before weaning from nephrocalcinosis. The other half of animals survive and appear healthy perhaps due to the upregulation of some alternative vitamin D-catabolic pathway (C-26 hydroxylation or 26,23-lactone formation). All CYP24-null animals exhibit abnormal bone histology, characterized by excessive unmineralized bone matrix and reminiscent of that previously observed in experiments involving exogenous 1,25-(OH)2D3 intoxication (136). This could be caused by an inability of target cells, in this case osteoblasts, to turn off the 1,25-(OH)2D3 signal in the absence of the C-24 oxidation pathway. An alternative explanation that CYP24 is needed for the synthesis of some essential 24-hydroxylated metabolite of vitamin D [e.g., 24,25-(OH)2D3] needed for a yet to be defined role in bone formation has been proposed (314) but seems unlikely for several reasons. Among the data that argue against such an essential role for 24-hydroxylated metabolites are the findings from the study of fluorinated analogs of vitamin D (149, 234, 338). These analogs are irreversibly blocked in the C-24 and C-23 positions of the side chain with one or more atoms of fluorine and therefore unable to undergo 24- or 23-hydroxylation. However, they are fully biologically active in all known in vivo functions of vitamin D. Furthermore, the biochemical machinery required for transducing the signal from a 24-hydroxylated metabolite has never been satisfactorily demonstrated in bone (or any other) cells. Thus it appears that 24-hydroxylation is not essential for vitamin D to fulfill its many biological roles in vertebrate biology. Therefore, evidence from the CYP24-null mouse seems to confirm our hypothesis that the C-24 oxidation pathway is a complex, self-induced mechanism for limiting the action of 1,25-(OH)2D3 in vitamin D target cells once the initial wave of gene expression has been initiated (see sect. III). This is discussed in greater detail in section V.
B. Role of Parathyroid Gland and Its Hormone
For many years it has been clearly recognized that the parathyroid gland is the calcium-sensing organ in the body (266, 270, 283). Thus, in response to even slight hypocalcemia, the parathyroid glands react within seconds to secrete the 84-amino acid peptide hormone PTH (302). This hormone then initiates the sequence of events that results in the mobilization of calcium to replace that which has been taken from plasma.
There has been a great deal of recent effort expended in the direction of the calcium receptor for the parathyroid glands. The calcium receptor is well known (39, 225) and appears to act in a cAMP-dependent mechanism to facilitate the secretion of PTH. This calcium receptor may play an important role in other tissues as well. Interested readers are directed elsewhere (301). The PTH has a lifetime in plasma that can be measured in minutes if not seconds (238). The receptor for the PTH is known and has been cloned (289). This receptor is found throughout the length of the nephron of kidney, is not found in the intestine, and is found in the osteoblasts but not osteoclasts of the skeleton (2). In the kidney, the PTH plays an important role in many functions (101). Well known is that it blocks reabsorption of phosphate causing a phosphate diuresis (39). In the proximal convoluted tubule cells, it activates the 25-hydroxyvitamin D-1-hydroxylase (25-OH-D3-1-OHase) that converts 25-hydroxyvitamin D3 to the active hormone, 1,25-(OH)2D3 (109, 298, 340). As already discussed in section I, the 25-OH-D-1-OHase has now been cloned by three research groups (298, 316, 332) that will undoubtedly result in our understanding of the molecular mechanism whereby PTH activates the 1-OHase. At the present time, it is known that PTH through cAMP (141) activates the 1-OHase by increasing the mRNA encoding for this important enzyme (298). Whether it acts at the transcriptional level or elsewhere remains to be determined. At the same time the PTH through cAMP activates the 1-OHase, it markedly suppresses the 25-OH-D-24-OHase, the major enzyme involved in destruction of the vitamin D hormone as described in section I (297). Again, the mechanism of suppression of 24-hydroxylase (24-OHase) by PTH remains unknown, although there is clearly a decrease in the mRNA encoding for the 24-OHase. These two actions result in a marked elevation of plasma levels of 1,25-(OH)2D3 (341).
E. Vitamin D Metabolites and Other Hormones
There has been considerable interest in other metabolites of vitamin D playing an important role in the regulation of calcium mobilization in suppression of hypercalcemia, or in bone growth. Of particular importance are the many studies carried out on 24,25-(OH)2D3 . This compound has been alleged to be important in the regulation of calcium homeostasis (245) or in the formation of the skeleton (33, 230, 255) or in counteracting the hypercalcemia activity of 1,25-(OH)2D3 (201). By now, extensive studies have been carried out with fluoro analogs, namely, 24,24-F2-25-OH-D3 to show that hydroxylation on the 24-position has no functional significance (38, 149). Thus animals grown for two generations with 24,24-F2-25-OH-D3 as their sole source of vitamin D illustrate that 24-hydroxylation is not required either for skeletal formation, maintenance of calcemia, or growth of bone. More recently, a knockout of the 24-hydroxylase has been reported, and the results are not at all clear as to whether 24-hydroxylation plays a role except in the destruction of the potent hormone 1,25-(OH)2D3 and its precursor, 25-OH-D3 (313). Further work using these models is required before any conclusions can be reached.
Other hormones such as estrogen and glucocorticoids have significant effects on bone and calcium metabolism, but they do not appear to be directly involved in regulating serum calcium concentration. This seems largely to be the role of the vitamin D hormone, the PTH, and calcitonin.
F. Intestinal Calcium and Phosphate Absorption
From a historical point of view, the role of 1,25-(OH)2D3 in intestinal absorption of calcium is perhaps best known. Orr et al. (256) discovered that vitamin D is required for intestinal calcium absorption many decades ago. This was reaffirmed by the work of Nicolaysen and Eeg-Larsen (239), who further demonstrated that the need for calcium increased the ability of the animal to absorb calcium. He postulated the existence of an endogenous factor that would inform the intestine of the skeletal needs for calcium. This basic observation was then shown to be primarily the vitamin D endocrine system, and 1,25-(OH)2D3 has been thought to be the agent that stimulates intestinal calcium absorption to meet the needs of the skeleton (34). It is, therefore, abundantly clear that intestinal calcium absorption remains as one of the basic functions of 1,25-(OH)2D3 . Quite independently of calcium is the role of 1,25-(OH)2D3 in stimulating intestinal absorption of phosphate (126, 180). Both are active calcium transport mechanisms, but they appear to be independent of each other (63, 126, 180). The molecular mechanism of action of 1,25-(OH)2D3 in stimulating intestinal calcium absorption and intestinal phosphate absorption remains unknown, despite many efforts by many investigators. 1,25-Dihydroxyvitamin D3 stimulates the production of calbindin D9k in mammals (342) and calbindin D28k in birds (68) to appear in the intestine. In the case of the 28k protein in birds, it is absent in deficiency and present in large amounts after stimulation by vitamin D (357). A vitamin D-responsive element has been demonstrated to be present in the calbindin D9k promoter in mammals (77) and the 28k mammalian gene (113) but that has not been shown for the calbindin D28k protein of birds. The exact molecular mechanism for initiating production of the calbindin D28k remains to be determined. Furthermore, if one studies the time course of appearance of the 28k in birds as related to calcium absorption, there is no clear-cut correlation (125, 312). The appearance of this protein and calcium transport coincide as a function of time in response to vitamin D or 1,25-(OH)2D3 . However, calcium absorption diminishes while calbindin D28k remains high in the gut. Therefore, there appears to be at least some discrepancy between calbindin D28k and calcium transport. It has suggested that some other protein or proteins are involved. This led to an analysis of a calcium pump in the basolateral membrane that is induced by 1,25-(OH)2D3 (357). However, the degree of induction in the time course of its appearance is not certain to account for the role of vitamin D in calcium transport. In short, the molecular mechanism of action of 1,25-(OH)2D3 in inducing intestinal calcium and phosphate transport is largely unknown. Wasserman and Feher (357) believe there are multiple sites of action of 1,25-(OH)2D3 in intestinal calcium absorption. Considerable work will be required before one can demonstrate the exact role of the calbindin proteins and the calcium pump in the vitamin D-induced calcium transport.
G. Vitamin D and Bone Calcium Mobilization
Even more poorly understood is the role of vitamin D in bone resorption or bone mobilization. The idea that vitamin D could result in the mobilization of calcium from bone was derived from the early work of Bauer et al. (14). A vitamin D-deficient animal on a zero-calcium diet will provide an increase in serum calcium at the expense of skeleton when given vitamin D. This mechanism requires the presence of PTH (110). Furthermore, 1,25-(OH)2D3 is clearly a stimulator of osteoclastic bone resorption in culture (269, 319). However, the osteoclast has neither a receptor to the PTH nor a receptor to the vitamin D hormone (223). Instead, a signal appears to arise from interaction of these two hormones with the osteoblast. This signal causes the osteoclast to resorb bone (221, 222, 328). There is also the possibility that upon 1,25-(OH)2D3 and/or PTH signaling, the osteoblast may cause the transport of calcium from the bone fluid compartment to the plasma compartment (333). The nature of the signal arising from stimulation by the PTH and by the vitamin D hormone on the osteoblast has not been determined.
On the other hand, a great deal of progress has been made in understanding the role of vitamin D and osteoclastic formation and differentiation (328). Through the work of Abe et al. (1) and Tanaka et al. (334) has come the discovery that 1,25-(OH)2D3 plays an important role in causing the differentiation of promyelocyte to the monocyte. The monocyte is considered the precursor of the giant osteoclast. Further differentiation of the osteoclast precursor is catalyzed by the osteoclast differentiation factor that is produced by the osteoblast in response to 1,25-(OH)2D3 (328).
The action of vitamin D in stimulating osteoclastic bone resorption may be to provide bone calcium for the plasma but more likely to cause bone resorption in preparation for coupled formation to complete the bone-remodeling process. This would argue that the vitamin D hormone is involved in this important function that strengthens bone and repairs microfractures that may have occurred during bone usage. At this stage, therefore, 1,25-(OH)2D3 may be viewed as an important hormone that not only plays a role in bone calcium mobilization when required but also plays an important role in initiating bone remodeling and modeling systems required for shaping bone and required for repairing bone (107, 319).
A. Overall Mechanism of Transcriptional Regulation by Vitamin D
The mechanism by which 1,25-(OH)2D3 exerts its effects on transcription is rapidly being uncovered. It is becoming clear that the vitamin D system shares many features with other ligand-activated nuclear receptors such as the retinoic acid receptor (RAR) and thyroid hormone receptor (TR) (95), including many of the same transcriptional cofactors. A summary of what is currently known about the activation process is shown in Figure 6.
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| FIG. 6.
Proposed mechanism for transcriptional upregulation of a target gene by vitamin D receptor (VDR). After ligand binding, receptor forms a heterodimer on a response element with retinoid X receptor (RXR). Binding of coactivator protein to heterodimer-DNA complex is followed by histone acetylation and subsequent release of histones from DNA. Transcription factors are then able to initiate transcription of target gene, resulting in production of corresponding protein. RNAP, RNA polymerase; CBP, calcium-binding protein; DRE, vitamin D response element.
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In the first place, activation of vitamin D target genes has been shown to require a specific nuclear receptor protein, the VDR. Target genes are upregulated through binding of the VDR protein to specific DNA sequences termed response elements in the promoter regions of these genes. As shown at the top of Figure 6, binding of 1,25-(OH)2D3 to the receptor increases heterodimerization of VDR with a cofactor, the retinoid X receptor (RXR), on a response element (169). Binding of the heterodimer to the response element induces a bend in the DNA of the promoter (151). Binding of 1,25-(OH)2D3 to the receptor also appears to change the conformation at the COOH terminus of the VDR, permitting a region termed the AF-2 domain to interact with other transcription factors, including coactivator proteins such as SRC-1 (216, 253, 279, 356). Recent exciting work indicates that coactivator proteins possess intrinsic histone acetylase activity (59, 345). These coactivators bind to transcriptional "integrators" such as calcium-binding protein (CBP) and p300 (59, 152, 165), which, in addition to other functions, have also been shown to possess histone acetylase activity (179). Thus recruitment of coactivators to a promoter by a liganded receptor appears to result in remodeling of DNA structure through acetylation of histones and their subsequent release from DNA. This in turn leads to opening of the promoter to the transcriptional machinery. The net result of binding of liganded receptor to an upregulated target promoter is therefore to increase the rate of transcription of the gene, leading to increased production of the corresponding protein. It must be stressed at this point that a number of the details of this proposed mechanism are unclear at this point, including the exact sequence of events after receptor binding to the promoter, so the order of events presented in Figure 6 is somewhat speculative.
In contrast to genes that are upregulated by vitamin D, several genes, including PTH (85, 251) and interleukin (IL)-2 (6), have been shown to be downregulated by 1,25-(OH)2D3 . The means by which this downregulation is carried out is not clear in all cases, and at least two possibilities exist. The first is that, as has been proposed for the IL-2 promoter, VDR may bind to a downregulatory response element and disrupt the binding of upregulatory transcription factors, leading to a decrease in transcription (6). For other downregulated genes, the situation may be quite different, and binding of VDR to an inhibitory response element may lead to interactions with repressor proteins that decrease transcription of the gene. Interestingly, corepressor proteins such as nuclear receptor corepressor (NCoR) (142) and silencing mediator for retinoic acid receptor and thyroid hormone receptor (SMRT) (60) have recently been found to bind, through intermediary proteins, to histone deacetylase enzymes (131, 233). Presumably in this situation the deacetylated histones then bind to the promoter of the downregulated gene and shut off transcription. The VDR has not yet been reported to bind to corepressor proteins, but given the rate at which such proteins are currently being discovered, it is possible that a corepressor that binds to VDR will be uncovered.
Phosphorylation of the receptor may also play a role in the induction of transcription by the VDR (73), perhaps through modulation of the affinity of VDR for the various cofactor proteins involved in transcription. Rapid phosphorylation of the VDR has been shown to occur in organ culture systems upon addition of ligand (41). The phosphorylated residues have been localized to the ligand-binding domain of the protein. The exact functional consequences of this phosphorylation have been difficult to determine. Estrogen receptor phosphorylation by mitogen-activated protein kinase has been shown to have a direct and measurable effect on transcription (167), but this phosphorylation was mapped to the NH2-terminal AF-1 region of the estrogen receptor, which is lacking in VDR. Effects of phosphorylation on activation of the AF-2 domain, at the COOH terminus of the protein, have not yet been clearly shown for the VDR. Indeed, even the kinase (or kinases) responsible for the phosphorylation in vivo has yet to be determined. Some studies have suggested that serine-208 of the VDR can be phosphorylated by casein kinase II and that phosphorylation of this residue may cause increased transcriptional activity (162). Other work has revealed that mutation of this serine failed to affect transcription, although alternate phosphorylation on adjacent serines was noted (135). The exact effects of phosphorylation must await determination of the precise amino acids phosphorylated in vivo and the functional consequences that follow from this.
B. Vitamin D Receptors
The VDR is a member of a superfamily of nuclear receptors (95). Within this family, the VDR has the highest similarity to the subfamily that includes retinoic acid, thyroid hormone, and peroxisome proliferator activator receptor (PPAR) receptors, to which it has sequence and structural resemblance. The VDR has been cloned from several species (12, 42, 92, 164, 219) and shows considerable similarity between species in size and sequence. In the rat, for example, the VDR protein consists of 423 amino acids, with a molecular mass of ~50 kDa, whereas in the human the protein has an additional 4 amino acids at the NH2 terminus, for a total of 427.
Like the other nuclear receptors, the VDR can be divided by function into several domains. An illustration of the different domains of the VDR is shown in Figure 7. At the NH2 terminus is a truncated A/B domain of ~20 amino acids, to which little function has yet been ascribed for the VDR. After this, the DNA-binding domain, termed the C domain, is located between amino acids 20 and 90. A D or hinge domain is located approximately between amino acids 90 and 130, followed by the COOH-terminal E or ligand-binding domain between amino acids 130 and 423. The ligand-binding domain of the protein is a complex region of the protein, responsible for high-affinity binding of ligand, for dimerization with RXR, and for binding to transcription factors (95). It should be noted that exact delineation of the division between the hinge region and the ligand-binding domain is somewhat uncertain and is based on deletion analysis.
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| FIG. 7.
Schematic illustration of structure of VDR protein, showing functional domains of which protein is composed. Regions of receptor thought to interact with transcription factors TFIIB and RXR are shown. COOH-terminal AF-2 domain is shown in black.
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Knowledge of the structural and functional properties of the receptor proteins has increased dramatically in recent years, after the advent of systems which allow expression and purification of large quantities of receptor protein from bacteria. This has allowed a piecemeal approach to determination of receptor domain structures; the DNA-binding and ligand-binding domains of several receptors have been expressed separately and purified, and structures have been determined by NMR or X-ray crystallography (32, 272). In contrast, to this point, information concerning the VDR has come primarily from site-directed mutagenesis, in which the receptor cDNA is mutated and the mutant protein studied by transfection into mammalian cells (236). No structural data are yet available concerning the VDR, but the other receptor domains may serve as models on which to base hypotheses about the VDR and its properties.
Structures for the DNA-binding domains of other receptors, including the RXR, have been obtained using both NMR and X-ray crystallography. The RXR structure showed that the DNA-binding domain, comprised of two zinc finger motifs, consists of two -helixes oriented at approximately right angles to one another (194). One helix, termed the orientation helix, thought to be critical for recognition of the receptor response element was proposed to fit into the major groove of the DNA and bind to a specific DNA sequence in the response element. These domains contain two zinc atoms tetrahedrally coordinated to eight conserved cysteine residues. Both the zinc atoms and the cysteine residues are necessary to maintain the three-dimensional structure required for response element recognition and DNA binding. The amino acid sequence of the DNA-binding domain is similar between members of the receptor superfamily, suggesting that these structures can be used as a model for that of the VDR.
There is as yet no direct evidence for the structure of the VDR-RXR complex bound to DNA. However, the crystal structure of the TR and RXR DNA binding domains complexed to a DR4 response element has been determined (272). With the use of this structure as a model, the VDR DNA-binding domain was hypothesized to have specific amino acid contacts with RXR: between the asparagine residue 14 of VDR and RXR residues glutamine-49 and arginine-52. In addition, lysine-68 and glutamate-69 of VDR were modeled to form salt bridges with RXR residues aspartate-39 and arginine-38, respectively. These interactions were thought to account for the optimal spacing of three residues between the direct repeats of the vitamin D response element (DR3).
The crystal structures of the ligand-binding domains of the RXR- (without ligand) (32), the RAR-
(with ligand) (279), and the TR-1 (with ligand) (356) have been determined. The ligand-binding domains of the RXR and RAR had been previously predicted to have a high content of -helix (64, 204), and this was confirmed by structural analysis. All three ligand-binding domains were found to share a common secondary structure of 12 -helixes, with a small content of 
-sheet. A comparison of the structures of the RAR and TR with that of the VDR is shown in Figure 8. The COOH-terminal portion of the proteins, termed the AF-2 domain, has been determined to be critical for transcription. Removal of this portion of the protein results in decreased ligand-binding affinity and loss of transcriptional activation. The structural data indicate that the helixes 11 and 12 of the RAR and TR may undergo a large conformational change in response to ligand binding, folding up around the ligand in what the authors of the RAR paper term a mouse trap action to form a hydrophobic pocket (279). This brings the amino acid residues on helix 12 which make up the AF-2 domain into position to interact with other transcription factors. The VDR shows sequence similarity to the RAR and TR in this region and may be expected to possess a similar three-dimensional structure and to undergo a similar conformational change upon ligand binding.
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| FIG. 8.
Sequence alignment of human retinoic acid receptor (RAR)-, rat thyroid hormone recetpor (TR)- and rat VDR. Sequences of ligand-binding domains of these proteins were aligned using program "Pileup" (Genetics Computer Group, University of Wisconsin-Madison). Gaps introduced into sequence to optimize alignment are denoted by dots. Numbering of amino acids of each ligand-binding domain (LBD) is to right of sequences and corresponds to numbering of full-length protein. -Helical regions of RAR and TR are underlined with solid lines; -sheet regions are underlined with dashed lines. Regions of amino acid identity are enclosed with boxes. Helixes are denoted by numbers in accordance with TR structure; sheet regions are denoted s1-s4, also according to TR structure. Amino acids in RAR and TR shown by X-ray crystallography to be in contact with ligand are shaded.
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The wild-type VDR binds its ligand, 1,25-(OH)2D3 , with extremely high affinity, in the range of 10
10 M (208, 282). Both the 1- and 25-hydroxyl groups are critical for high-affinity binding, and the absence of either results in approximately a 500-fold decrease in affinity of ligand for the receptor (82). The exact amino acid residues in the receptor that contact the ligand remain unknown, although some work has been done recently on affinity labeling of the binding site (273). Previous work with transfected cells has shown that deletion of the NH2-terminal 116 amino acids of the VDR left a protein with measurable ligand binding activity, whereas deletion of the NH2-terminal 160 amino acids did not (220). More recent work with fusion proteins has shown that deletion of the NH2-terminal 124 amino acids gave rise to a functional ligand-binding protein, whereas deletion to amino acid 172 did not (242). From the other end, removal of the COOH-terminal 20 amino acids of the VDR resulted in a 10-fold loss of affinity for ligand, whereas removal of more than this number resulted in complete loss of ligand binding (236). Thus a core domain of ~300 amino acids was shown to be required for the protein to bind ligand with wild-type affinity. This is in accordance with the ligand-binding domains of the related receptors that have been crystallized, which were all expressed in bacteria as proteins of between 250 and 300 amino acids.
An alignment of the VDR ligand-binding domain sequence with that of the RAR and TR supports the possibility that amino acids in the VDR directly in contact with ligand begin at approximately amino acid 220. In Figure 8, amino acids in contact with ligand in the RAR and TR crystal structures are shaded. Inspection of this figure shows that the contact amino acids begin at helix 3 in both the RAR and TR. Based on sequence alignment, the same region of the VDR begins at approximately amino acid 220, with a leucine residue that is conserved in all three proteins. Interestingly, work in which 10 amino acid segments of the VDR were sequentially deleted found that impairment of ligand binding did not occur until approximately amino acid 230, which is in accordance with this possibility (154).
Site-directed mutagenesis of the VDR ligand-binding domain has been performed recently on several of the cysteine residues in the human protein (235). Alteration of cysteine-288 to glycine resulted in severe attenuation of ligand binding at room temperature, whereas the same mutation at cysteine-337 resulted in a smaller decrease in affinity. The exact significance of this result is uncertain, although a contact between cysteine-237 and carbon-13 of retinoic acid was noted in the binding of retinoic acid to the RAR- (279). Interestingly, in Figure 8, the corresponding cysteine residue (cysteine-284) of the rat receptor aligns exactly with ligand-contacting amino acids in the RAR and TR, suggesting that this cysteine may in fact directly contact the ligand.
The VDR is generally expressed at relatively low levels in vivo. Target tissues, such as bone, kidney, and especially intestine, may have relatively high levels of receptor (3,000-6,000 fmol/mg protein), but in other tissues, the levels are generally much lower (72). The receptor has been shown to be present in most tissues that have been examined, including activated immune cells such as T cells, where it may play a role in modulating the levels of cytokines such as IL-2 (6). In contrast to other receptors, such as the glucocorticoid receptor, which is associated with a number of proteins, including heat shock proteins, in the cytoplasm before hormone binding (146), studies with radiolabeled 1,25-(OH)2D3 have shown that the VDR is predominantly nuclear (325). Little information is available concerning whether any proteins associated with the VDR before DNA binding. There are some data suggesting that many hormone-binding receptors, including VDR, contain a binding site for calreticulin in the DNA-binding domain (43). Some reports indicate that calreticulin may bind to VDR and inhibit activation of vitamin D target genes (358), but the physiological significance of this is as yet unclear.
There have been numerous reports in recent years of rapid nongenomic effects of vitamin D on calcium transport, termed transcaltachia (241). The physiological importance of these data is unclear. Whether these effects are mediated by the VDR or a different protein is also unclear. The development recently of a mouse model in which the VDR has been ablated (365) may provide some insight into the relevance of these reports, and into whether the VDR plays any role in them.
Recently, the promoters of the mouse (150) and human (226) VDR have been isolated. Some studies have suggested that the VDR is upregulated by treatment with agents such as forskolin, which induce protein kinase A (183). Further study of these promoters will shed more light on the means by which VDR protein levels are regulated inside target cells.
C. Retinoid X Receptors and Other Coactivators
In the absence of cofactor proteins, the VDR is unable, at physiological concentrations of protein, to bind to most response elements that have been described. This has become clear from both in vitro gel retardation assay experiments and from experiments involving receptor expression in yeast (155, 231, 310). Work with the VDR and with related receptors indicated that the RXR is the required cofactor.
The RXR was isolated several years ago, and initially its ligand and functional importance were unknown (211). Subsequent work identified 9-cis-retinoic acid as a ligand for RXR (134). It also became clear that RXR plays a critical role in binding to DNA of several different receptors, including the VDR, the TR, the RAR, and the PPAR, among others (98, 173). In the absence of RXR, it appears that none of these receptors binds efficiently to their response elements. Like other receptors, the RXR can be divided into functional domains, including an NH2-terminal A/B domain, a C domain which binds to DNA, and a COOH-terminal DE domain that binds ligand and activates transcription (58, 210). Heterodimerization with other receptors appears to be mediated through two interfaces: one in the DNA-binding C domain (366) and another in the ligand-binding E domain (359). Interestingly, in structural studies, the RXR ligand-binding E domain was found to crystallize as a dimer, and the dimer interface occurred along helices 9 and 10, exactly where mutagenesis studies suggest RXR heterodimerizes with other receptors (32, 261). This region of RXR from amino acids 389 to 429, termed the I box, is sufficient for strong interaction between RXR and TR, but the same sequence provides only weak interaction with VDR. Additional NH2-terminal RXR sequence is required for strong interactions with VDR (261), indicating a potential distinction between VDR and related receptors.
Most vitamin D response elements consist of two half-sites of the sequence AGGTCA separated by three bases (see sect. IIID). Because RXR is required for binding to the response element, the question arose as to whether one half-site was preferred by RXR, and if so, whether this preference was for the upstream or downstream site. For VDR, as for the other receptors that heterodimerize with RXR, it appears that RXR binds to the 5'-half-site and VDR the 3'-half-site (189, 366), although there may be some exceptions to this rule. The polarity of this binding has been shown to be important for maximal gene activation, with significantly lowered transcription rates occurring if the response element orientation is reversed relative to the promoter start site. Binding of the VDR-RXR heterodimer to a response element is greatly increased by 1,25-(OH)2D3 when salt concentrations are in the physiological range, i.e., 100-150 mM (169). Interestingly, for many of the receptors that heterodimerize with RXR, the natural ligand of RXR, 9-cis-retinoic acid, may not enhance DNA binding. Some findings indicate that RXR may be unable to bind ligand while in a complex with another receptor (98). In addition, some in vitro reports indicate that the VDR-RXR complex is disrupted by addition of 9-cis-retinoic acid (205). This contrasts with work in transfected cells indicating that both 1,25-(OH)2D3 and 9-cis-retinoic acid enhanced reporter gene expression from a vitamin D-responsive promoter (291). The reason for these conflicting results is unclear; it may be an experimental artifact, or it may be that the additional proteins that interact with the VDR-RXR heterocomplex in vivo, such as the coactivator/corepressor proteins, affect the conformation of the RXR and permit ligand to bind.
The VDR has been shown to interact directly with a growing number of other transcription factors, including transcription factor IIB (TFIIB). The interaction of VDR with TFIIB has been characterized in several reports (27, 206). TFIIB is a 30-kDa protein that was originally isolated as a cofactor associated with TATA-binding protein (120). A region of VDR of ~70 amino acids in the D domain has been reported to bind to a 43-residue NH2-terminal region of TFIIB (27, 203). The sequence of TFIIB in this region is similar to that of a zinc finger, suggesting that the zinc finger motif may function in protein-protein interactions as well as DNA binding. Interestingly, one report suggests that binding of ligand to VDR causes dissociation of TFIIB from the receptor, which may indicate a complex interaction between proteins before the initiation of transcription (217).
Interactions between the VDR and other factors are less well characterized, although this work is progressing, particularly with regard to the coactivator proteins. Recent work indicates that VDR binds to SUG1/TRIP1, a nuclear protein that also binds to other receptors (355). It should be noted that the function of SUG1 in VDR-mediated transcription is unclear. SUG1 binds to the AF-2 domain of the VDR (216, 355), but because SUG1 has been found to be a component of the proteasome complex in the nucleus, it may function primarily in receptor degradation.
The VDR has also been shown to bind to a growing number of coactivator proteins such as SRC-1 and TIF-1 (216, 355). Other coactivator proteins that have been discovered recently, including ACTR (59) and p/CIP (345), join the rapidly enlarging group of such proteins. The coactivators seem to fall into a family with many features in common, including size (~1,400 amino acids), structure (NH2-terminal Per-Arnt-Sim/basic helix-loop-helix domains, central receptor interaction domains) and activity (COOH-terminal histone acetylase domains). It seems likely that most, if not all, of the coactivator proteins will be found to interact with VDR and activate transcription. For example, both SRC-1 and TIF-1 have been shown to bind to the VDR (216, 355), whereas nuclear receptor coactivator protein (ACTR) has been shown to enhance transcription from a vitamin D target gene in transfected cells (59). However, it is not yet clear whether all coactivators interact with VDR equally well; some may function more effectively than others in the vitamin D system. An additional possibility is that tissue-specific expression may be a significant factor in determining which coactivator works with the VDR. For example, the coactivator ACTR was shown to be expressed at relatively low levels in kidney, an important vitamin D target tissue (59). This may indicate that other coactivators play a more important role in transcriptional activation of vitamin D-controlled genes, at least in this tissue.
Whether VDR interacts with corepressor proteins to bring about downregulation of target genes is currently unclear. Like the coactivator proteins, the corepressor proteins appear to have many features in common. The corepressors SMRT and NCoR (142), for example, have considerable sequence similarity, although NCoR possesses a large NH2-terminal region lacking in SMRT. In addition, both proteins appear to repress transcription through interactions with the Sin3 proteins, which bind to histone deacetylase enzymes (131, 233). However, unlike related receptors such as retinoic acid and TR, it is unclear whether the VDR is bound by corepressor proteins. At least one corepressor protein, NCoR, has been reported not to bind to VDR at all. NCoR binds to a conserved region of the TR and RAR, termed the CoR box, to exert its effects; binding of ligand to the TR or RAR causes dissociation of NCoR from receptor. NCoR does not bind to the CoR box region of the VDR, despite the similarity of the VDR sequence in this region to that of TR and RAR (142). This may explain why the VDR does not seem to exhibit dominant negative silencing seen with both the TR and RAR (262, 286). The possibility remains that other corepressor proteins will be found to bind to the VDR and that this interaction is responsible for silencing at least some of the genes that are downregulated by vitamin D.
-Hydroxylation
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References
The important reactions that occur to the vitamin D molecule and the important reactions involved in the expression of the final active form of vitamin D are reviewed in a critical manner. After an overview of the metabolism of vitamin D to its active form and to its metabolic degradation products, the molecular understanding of the 1
