Lennarz W.J., Lane M.D. (eds.) Encyclopedia of Biological Chemistry. Four-Volume Set . V. 4

Подождите немного. Документ загружается.

transcription factor. The AF-2 domain exists at the

extreme COOH terminus while the AF-1 domain is

located in the N-terminal region (or A/B domain) of the

receptors (Figure 2). The AF-1 domain is a constitutive

(i.e., hormone-independent) activation domain. In the

VDR, the N-terminal A/B domain is truncated compared

to other nuclear receptors and thus an analogous

constitutive AF-1 domain is lacking in the VDR.

In order for VDR to regulate transcription of target

genes, it must recognize and bind to DNA in the

promoter regions of vitamin D-responsive genes. It does

so through a specialized DNA-binding domain (DBD)

located near the amino terminus of the receptor

(Figure 3). This DBD is required for the VDR to bind

selectively and with high afﬁnity to speciﬁc DNA

sequences termed vitamin D-responsive elements

(VDREs). The minimal DBD of the VDR that mediates

VDR–DNA interactions resides between amino acid

residues 22 and 113 in the human sequence. There are

nine cysteine residues within the DBD that are conserved

throughout the family members. The ﬁrst eight of these

cysteines (counting from the N terminus) tetrahedrally

coordinate two zinc atoms to form two zinc ﬁnger DNA-

binding motifs. Mutation of the ﬁrst eight of the nine

cysteine residues to serines eliminates VDR binding to

both nonspeciﬁc and speciﬁc DNA sequences and

eliminates VDR-mediated transactivation. Indeed, inac-

tivating mutations within the DBD of the human VDR

are responsible for the rare inherited disorder termed

vitamin D-resistant rickets type II. These patients

express a nonfunctional VDR that cannot bind to

DNA, and they exhibit classical symptoms of vitamin

D deﬁciency that are not corrected by providing an

external source of vitamin D.

LIGAND-BINDING DOMAIN (LBD)

The large carboxyl-terminal ligand-binding domain

(LBD) of VDR is organized into 12

-helices. As the

name suggests, the LBD is responsible for high-afﬁnity

binding of the 1,25(OH)

ligand, exhibiting equili-

brium binding constants of the order of 10

210

SRCs

LXXLL

Med

220

RNA Pol II

TBP

LXXLL

VDRE

p300

SRCs

p300

NCoA62/

SKIP

RXR

VDR

VDRE

RXR

VDR

NCoA62/

SKIP

VDR

VDRE

RXR

VDR

RXR

VDR

RXR

SRCs

p300

FIGURE 1 Mechanism of action of the vitamin D receptor. VDR

binds to its ligand, 1,25(OH)

, heterodimerizes with RXR, and

binds to a DR-3 type VDRE in the promoter of 1,25(OH)

responsive genes. VDR occupies the proximal or 3

half-site while RXR

occupies the distal or 5

half-site. VDR/RXR then recruits coactivators

such as SRCs and p300/CBP. The HAT activity of these coactivators

loosens the chromatin structure, allowing for a more transcriptionally

permissive environment. SRCs and p300/CBP dissociate, allowing

for binding of NCoA62/SKIP and the Mediator-D multimeric

complex. This latter complex is thought to recruit RNA Pol II and

the core transcriptional machinery to initiate active transcription of

the target gene.

VITAMIN D RECEPTOR 379

211

M. Both the 1-hydroxyl and 25-hydroxyl moieties

are crucial for efﬁcient recognition by VDR, and the

nonhydroxylated, inactive parent vitamin D

compound

does not bind appreciably to the VDR.

In addition to hormone binding, the LBD is required

for several other aspects of receptor function, particu-

larly in mediating protein –protein interactions. One

important protein– protein contact is the heterodimer-

ization of VDR with the retinoid X receptor (RXR). As

mentioned above, VDR– RXR heterodimer formation is

generally required for high-afﬁnity interaction of the

receptor with VDREs and an extensive heterodimeriza-

tion interface that coalesces around helices 10 and 11

in the LBD of VDR mediate protein–protein contacts

with RXRs.

The LBD also mediates association of VDR with

comodulatory proteins. Many of these interactions

require at the extreme C-terminus of the receptor,

where the AF-2 domain is located (Figure 2). The AF-2

domain is highly conserved throughout the hormone

receptor superfamily and its main structural feature is

that of an amphipathic

-helix. Removing 25 amino

acids from the C-terminus of hVDR (D403–427), which

contains the AF-2 domain, results in a complete loss of

1,25(OH)

/VDR-activated transcription. This loss of

function is not due to altered binding of RXR, VDRE, or

hormone and the mutant receptor was appropriately

targeted to the cell nucleus. Thus, the AF-2 domain of

VDR, corresponding to helix 12, plays a central role in

1,25(OH)

-activated transcription mediated by VDR.

Molecular Mechanism of

Transcriptional Control by VDR

VDR INTERACTION WITH VITAMIN D

ESPONSIVE ELEMENTS

VDR modulates transcription by binding to speciﬁc

VDREs, in the promoter regions of responsive

genes. VDREs from a variety target genes have been

identiﬁed and on this basis a consensus VDRE may be

generally described as an imperfect direct repeat of a

core hexanucleotide sequence,G=AGGTG=CA; with a

spacer region of three nucleotides separating each half

element (also termed DR-3 for direct repeat with a

three nucleotide spacer). The mechanism of VDR

binding to VDREs is reﬂected in the direct repeat

nature of the element. For example, puriﬁed VDR

alone does not bind to a VDRE with high afﬁnity.

Rather, RXR is required for high-afﬁnity binding of

VDR to VDREs. The asymmetry of the directly

repeating motif causes the VDR–RXR heterodimer

to bind to the VDRE with a deﬁned polarity, with

RXR occupying the 5

half site and VDR occupying

the 3

half site.

The natural VDREs identiﬁed thus far provide only

a snapshot of the DNA sequences that mediate the

transcriptional effects of the VDR. Variations on

the DR-3 motif for VDREs have been identiﬁed in

the elements that mediate vitamin D responsiveness

in the calbindin D

and calbindin D

28k

genes.

Moreover, several synthetic elements with large

spacer regions and inverted arrangements can

mediate vitamin D responsiveness under certain

conditions. It is likely that the afﬁnity of VDR for

these atypical elements may vary from that of the

classic DR-3 motif adding yet another level of

regulatory complexity to the process of VDR-mediated

gene expression.

FIGURE 2 Functional domains of the VDR. A/B, amino terminal

region; DBD, DNA- binding domain containing two zinc-ﬁnger motifs;

LBD, ligand-binding domain; AF-2, activation function-2 domain

encompassing helix 12.

Helix II

Helix I

Helix III

T-box

P-box

D-box

DPLSTSAAMAEM NH

DRNVPRI

KGFFRRSMKRKALFT

RLKRCVDIGMMKEFILTDEE

FIGURE 3 Schematic of the DNA-binding domain of the VDR. The cysteines responsible for coordinating the zinc atoms are shown in red.

380 VITAMIN D RECEPTOR

One important role for the 1,25(OH)

ligand in

the transactivation process is to dramatically enhance

both the formation of the VDR–RXR heterodimer and

the subsequent interaction of the heterodimer with the

VDRE (Figure 1). Upon ligand binding, there is a clear

1,25(OH)

-dependent decrease in the formation of

VDR homodimers and a concomitant increase in VDR

heterodimerization with RXR. These ligand-induced

changes in VDR–RXR interactions are likely due to

altered conformations of the VDR that disrupts weak

homodimers of unliganded VDR and promotes

liganded VDR heterodimerization with RXR. The

interaction of VDR and RXR generates a heterodimeric

complex that is highly competent to bind DR-3 like

VDREs and subsequently affect the transcriptional

process. Thus, the VDR–RXR heterodimer is the

primary active complex involved in controlling DR-3

VDRE-driven promoters.

COMMUNICATION BETWEEN VDR AND

THE

TRANSCRIPTIONAL MACHINERY

The regulation of VDR-mediated transcription involves

a complex series of macromolecular interactions occur-

ring in a temporally coordinated fashion (Figure 1).

Other transcriptional components that associate with

the liganded VDR/RXR heterodimer may be classiﬁed

into two general categories: general transcription factors

and the comodulatory proteins. The interaction of VDR

with the ﬁrst group results in direct contacts with the

preinitiation complex (PIC), which may facilitate

assembly or recruitment of the PIC and thereby

stimulate transcription by RNA Pol II. Transactivator

interaction with the PIC may occur via adapter proteins

such as the TBP-associated factors (TAFs) or via direct

interaction with other core transcription factors. In this

regard, TFIIB may be a key target since it is known

to interact directly with VDR and augment vitamin

D-activated transcription in transient gene expression

studies. Indeed, differences in the activities of NH

terminal VDR isoforms are attributed to differential

interactions with TFIIB thus, supporting an important

role for the VDR–TFIIB interaction in determining the

overall transactivation potential of the liganded VDR.

In addition to direct contacts with the general

transcription machinery, the liganded VDR is also linked

to the transcriptional PIC by the NR comodulatory

factors. The general functional properties of the NR

comodulators are their ability to interact with nuclear

receptors and control their transcriptional responsive-

ness to ligand. NR comodulators are classiﬁed either as

coactivators or corepressors, and they aid in the

induction or repression, respectively, of ligand/recep-

tor-mediated transcription. NR coactivators may func-

tion as macromolecular bridges between the receptor

and the transcriptional machinery that aid in the

assembly or promote the stability of the preinitiation

complex. Moreover, several coactivator proteins either

possess intrinsic histone acetyltransferase (HAT) activi-

ties, or recruit other proteins that possess HAT activity

(e.g., CBP/P300). Acetylation of histones near the

promoter presumably results in a loosening of chroma-

tin structure and greater accessibility of the promoter for

the transcriptional machinery (Figure 1). The best-

characterized coactivators are the steroid receptor

coactivator (SRC) family of nuclear receptor coactiva-

tors that includes three members at present: SRC-1

(NCoA-1), SRC-2 (GRIP-1, TIF2, NCoA-2), and SRC-3

(pCIP, RAC3, ACTR, AIB-1, TRAM-1).

Structural studies of related receptors provide insight

into the mechanism of ligand-induced interaction of

VDR with SRC coactivators. It is hypothesized that the

1,25(OH)

ligand promotes coactivator interaction

by inducing a repositioning of the AF-2 activation helix,

or helix H12 (Figure 4). In the unliganded state, the AF-2

domain (helix H12) projects out away from the globular

core of the LBD, while in the liganded state the AF-2

domain is folded over onto the LBD globular core

domain. One outcome of helix H12 folding is

the creation of a platform or protein interaction surface

through which coactivator proteins such as SRCs

effectively dock with the VDR. The domain in the

coactivator proteins, which is responsible for this

interaction is termed the nuclear receptor or NR box

andisanamphipathic

-helix composed of the

consensus core sequence LXXLL. This interaction is

essential for transactivation mediated by the VDR.

A large multiprotein complex called Mediator D is

another coactivator required for VDR-mediated tran-

scription (Figure 1). Mediator D is also known as

vitamin D receptor interacting protein (DRIP), thyroid

receptor activating protein (TRAP) complex, and the

mammalian mediator complex. The Mediator D com-

plex is composed of at least ten different proteins

anchored by Med220, which interacts directly with

ligand-activated VDR/RXR heterodimers through one

of two LXXLL motifs. DRIP is essential for in vitro

VDR-mediated transcription from chromatinized tem-

plates. However, as the DRIP complex does not contain

any SRCs and does not possess HAT activity, it appears

to potentiate NR-mediated transcription through dis-

tinct mechanisms. In particular, DRIP directly recruits

the RNA polymerase II holoenzyme to 1,25(OH)

activated VDR, indicating that DRIP may serve as a

bridge between VDR and the core transcriptional

machinery. Indeed, chromatin immuno-precipitation

studies show that DRIPs enter transcriptional complexes

at a later time than SRC coactivators, supporting

the nonredundant roles of these coactivator classes in

NR-mediated transactivation.

Another protein named NCoA62/SKIP was identiﬁed

as a VDR and NR coactivator, and as such, it augments

VITAMIN D RECEPTOR 381

VDR- and other NR-activated transcriptional processes

in transient reporter gene assays. On the basis of its

primary amino acid sequence and its function,

NCoA62/SKIP is a unique protein. One key difference

is the lack of LXXLL motifs which are characteristic of a

large variety of coactivators including the SRCs,

CBP/P300 and the Mediator D coactivator complex. In

addition, NCoA62 interacts with VDR in a ligand- and

AF2-independent manner. Although NCoA62/SKIP

does not interact directly with SRC coactivators,

NCoA62/SKIP, VDR, and SRCs form a ligand-depen-

dent ternary complex. NCoA62/SKIP also functions

cooperatively with SRC coactivators to augment VDR-

activated transcription and it is recruited to vitamin D

responsive target genes in a distinct, temporal manner

from the SRC coactivators. Presently, the mechanisms

involved in this synergistic action of these two coacti-

vator classes are unknown.

Summary

Research spanning the 1980s and 1990s reveals that

1,25(OH)

-mediated transcription is more complex

than the simple binding of the receptor to DNA and the

recruitment of RNA Pol II to initiate transcription.

VDR/RXR-activated transcription involves complex

interactions that may occur in a spatially distinct and

temporally coordinated fashion to increase the rate at

which 1,25(OH)

-responsive genes are transcribed

and at which the resulting RNA transcript is processed.

A model for VDR-mediated transcription is proposed

which incorporates numerous properties of VDR that

were discussed in this section (Figure 1). The initial event

in this model is high-afﬁnity binding of the 1,25(OH)

ligand to the VDR. Ligand binding induces VDR/RXR

heterodimerization and the heterodimer speciﬁcally

binds VDREs in the promoter regions of vitamin D

responsive genes. The heterodimer then recruits coacti-

vator molecules that acetylate core histones to make the

DNA more accessible to the transcriptional machinery.

Meanwhile, other coactivators and the PIC are recruited

to the site and activated transcription proceeds. Under-

standing the complex interplay that occurs between

these various factors is crucial to unraveling the

complexities of activated or repressed transcription

mediated by vitamin D and the VDR.

FURTHER READING

Feldman, D., Glorieux, F. H., and Pike, J. W. (eds.) (1997). Vitamin D.

Academic Press, San Diego.

Haussler, M. R., Whitﬁeld, G. K., Haussler, C. A., Hsieh, J. C.,

Thompson, P. D., Selznick, S. H., Dominguez, C. E., and

Jurutka, P. W. (1998). The nuclear vitamin D receptor: Biological

and molecular regulatory properties revealed. J. Bone Miner. Res.

13, 325–349.

Jones, G., Strugnell, S. A., and DeLuca, H. F. (1998). Current

understanding of the molecular actions of vitamin D. Physiol.

Rev. 78, 1193–1231.

Jurutka, P. W, Whitﬁeld, G. K., Hsieh, J. C., Thompson, P. D.,

Haussler, C. A., and Haussler, M. R. (2001). Molecular nature of

the vitamin D receptor and its role in regulation of gene expression.

Rev Endocr. Metab. Disord. 2, 203–216.

Malloy, P. J., Pike, J. W., and Feldman, D. (1999). The vitamin D

receptor and the syndrome of hereditary 1,25-dihydroxyvitamin D-

resistant rickets. Endocr. Rev. 20, 156–188.

McKenna, N. J., Lanz, R. B., and O’Malley, B. W. (1999). Nuclear

receptor coregulators: Cellular and molecular biology. Endocr. Rev.

20, 321–344.

Norman, A. W., Mizwicki, M. T., and Okamura, W. H. (2003). Ligand

structure–function relationships in the vitamin D endocrine system

from the perspective of drug development (including cancer

treatment). Recent Results Cancer Res. 164, 55–82.

Rachez, C., and Freedman, L. P. (2001). Mediator complexes and

transcription. Curr. Opin. Cell Biol. 13, 274–280.

VDR

AF-2

1,25 D

SRC

FIGURE 4 The vitamin D receptor undergoes a conformational

change upon binding hormone. The VDR binds ligand and this induces

a conformational change in the AF-2 domain to trap the ligand in the

binding pocket. This change also creates a hydrophobic cleft or surface

on VDR that LXXLL motifs in coactivator proteins use for docking to

the VDR.

382 VITAMIN D RECEPTOR

Sutton, A. L., and MacDonald, P. N. (2003). Vitamin D: More than a

“bone-a-ﬁde” hormone. Mol. Endocrinol. 17, 777–791.

BIOGRAPHY

Paul MacDonald and Diane Dowd are Faculty Members in the

Department of Pharmacology at Case Western Reserve University.

They are actively involved in basic research on the molecular, cellular,

and physiological roles of the vitamin D receptor in normal and

pathological states. Dr. MacDonald holds a Ph.D. in biochemistry

from Vanderbilt University and received his postdoctoral training

at the University of Arizona. Dr. Dowd received her Ph.D. in

biochemistry from Vanderbilt University and her postdoctoral training

at the Arizona Cancer Center and University of Arizona.

VITAMIN D RECEPTOR 383

Vitamin D

Hector F. DeLuca and Margaret Clagett-Dame

University of Wisconsin, Madison, Wisconsin, USA

This article discusses the chemical identity and production of

vitamin D. Its endocrine system, the nuclear receptor, and its

mechanism of action are treated, along with the degradation of

vitamin D hormone and its roles.

Chemical Identity, Natural

Occurrence, and Production

The vitamin Ds are secosterols in which the B-ring of the

steroid nucleus is replaced by a diene bridge. The two

most common forms are vitamin D

(ergocalciferol) and

vitamin D

(cholecalciferol). The structures of these two

nutritionally important compounds are illustrated in

Figure 1.

Although it is classiﬁed as a vitamin because of the

nature of the discovery process, vitamin D in actual fact

should not be considered a true vitamin for the following

reasons. First and foremost, vitamin D utilized by higher

organisms is manufactured in the epidermis of skin by

photolysis of 7-dehydrocholesterol, a side reaction

product of cholesterol synthesis. Secondly, vitamin D is

nearly absent from the food supply for the most part.

Vitamin D is found in ﬁsh liver oils and in egg yolk but is

not found in virtually all plant materials, in skeletal

meats, seeds, fruits, and vegetables. In fact, very little is

found in an expected source, milk. We consider milk an

important supply of vitamin D in many countries,

primarily because it is fortiﬁed by the addition of

vitamin D made artiﬁcially by man.

The chemical process whereby 7-dehydrocholesterol

is converted to vitamin D

is well known. The 5,7-diene

portion of either ergocalciferol or 7-dehydrocholesterol

will absorb 280– 310 nm ultraviolet light. This causes an

isomerization to form a compound, previtamin D

which in itself is biologically inactive and remains in

skin. Previtamin D is in equilibrium with vitamin D

. The

plant sterol, ergosterol, is converted to vitamin D

by an

identical process. For many years, vitamin D

was the

major synthetic form of vitamin D used in vitamin

capsules, animal feeds, and in the fortiﬁcation of foods.

Because of improved synthetic methods, vitamin D

has

become the major form used for both fortiﬁcation and

nutrition of domestic animals. The photolysis of

7-dehydrocholesterol is quite efﬁcient in skin, and

summer sun on hands and face and will in 10 minutes

produce the daily requirement of vitamin D (currently

believed to be 10–20 mg per day). Winter sun is unable to

induce production of vitamin D because of the angle of

the sunlight and the ﬁltration of the critical wavelengths

that cause vitamin D production. It is generally accepted

that skin production of vitamin D

is quantitatively the

most important source of the vitamin for human health.

Conversion of Vitamin D

to its Hormonal Form

The vitamin D

that is produced in skin or that is

absorbed from the intestine from vitamin pills or

fortiﬁed foods is biologically inactive as such. The

body must process vitamin D to form a ﬁnal vitamin D

hormone that is believed to carry out all of the known

functions of vitamin D. Vitamin D

is ﬁrst hydroxylated

in the liver to form the circulating form of vitamin D

25-hydroxyvitamin D

(25-OH-D

). Interestingly, this

compound is also biologically inactive and must be

further modiﬁed before function. The second step occurs

in the proximal convoluted tubule cells of the kidney

where a 1

-hydroxyl group is added to the molecule to

produce the ﬁnal vitamin D hormone, 1

,25-dihydroxy-

vitamin D

(1,25-(OH)

). The reaction steps that

result in this transformation are shown in Figure 2.

The ﬁrst step in conversion of vitamin D to the

circulating form occurs in the hepatocytes of liver but

not exclusively so. At least two enzymes are known to

make this conversion, a mitochondrial enzyme and a

microsomal enzyme, both of which have been cloned.

No natural human mutation blocking this initial step has

yet been discovered. However, the second step occurring

in the proximal convoluted tubule cells of the kidney is a

highly regulated one. This 1

-hydroxylase, also known

as CYP27B1, has also been cloned and natural

human mutants of this enzyme are now well known.

These mutations result in a disease called vitamin

D-dependency rickets type I in which children present

vitamin D-deﬁciency diseases, i.e., rickets, hypocalce-

mia, and hypophosphatemia, even though they are

naturally irradiated with sunlight or are provided

vitamin D as a supplement. These children are cured by

the administration of physiologic amounts of the natural

vitamin D hormone, 1,25-(OH)

, establishing clearly

the two-step activation process of vitamin D for man.

The Role of 1,25-(OH)

in Calcium, Phosphorus,

and Bone Metabolism

The basis for discovery of vitamin D in the ﬁrst place is

the disease rickets in children. This disease is also

accompanied by hypocalcemia (low blood calcium)

causing convulsive tetany. In adults, the deﬁciency of

vitamin D causes the disease, osteomalacia. The

diseases, rickets and osteomalacia, result from a failure

to mineralize the organic matrix of newly synthesized

bone. Early investigations to understand how vitamin D

functions were, of course, directed toward the formation

of mineralized bone. However, it is now abundantly

clear that 1,25-(OH)

does not act directly on the

mineralization process in the healing of rickets or

osteomalacia but rather acts on intestine, kidney, and

bone to provide calcium and phosphorus in the plasma

required for the mineralization of skeleton and preven-

tion of the convulsive disease, hypocalcemic tetany.

The essence of vitamin D action, therefore, in curing

rickets and osteomalacia is the elevation of plasma

calcium and phosphorus to supersaturating levels that

are required for normal mineralization. The vitamin D

hormone acts to provide this calcium by stimulating the

enterocyte of the small intestine to absorb calcium as

well as phosphate by an independent mechanism both of

which require active metabolic energy.

Because of the constant need for calcium to support

normal neuromuscular function, calcium must be

available in the plasma even when it is not available

in the diet. Thus, the vitamin D hormone, i.e., 1,25-

(OH)

acting together with parathyroid hormone will

cause the mobilization of calcium from the skeletal ﬂuid

compartment into the plasma compartment. Thus, the

skeleton serves not only a structural function but also as

a source of calcium and phosphorus. A ﬁnal point is

that 1,25-(OH)

works in concert with the parathyr-

oid hormone to induce the distal nephron of the kidney

to reabsorb the last 1% of the ﬁltered load of calcium

into the plasma compartment. These then work to

maintain plasma calcium and phosphorus at levels that

support both bone formation and prevent hypocalcemic

tetany. A diagram representing these functions is

shown in Figure 3.

The Vitamin D Endocrine System

One of the most elegantly regulated substances in the

body is the plasma calcium level. The parathyroid glands

continuously monitor serum calcium levels and a

calcium receptor therein responds when calcium falls

even slightly below the normal level. By a G protein

FIGURE 1 The nutritionally important forms of vitamin

D. Vitamin D

is the form manufactured in skin and vitamin D

the form produced by the irradiation of ergosterol. In mammals, both

are equally active and both appear to be metabolized by almost

identical routes.

FIGURE 2 The required metabolism of vitamin D for function. Shown here is the metabolism of vitamin D

to the major blood form of vitamin

D, 25-hydroxyvitamin D3 and its subsequent metabolism in the kidney to form the ﬁnal vitamin D hormone, 1

,25-dihydroxyvitamin D

. It is this

form of vitamin D

that carries out all of the known functions of the vitamin. Vitamin D

not shown here is metabolized in an almost identical

fashion to its active form, 1

,25-dihydroxyvitamin D

VITAMIN D 373

mechanism, the calcium receptor stimulates the

secretion of parathyroid hormone, a peptide hormone,

that is the body’s signal requesting calcium. It binds to

the entire nephron of the kidney and to the osteoblasts of

bone. Among its many functions in the kidney is to

stimulate the 1

-hydroxylase (CYP27B1) to produce

the vitamin D hormone in substantial quantities.

The vitamin D hormone then acts on the bone, kidney,

and intestine as described earlier to mobilize calcium as

needed to raise blood calcium into the normal range,

clearing the set-point of the parathyroids shutting down

parathyroid secretion. Overshoot of calcium results in

the C-cells of the thyroid secreting calcitonin, a 32

amino acid peptide hormone that blocks calcium

mobilization from the skeleton. Calcitonin also has a

stimulatory effect on the 1

-hydroxylase to produce the

vitamin D hormone in small amounts to support its

noncalcemic activities.

The Vitamin D Receptor

1,25-(OH)

is a true steroid hormone that acts

through a nuclear receptor called the vitamin D receptor

(VDR). The human receptor is a 427 amino acid protein

that serves as a ligand-dependent transcription factor.

It is a member of the nuclear receptor superfamily of

receptors. Other members of this superfamily include

the retinoic acid, thyroid, estrogen, testosterone, gluco-

corticoid, and other steroid hormone receptors. VDR is

the smallest of the steroid hormone receptors and largely

acts in a fashion quite similar to other steroid hormone

receptors. Unlike other nuclear receptors, there is only a

single VDR in all tissues. No subtypes are known and it

is this receptor through which all vitamin D actions

occur. Natural mutants of the human VDR are known

and produce a disease called vitamin D-dependency

rickets type II. In this disease, high blood levels of

1,25-(OH)

occur in the face of severe rickets and

severe hypocalcemia and hypophosphatemia. VDR null

mutant mice have been produced and are available as

experimental tools. The vitamin D-dependency rickets

type II patients present a variety of diseases depending

upon where the mutation has occurred. If there is a

complete absence of a functional receptor, then very

brittle vitamin D-resistant rickets occurs and can only be

treated by the infusion of calcium and phosphorus into

the blood stream to mineralize the skeleton but many of

the other functions of vitamin D go unmet. The fact that

VDR null mutant mice are normal at birth shows that

vitamin D is not an embryonic developmental factor but

functions primarily after parturition.

There are reports of nongenomic functions of

vitamin D. These have been largely cellular-based

observations, which have yet to be shown to occur

in vivo. In fact, the administration of large amounts of

1,25-(OH)

to receptor null mutant mice produces no

signiﬁcant phenotype, suggesting that the only mechan-

ism of action of vitamin D is through its receptor under

physiologic circumstances.

Molecular Mechanism

Vitamin D-response elements (VDE) are found in the

promoter region of target genes. These are hexameric

repeats separated by three nonspeciﬁed bases. There can

be as many as three repeat elements in a responsive

element system. The VDR must heterodimerize with

another transcription factor, the retinoid X receptor

(RXR), in order to bind to the responsive elements. The

RXR binds to the 5

repeat and the VDR to the 3

repeat.

Figure 4 provides a cartoon to illustrate what is currently

known about the mechanism of action of the vitamin D

hormone working through its receptor to regulate

expression or suppression of target genes. As with

other steroid hormones, it is anticipated that the

unliganded receptor may be retained in an inactive

state by being bound to a corepressor. This has not been

speciﬁcally shown for the VDR but is known for other

class II nuclear receptors. There is increasing evidence

that such a corepressor might be identiﬁed for the VDR.

In this model, the corepressor that interacts directly with

the VDR would be released upon binding of the ligand

to the receptor probably because of a change in VDR

conformation. The receptor would then be free to

interact with a coactivator protein that is believed to

be involved in chromatin remodeling and to link the

complex bound at the hormone response element to the

basal transcription machinery. Among the known

coactivators believed to interact directly with the VDR

FIGURE 3 A diagrammatic representation of how the vitamin D

hormone 1

,25-(OH)

) functions to maintain plasma calcium at

supersaturating levels which are required for prevention of hypocal-

cemic tetany (neuromuscular) and for the prevention of rickets and

osteomalacia (mineralization of bone).

374 VITAMIN D

are SRC 1, 2, 3, and DRIP 205. Likely other participat-

ing coactivators will be discovered. A phosphorylation

does occur on serine 205 of the VDR but whether this is

of functional importance remains to be determined.

Metabolism and Degradation

The vitamin D hormone is degraded speciﬁcally by

an enzyme it induces. That enzyme is the CYP24

(24-hydroxylase), which carries out a series of reactions

on the side chain of the vitamin D hormone and its

precursor, 25-OH-D

. It ﬁrst causes hydroxylation in the

24R-position followed by oxidation of that same

hydroxyl to a ketone. The next step again carried out

by the same enzyme is a 23-hydroxylation followed by

cleavage and oxidation giving rise to the biliary

excretion product called calcitroic acid. Calcitroic acid

is biologically inactive. Thus, the vitamin D hormone,

an extremely potent biological substance, ensures

its limited activity by inducing its own destruction.

Other metabolism of vitamin D is known including the

formation of the 26,23-lactone derivative, 26-hydroxy-

lation, but quantitatively the most important reaction

regulating the level of vitamin D hormone is the CYP24.

The CYP24 is regulated in a negative fashion by the

parathyroid hormone causing an instability of the

mRNA encoding for that enzyme. Thus, the parathyroid

hormone not only stimulates the 1

-hydroxylase but

also stimulates the destruction of the enzyme that

destroys the hormone, thereby ensuring that large

amounts of the vitamin D hormone are available to

carry out its calcium functions.

New and Nonclassical Roles

of the Vitamin D Hormone

With the discovery of the VDR came the observation

that it is found in tissues not previously appreciated as

target tissues of vitamin D action. The discovery of

FIGURE 4 A cartoon representing the molecular mechanism of action of the vitamin D hormone (1,25-(OH)

) in regulating transcription of

target genes in the target tissues. Binding of the ligand, i.e., 1,25-(OH)

to the VDR causes a conformational change and a rejection of the

corepresssor. It also allows binding of a coactivator and formation of a heterodimer with RXR on the vitamin D responsive elements. Several

proteins are required to form the transcription complex, all of which have not yet been identiﬁed. There is a bending of the DNA and a

phosphorylation on serine 205 but their exact role in either suppression or activation of transcription remains unknown.

VITAMIN D 375

the VDR in the intestine, kidney tubules, and osteoblasts

was not unexpected. However, its presence in the

parathyroid gland, the islet cells of the pancreas, the

immune cells such as T-cells, macrophages, and kerati-

nocytes made it possible that the vitamin D hormone

had important functions besides raising blood calcium

and phosphorus and mineralizing the skeleton. It is now

clear that an important function of the vitamin D

hormone is to suppress the growth of parathyroid glands

and to suppress production of the parathyroid hormone.

This basic function has been utilized by physicians using

1,25-(OH)

and its analogues to treat secondary

hyperparathyroidism in kidney failure patients.

Another important VDR site are the cells of the

immune system and in the promyelocytes which are the

precursors of the monocyte. The vitamin D hormone

stimulates the formation of the monocyte by causing

the production of RANKL by osteoblasts and stromal

cells. This results in a coalescence of the monocytes to

form giant osteoclasts which are further activated by

RANKL. Thus, the formation of the giant osteoclast

that causes bone turnover is a vitamin D function.

Furthermore, vitamin D also functions to cause the

formation of new bone, thus playing an important role

in the bone remodeling and modeling systems.

The role of the vitamin D hormone in the keratinocyte

has also become of importance therapeutically. The

vitamin D hormone added to the keratinocytes in cultures

or added topically will cause a cessation of proliferation

of the keratinocyte and will cause its differentiation.

Thus, 1,25-(OH)

and analogues have been success-

fully used in the topical treatment of psoriasis.

Finally, attention must be focused on the immune

system where the vitamin D hormone is now known to

cause important immuno-modulatory effects. The vita-

min D hormone can be used to block certain animal

models of autoimmune disease. For example, it can be

used to prevent experimental autoimmune encephalo-

myelitis (EAE), a model of multiple sclerosis in mice.

However, in so doing, it does induce an abnormal rise in

serum calcium. 1,25-(OH)

can be used to prevent

diabetes in the nonobese diabetic mouse model of type 1

diabetes. It can be used to prevent rheumatoid arthritis,

systemic lupus, and inﬂammatory bowel disease.

Thus, the immuno-modulatory role of the vitamin D

hormone under normal circumstances is only now

becoming realized.

Therapeutic Uses of 1

,25-(OH)

and its Analogues

As discussed earlier, besides the treatment of vitamin

D-dependency rickets and vitamin D-resistant rickets of

many types, the vitamin D hormone and its analogues

are certainly central actors in the treatment of bone

disease secondary to kidney failure. This is primarily

by suppression of parathyroid proliferation and by

suppression of parathyroid hormone production.

1,25-(OH)

and its analogue, 1

-OH-D

has been

successfully used in certain parts of the world to treat

postmenopausal and age-related osteoporosis, giving a

modest rise in bone mass along with a clear reduction in

fracture rate. The danger of hypercalcemia has limited

its use in the treatment of this disease. However, newer

vitamin D analogues with greater bone anabolic activity

than 1,25-(OH)

are currently under development

and may make it possible to safely treat osteoporosis in

the future.

An analogue of 1,25-(OH)

called Dovonex is

currently marketed for the topical treatment of the

hyperproliferative skin disorder, psoriasis. Improved

analogues will likely appear for treatment of psoriasis.

There are potential new uses for the vitamin D

analogues in the autoimmune diseases as described

above or in the treatment of malignant cancer growth.

The vitamin D hormone has been found to suppress

growth of malignant cells in vitro and to cause their

differentiation into functional cells. Thus, the use of

vitamin D compounds as a potential treatment of

metastatic neoplastic disease has been exciting but it

has not yet yielded a therapeutic agent. The major

problem with the use of 1,25-(OH)

and many of its

analogues for treatment of disease is that its primary

purpose is to raise blood calcium and phosphorus

which means that is a major side effect that must be

dealt with before it can be used to treat noncalcemic

diseases. One approach to this is the synthesis of

analogues that retain the ability to suppress cancerous

growth but do not raise blood calcium. New analogues

with these desirable characteristics are currently

under basic study, but are not yet available for

pharmaceutical use.