Should We Put a Title Here?Synthesis and Function of Vitamin D and Vitamin D

Receptor

Jeffrey Lee, John Noh, and Jonathan Yu

And our names?

Introduction

Despite the cancerous nature of sunlight due to UV radiation, we are dependent

on sunlight as a natural means of obtaining vitamin D, a crucial compound that plays an

important role in maintaining bone health. However, most people do not realize that new

research has even shown a positive correlation between low vitamin D levels and risk of

cancer. (1). We can also consume vitamin D through supplements, which provide the same

Supplemental sources of vitamin D are also sold. benefits as the synthesis of vitamin D

through UV radiation. Once consumed or absorbed into the body, vitamin D goes through

a complex series of chemical reactions in the liver and kidney to form the physiologically

active 1α,25-dihydroxyvitamin D [ 1,25(OH)2D], also known as calcitriol. Active vitamin

D is functionallyvery dynamic; it can induce genomic response by altering transcription

via vitamin D receptors, but it can also facilitate absorption of phosphate and magnesium

ions (among other capabilities). These aspects of vitamin D metabolism are explored in

the paper, along withas well as future implications of this powerful vitamin and hormone,

are explored in this paper.

Subcutaneous Synthesis of Vitamin D

Vitamin D comes in twohas two forms: chlolecalciferol (vitamin D3) and

Ergocalciterol ergocalciferol (vitamin D2). While vitamin D3 increases serum [25(OH)D]

levels, vitamin D2 does not achieve the same resultsBoth are precursors to 1,25(OH)2D,

which is the active form utilized by the human body as a hormone, but vitamin D3 is

significantly preferred. (2). For this reason, vitamin D3 is the form of vitamin D that will

be discussed. The major source of vitamin D3 for people comes from the exposure of the

skin to ultraviolet B (UVB) radiation (280–320 nm). The synthesis of Vitamin D3 begins

when light energy (UVB rays) strikes the precursor molecule 7-dehydrocholesterol.3 The

effectiveness of UVB on formation of previtamin D3 in the skin is influenced by UVB-

absorbing molecules such as chromophores in the skin, consisting of melanin,

deoxyribonucleic acid (DNA), ribonucleic acid (RNA), proteins, and 7-

Dehydrocholesterol (7-DHC). 7-DHC absorbs UV radiation between 290 nm and

315 nm, causing it to isomerize, resulting in a bond cleavage between carbons 9 and 10 to

form the 9,10-seco-sterol previtamin D3.4

Dependent on temperature and timeP, previtamin D3 undergoes nonenzymatic

isomerization, which is

dependent on temperature and

time, to form vitamin D3

(cholecalcioferol) as seen in

Figure 1. In contrast to 7-DHC,

which is a 5,7-diene, vitamin D3

is a 5,7,19-triene with three

conjugated double bonds typical

Figure 1: The subcutaneous synthesis of vitamin D3 with the help of UVB and thermal energy. Also shows alternate paths available for the compounds used in the synthesis that may

occur in the dermal layer.

for vitamin D molecules.3 Around 50% of the previtamin D3 can isomerize to vitamin D3

within 2.5 hours in the skin. Within 12–24 hours after UVB exposure, the circulating

concentrations of vitamin D3 are at their maximum levels.3 If previtamin D3 is formed in

the skin, it can also undergo either photoisomerization to lumisterol, tachysterol, and

toxisterols, or it can be retransformed to 7-DHC.3 A broad overview of this process can

be seen in Figure 1.

Figure 1: The subcutaneous synthesis of vitamin D3 with the help of UVB and thermal

energy. Also shows alternate paths available for the compounds used in the synthesis that

may occur in the dermal layer.

Activation of Vitamin D3 in Hepatocyte (Liver) and Kidneys

A At this point, the vitamin D is

Figure 2: Pathway from vitamin D to the active form (calcitriol, bottom) and inactive form (right) through processing

by cytochromes.

biologically inactive and must be activat ed in the li ver and the kidneys. Thi

s is als o the form vitamin D comes in through consuming supplements.The vitamin D fou

nd in vitamin supplements is also in this inactive form. (consumption). Afte r the ina

ctive vit amin D binds to the carrier proteins, vitamin D-binding protein (DBP), itI

nactive vitamin D is transp orted to the liver where it is enzymatically hydroxyla

ed to 25-hydroxyvitamin D [25(OH)D].5 The hydroxy ation  nto 25-hydroxyvitamin D [

25(OH)D] is cat  lyzed b y microsomal cytochrome P450 enzyme CYP2R1 and/or the mitochondr

al cytochrome P450 CYP27A1 (see Figure 2),; both of which are constitutively expressed

.3,6 Cytochromes a  e h  moprote ins that supply ATP via electron transport to the mo

ecules. Besides the CYP2R1  a d CYP2 7A , ther e are  lso se veral other cytochrome P

50 mixed function oxidases (CYP2C11,, CYP3A4,, CYP2D25, and CYP2J3) that exhibit vitam

in D 25-hy  roxyla se act ivi  ies.3 T he normal circulating levels of 25(OH)D i  the b

loo  are between 25 nmol/L and –200 nmol/L.3 Currently, vitamin D l

25-hydroxyvitamin D [25(OH)D] , bound to DBP, is then transported to the

kidneys and is finally hydroxylated by CYP27B1 (25-hydroxyvitamin D-1α-hydroxylase

or; 1αOHase) at the C1α position to hormonally active 1α,25-dihydroxyvitamin D

[1,25(OH)2D].3 The overall activation of vitamin D3 can be seen in Figure 2 where there

are two separate pathways 25(OH)D can take to form two different active forms of

vitamin D or return to the inactive form as 24,25-(OH)2D.5 Calcitriol has biological

effects oin the kidneys but is usually sent into the bloodstream to be used by other parts

of the body.

Figure 2: Pathway from vitamin D to the active form/inactive form through the use of various

cytochromes. Also shows where the cytochromes take action (liver, kidney) and shows the chemical

composition of the product of the chemical pathway.

Distribution of Vitamin D to the Rest of the Body

Once in the bloodstream, vitamin D in the form of active 1,25(OH)2D 1,25-OHD3

(1,25D) or 25(-OH)D3 (25D) often binds to a protein called

gc-globulin (group-specific component of serum), also known as vitamin D binding

protein (DBP) (see Figure 31).7 1 This protein can be found in plasma and cerebrospinal

fluid, where it binds to vitamin D metabolites and transports them to target organs. DBP

belongs to the family of albumin proteins and is encoded by the GC gene residing on

chromosome 4.1 DBP consists of 458 amino acids, including numerous cysteine residues,

which form multiple disulfide bridges within the protein. The three domains of the

protein have many α-helices; six of them on the first domain form the binding site for

vitamin D ligands.7

DBP plays an important role in maintaining stable supplies of

vitamin D for the body. When researchers knocked out the gene coding for

DBP in mice, the knockout mice did not seem to have any physiological

defects. However, when fed a vitamin D deficient diet, the knockout mice

showed signs of bone disease (a common symptom of vitamin D deficiency)

sooner than the wild-type mice, suggesting that they were less able to cope

with vitamin D depletion.1 Another study showed that the kidneys recover vitamin-D

bound DBP from urine, demonstrating the mechanism by which DBP aids retention of

vitamin D.1 7

Cells in the body have several ways of accessing the body’s supply of vitamin D

[(1,25(OH)2D 1,25D and 25(OH)D]).

One way is by simple diffusion of free

vitamin D across the cell membrane.

The level of free vitamin D in the body

is governed by the levels and affinity of

Figure 42: Binding of 1,25D to CYP24A1 located in the inner mitochondrial membrane. The heme group (red dotted

sphere) reduces oxygen to hydroxylate 1,25D

DBP. Vitamin D can also enter the cell while still bound to DBP through active-receptor-

mediated uptake. The DBP-vitamin D complex binds to a cell surface receptor called

megalin (LRP2) and is internalized in a vesicle, where vitamin D is released and DBP is

denatured.7 1 Once inside cells, 1,25(OH)2D1,25D directs vitamin D-dependent gene

regulation through the vitamin D receptor (VDR), while 25(OH)D is first converted into

active 1,25(OH)2D 1,25D through hydroxylation of the 1-α carbon by cytochrome p450

27B1 (CYP27B1, also known as 1-α hydroxylase), usually located in the inner

mitochondrial membrane.1,27,8 This oxidation reaction of 25(OH)D occurs when NADPH-

CYP reductase captures an electron pair from the conversion of NADPH to NADP and

transfers it to CYP27B1, which has high specificity for 25-hydroxylated steroids (i.e.

25(OH)D) and reduces oxygen via the heme group in its active site to hydroxylate

25(OH)D (see Figure 2).2,36,8 The mechanism is likely similar to the reduction of oxygen

to water, which involves heme D, the site of oxygen reduction in many types of

bacteria.20 Another cytochrome, CYP24A1, operates in a similar fashion to catalyze the

hydroxylation of the 24 carbon of 1,25(OH)2D (see Figure 4) 1,25D to form inactive

24,25(OH)2D24,25-OHD, which is later excreted in urine.6

Vitamin D in the Brain

About 50 years after the discovery of vitamin D, researchers began to find

evidence of vitamin D in the brain. One crucial discovery was that of the expression of

CYP27B1 in human and rat brains. Immunohistochemistry revealed the distribution of

CYP27B1 and VDR in the brain. Researchers found that microglial cells (macrophages in

the brain), glial cells, and Purkinje cells (located in the cerebral cortex) in the brain)

actively produced 1,25(OH)2D1,25D, the active form of vitamin D. This was done via

CYP27B1, which seemed to be restricted to just the cytoplasm of those cells.5 9 In

addition, VDR was also shown to be expressed extensively throughout the human and rat

brain of both neurons and glial cells. Unlike CYP27B1, VDR was found solely in the

nucleus of brain cells.6 10 The supraoptic and paraventricular nuclei of the hypothalamus

and the substantia nigra, which is located in the midbrain and important to the body’s

reward system, showed the most substantial expression of CYP27B1 and VDR; this same

pattern of distribution is seen with other neurosteroids.5,69,10 Most locations in the brain

that had CYP27B1 also had VDR. Interestingly, the distribution of VDR in the brain was

strikingly similar in both humans and rodents.6 10 CYP24A1 was also found in glial cells

hydroxylating and inactivating 1,25D.5 9 The presence of both CYP27B1 and CYP24A1

in brain cells reveals that the brain is capable of regulating the amount of active vitamin

D in the brain. Another study has shown that vitamin D metabolites are able to cross the

blood brain barrier. However, the mechanism by which it they does so areis still

unknown.711

This growing body of evidence demonstrates the presence of vitamin D in the

brain; however, the effects of vitamin D are still being discovered. In recent years, the list

of supposed benefits of vitamin D has grown – many studies claim that it can lower blood

pressure, boost the immune system, or even help prevent cancer – so has the list of

vitamin D’s effects on the brain. Looking through the literature, there are studies showing

that vitamin D can alter dopamine, acetylcholine, and noradrenaline neurotransmission;

helps prevent onset of Parkinson’s, schizophrenia, depression, and other mental illnesses;

and plays a multifaceted role in brain development.5,79,11

Function of VDR: Vitamin D Receptor

Like all molecules in the body, vitamin D needs other cofactors and receptors in

order to function properly. In 1969, more than forty years after vitamin D was

discovered, the nuclear vitamin D receptor (VDR) was also found. VDR proved to play

an incredible role in the bodyin the body, as it was discovered in more than thirty tissues

and organs. of the human body. As a result of this flurry of research, it appears that there

are two main categories of action carried out by the so-called “VDS-VDR”

conformational ensemble model. Not only can the vitamin D receptor carry out important

genomic functions, as evidenced by the presence of VDR in the immune system, bone

marrow, adipose cells, etc., but it can also carry out rapid responses (RR) that could occur

within minutes to an hour. This This source of rapid responses comes fromis derived

from the knowledge that VDR regulates gene transcription. Once again, structure can

yield insights into function; the structural and stereospecific aspects of the VDS-VDR

model can explain how vitamin D regulates both nongenomic and genomic response via

specific ligand-binding pockets.

VDR fits into the nuclear receptor

superfamily, which isare a class of

transcriptional regulators in animals.

Nuclear receptors are ligand-activated; in

the case of VDR, vitamin D would be the Figure 4: Shapes of the optimal ligands for VDR-mediated responses and for RR, as well as for vitamin D binding protein (DBP). There is a characteristic ligand

shape for each type of response.

ligand that “activates” transcription. Tissues that contain VDR (over 37) define specific

locations where vitamin D can initiate biological responses. Some of these responses

include the classic calcium homeostasis system, along with five other systems, including

the brain, whichh we will be focused on later. Thee ligand-receptor complex is what

produces the biological reactions.

VDR Structure and Function:

Furthermore, Vitamin D is considered a conformationally flexible molecule; the

side chain that contains five single carbon-carbon bonds is the source of this flexibility

(see figure 54 - A).812 . Furthermore,

the cyclohexane ring has the ability

to interchange rapidly between alpha

and beta chair conformations (B).

Probably the most practical

observation is that the three different

ligand shapes that appear in nuclear

localized VDR, membrane-caveloae

localized VDR, and plasma DBP (F).

Ultimately, the conformational

flexibility enables vitamin D to carry

out a of variety functions via VDR.

The two major classes are the rapid

Figure 5: Shapes of the optimal ligands for VDR-mediated responses and for RR, as well as for vitamin D binding protein (DBP). There is a characteristic ligand shape for each type of

response.

cytoplasmic or membrane responses (kinetically favored) and the slow genomic

responses (thermodynamically favored).

The first class of response that can

be induced isare the traditional genomic

responses. VDR is a DNA-binding

transcription factor consisting of a

heterodimer (two different molecules

bound together, usually macromolecules –

in this case, the VDR with the vitamin D

ligand, as well as an unoccupied retinoid X

receptor (RXR – see figure 65)8.13 After

the ligand binds to the VDR genomic

pocket (GP), there is a conformational

change to allow it to serve as a platform for

coactivator binding. The coactivator allosterically stabilizes the VDR-RXR heterodimer,

then allowing it to be phosphorylated by serine protein kinases. This new complex can

positively and negatively regulate gene transcription by recognizing vitamin D response

elements (VDREs) in DNA. The VDR-RXR then recruits additional comodulators to help

initiate transcription. There are many hypotheses concerning how exactly thisof how

exactly happensthis happens; Dr. Haussler’s team proposes that there is a simultaneous

binding of multiple factors in a supercomplex at the promoter, based on the model

RANKL gene promoter. Activated VDR can also interact with transcriptional

coregulators to control gene expression. VDR consists of 427 amino acids with two main

Figure 65: Structure-function relationships and proposed mechanism of gene induction and repression

by VDR.

functional groups: a zinc finger DNA binding domain near the N-terminus, and a vitamin

D ligand binding domain near the C-terminus. A structure consisting of 12 α-helices

allows VDR to heterodimerize with the retinoid X receptor.13

The practical implications come from finding the genes that are directly regulated

by this VDR complex. So far, at least eleven genes that encode bone and mineral

homeostasis (the traditional target of VDR) have been found, including gene products

that facilitate intestinal calcium intake. Another network that has been found to be

regulated by VDR is theare encoding factors that impact cell life/cancer, the immune

system, and metabolism. These come from inducing and repressing various genes

involved in diseases such as type I diabetes, multiple sclerosis, and arthritis. It has even

been found to blunt various genes involved in inflammatory responses, thus reducing the

risk of heart disease and Alzheimer’s. It is clear that there are many areas regulated by

VDR, and there will certainly be more to come.

Vitamin D also plays a role in a second category of responses: rapid responses.

This cannot be explained by VDR-mediated gene transcription, as was shown in the

classical genomic responses. This is a relatively new area of research; the first rapid

responses were discovered in the 1980s from the rapid hormonal simulation of intestinal

calcium absorption in chicks. The transfer of calcium to the intestine was noticed only

after 4-5 minutes after transfer of vitamin D to the celiac artery. The main difference that

separates genomic and rapid responses is the time delay; genomic responses often take

days while the rapid

response pathway

takes mere

minutes. However, rapid responses are also often induced through a different mechanistic

pathway. The first clue came after it was noticed that only the 6-s-cis locked and not the

6-s-trans locked analog was capable of producing rapid responses in the chick model.

This also means that the VDR also can adopt different conformers – the so called “VDR-

GP” for genomic responses, the membrane caveolae localized “VDR-AP” for alternative

binding, and the plasma vitamin D-binding protein (DBP).

In particular, it has been found that the caveolae is the source of many rapidly

responding signal transduction pathways. Caveolae are located in the plasma membrane

and are enriched in sphingolipids and cholesterol. It was demonstrated both that vitamin

D showed the same binding

affinities to VDR in the

caveolae as observed with nuclear VDR, and that vitamin D localized in vivo in the

plasma membrane caveolae9.14

Finally, the “VDR AP” site was proposed to resolve the VDR paradox (see Figure

7).23 Traditionally, only

a single ligand binding

domain has been

recognized – the one

that binds only the 6-s-

trans shape. However,

the rapid response

conformer is not able

to dock to this specific binding site. Computational work showed that there is an

Figure 6: Different mechanisms by which vitamin D and VDR can induce chemical responses in the body. On the left, the caveolae-related pathway leads to activation of the

secondary messenger system to elicit short term responses. On the right, 1,25D can interact with VDR localized in the cell nucleus to produce genomic responses through

gene transcription.

Figure 7: The proposed VDS-VDR conformational ensemble model. The left panel shows the conformational flexibility of VDS, the middle panel shows the different binding sites on VDR, with the yellow oval showing overlap between the two regions. The right panel shows specific

conformational dynamics of VDR AF2 domain; the Boltzmann distribution is altered depending on the nature of the ligand, changing the energy landscape of VDR ensemble members to bias a

specific downstream event.

Figure 7: The proposed VDS-VDR conformational ensemble model. The left panel shows the conformational flexibility of VDS, the middle panel shows the different

binding sites on VDR, with the yellow oval showing overlap between the two regions. The right panel shows specific conformational dynamics of VDR AF2 domain; the

Boltzmann distribution is altered depending on the nature of the ligand, changing the energy landscape of VDR ensemble members to bias a specific downstream event.

alternative binding site available. This conformational model was proposed by Dr.

Haussler and his team, whereby the VDR could accommodate differently shaped ligands

to initiate both genomic and rapid responses. The steroid hormone would essentially “test

the waters” and form a receptor-hormone complex with the receptor species that provided

the highest affinity and most stability. Figure 86 shows the main differences between the

genomic and rapid

response

pathways.15 0. It

seems as though the

two categories are

vastly different, but

a small portion of

VDRs at the

membrane is now believed to regulate the expression of genes, thus regulating the

activity of many kinases, phosphatases, and ion channels. However, more research needs

to be done to further elucidate the mechanisms behind this process.

Implications of Vitamin D/VDR and Sleep:

Since VDR has recently been found in the brain, some interesting new hypotheses

have emerged concerning the function of vitamin D in the brain.18 1. One of these concerns

the role of vitamin D in sleep. Normally, sleep is highly organized. Humans typically go

to sleep at the same time every night, going through specific phases, such as REM, slow-

wave, etc. Waking up from sleep is involuntary and also occurs in a fairly stable manner.

Figure 8: Different mechanisms by which vitamin D and VDR can induce chemical responses in the body. On the left, the caveolae-related pathway leads to activation of the secondary messenger system to elicit short term responses. On the right, 1,25(OH)2D can

interact with VDR localized in the cell nucleus to produce genomic responses through gene transcription.

This seems to imply that sleep is not caused by a buildup of sleep-inducing hormones and

substances; rather, it is a result of a circadian rhythm type function where sleep is highly

coordinated by the time of day. This implies that one of the reasons for sleep problems

stems from brain chemistry.

Normally, many different hormones are secreted before sleep, leading to

drowsiness – a warning, so to speak. Antidiuretic hormone is produced to limit urine

production, melatonin levels increase, and so on. The brain also induces paralysis as deep

sleep arrives, activating specific neurotransmitters to turn off the signals responsible for

wakefulness. These two categories, timing and paralysis, are essential to sleep. Saper and

colleagues suggest an on-off switch mechanism, which is responsible for sleep; one part

of the brainstem is active while the other is suppressed.16 2 Specifically, the hypothalamus

is thought to be involved because the stimulation of the posterior hypothalamus induces

arousal, while stimulation of the anterior hypothalamus and adjacent basal forebrain

region causes sleep.1317 . Now, how does this tie in with vitamin D? Vitamin D targeted

neurons have been discovered in specific brain and spinal cord locations in multiple

animals. This suggests a possible role of vitamin D in regulating sleep. A 2-year

uncontrolled trial of vitamin D supplementation in 1500 patients with neurological

complaints and sleep problems saw improvements in both these functions.14 18 Further

research needs to be done in this area, as sleep is also influenced by sociological and

psychological factors. For example, pain has been shown to influence the quality of sleep,

but pain has also been linked to vitamin D.19 5 Therefore, vitamin D may ameliorate the

quality of sleep through a multitude of factors – not only through chemical pathways in

the brain, but also through alleviating pain. There is a delicate balance between these

factors; vitamin D could directly impact sleep, which could then improve feelings of

pain. Vitamin D could also improve a multitude of variables, including mood, quality of

life, etc. which could also improve pain (a subjective feeling that could be influenced by

psychology). Further research needs to be done to elucidate the function of vitamin D in

these processes.

Vitamin D has clearly grown in importance over the last half century. The

involvement of vitamin D in vital bodily processes, from bone health to even regulating

gene expression, reveals how potent this single hormone is to human health. New models

to elucidate the nature of vitamin D and VDR binding will further this cause and may

even reveal new routes for drug development and helping curethe curing of diseases, one

of the most fundamental concerns for the human race.

References

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Figure 4 source: http://www.sciencedirect.com/science/article/pii/S002228360901451X