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Evidence for a receptor bindiDg 24R,2S-dihydroxyvitamin D3 indeveloping bone

by

ALYSONBYRD

Department of Surgery, Division of Experimental Surgery

MeGUI University, Montreal

March 1999

A thesis submitted to the Faeulty of Graduate Studies and Research in partialfulfUment of the requiremenfs of the degree of

MASTER OF SCIENCE

e ALYSON BYRD, 1999

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TABLE OF CONTENTS

Sections Page

LIST OF FIGURES 4

~<:~O~IJH(J~S 5

ABBREVIATIONS 7

1. ~STRAcr 10

II. RÉsUMÉ... .. ... . .. . .... . ... .. .. . .. . .. .. .. Il

ID. INTRODUCTION 13

A. The Vitamin D Endocrine System 13

B. Bone Histogenesis 16

C. Nuclear Hormone Receptors. .. .. . . . . . . . . .. . .. . . . . . . . . . . . . . . . ... . . .. .. 18

D. 24R,25-dihydroxyvitamin D3...........................•..•........... 22

IV. HYPOTlŒSIS 28

v. OB~~~~1rl{)~~ ...............................•..•...........3()

VI. MATE~AND METHODS 32

A. Preparation of Nuclear and <:ytosol Extracts 32

B. <:rode Extracts 33

C. <:ompetition Assays 34

D. Saturation Analysis Experiments 35

E. Sucrose Gradient Sedimentation Experiments 36

F. mRNA Extraction 37

G. Two..Hybrid cDNA Library Constnlctïon 38

H. DNA-Binding Domain Vector <:onstIUction 38

2

IX.

X.

XI.

•

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L Yeast Two-Hybrid Screening 39

J. PCR Screening of the Yeast Two-Hybrid Libraries 39

vu. RESULTS 42

A. Ligand Saturation Analysis of Nuclear Extracts 42

B. Competition Analysis of Nuclear and Cytosol Extraets

and of DBP 42

C. Sucrase Gradient Sedimentation of Nuclear Extraets 45

D. Tissue Specificity 47

E. Yeast Two-Hybrid Screening 48

F. PCR Screening of the Yeast Two-Hybrid Libraries 49

Vill. DISCUSSION 50

A. Ligand Saturation Analysis of Nuclear Extracts 50

B. Competition Analysis of Nuclear and Cytosol Extraets

and of DBP 52

C. Sucrose Gradient Sedimentation of Nuclear Extraets 55

D. Tissue Specificity 57

E. Yeast Two-Hybrid Screening 59

F. PCR Screening of the Yeast Two-Hybrid Libraries 62

G. Alternative Strategies to Clone the 24R,2S(OH)2D3 Receptor 64

S~YJ\lS[[) CONCLlJSIONS 69

ORIGINAL CONTRIBUTION TO KNOWLEDGE 71

REFERENCES 72

3

•Figure

LIST OF FIGURES

Following page

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Figure 1: Simplified schematic of vitamin D metabolism 13

Figure 2: Nuclear hormone receptor superfamily structure and classification... 19

Figure 3: VDR-mediated transcription 20

Figure 4: Abnormal bone fonnation in 24-0Hase-deficient mice 25

Figure 5: Saturation analysis of eH]-24R25(OH)2D3 binding bymandiblelcalvaria nuclear extraet. 43

Figure 6a: Specificity of eH]-24R2S(OHhD3 binding bymandiblelcalvaria nuclear extract. 44

Figure 6b: Specificity of eHl-24R25(OH)2D3 binding bymandiblelcalvaria cytosol extract 44

Figure 6c: Specificity of eH]-24R2S(OHhD3 binding by liver nuclear extract 44

Figure 7a: Specificity of eH]-24R25(OHhD3 binding to bovineGlobulin Cohn Fraction IV (Sigma) as a source of DBP 45

Figure Th: Specificity of eH]-24R25(OH)2D3 binding to Gc Globulin(Sigma) as a source of DBP 45

Figure 8a: Complex fonnation between DBP7 actin, and DNase 1. 46

Figure 8b: Sucrose gradient sedimentation of liver nuclearextract after labeling with IoM eHl-24R25(OH)203 46

Figure 9a: Complex fonnation between OBP, actin and anti-actin antibody 48

Figure 9b: Sucrose gradient sedimentation of Iiver nuclear extractincubated with eH]-24R25(OH)2D3 and anti- actin antibody 48

Figure 10: Tissue specificity of the eHl-24R25(OH)2D3 binding- proteinin nuclear extraets 48

4

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ACKNOWLEDGEMENTS

To Dr. René St-Arnau~ my thesis supervisor, without whom 1 could never bave

accompüsbed my goals. He bas offered me endIess advice, encouragement, and patience,

both in this project and thesis, as weU as in my pursuit of extra-eurricular interests. 1

would like to thank him for providing me with top of the line scientific and personal

guidance, bath to which 1 am indebted.

To Janet Moir-Brazeau, a coUeague who bas endured, with exceptional patience, my

countless questions and worries. 1 thanle her for taking me througb Many of the

procedures 1 was performing for the fml time, offering sound suggestions and great

companionship.

To Josee Prud'homme, also a cO-worker and always full of encouragement and good

humour. 1 appreciate her belp with setting up the mice matings, the genotyping and the

dissections.

To Serge Messerlian, a feUow graduate student with wbom 1 bave shared a myriad of

frustrations and celebrations and from whom 1have sougbt advice and answers to infinite

queries. 1 thank him for bis companionsbip, understanding, and more formaUy, for bis

contribution of the VDR primers.

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To Alice Arabian, my co-worker, 1 owe mucb to ber expert instruction and ber sound

reasoning. 1 thanle ber for ber belp with perfonning and interpreting the ligand binding

assays.

To Louise Marineau and Mia Esser, the animal teehnicians, 1 thank them both for aIl their

help with the mice matings and dissections.

To Jane Wishart, the MediCal imager, 1 wisb to thank ber for her help in preparing the

figures in the Introduction.

To Dr. Xiangming Gao, Dr. Olivier Dardenne, Dr. Isabelle Quelo and Dr. Gourgen

Ambartsoumian, my colleagues, 1 very much value aIl of their advice in overcoming my

experimental difficulties, as weIl as their indispensable camaraderie. 1 truly could not

have found a better group of people to work with and 1 hopc the friendships forged

remain strong, despite our diverging paths.

Finally 1 would like to give my appreciation to my Mother and Father, and Tom, for

supporting me in my decisions and allowing me the freedom to choose my own direction.

This research was conducted thanks to funding from the Shriners of North America.

6

• ABBREVIATlONS

25(0H)D3 25-hydroxyvitamin D3

1a,25(OH)2D3 1a,25-dihydroxyvitamin D3

24R925(OH)2D3 24R925-dihydroxyvitamin D3

la-ORase 25-hydroxyvitamin D la-hydroxylase

24-0Hase 25-hydroxyvitamin D 24-hydroxylase

25-0Hase vitamin D 2S-hydroxylase

AF-2 activation function-2 domain

AR androgen receptor

3-AT 3-amino-1 92A-triazole

• ATP adenine triphosphate

bp base pairs

Bmax maximal binding

BSA bovine senun albumin

CAT chloramphenicol acetyltransferase

cDNA complementary deoxyribonucleicacid

CKB creatine kinase brain isoform

CMV cytomegalovirus

C-tenninal carboxy-terminal

DBD DNA-binding domain

DBP vitamin D binding protein

• dCTP deoxycytosine triphosphate

7

• DNA deoxyribonucleicacid

DR3 direct repeat separated by 3 base pairs

dNTP deoxy N triphosphate

DIT dithiothreitol

E17.5 embryonic clay 17.S

EDTA ethylene diamine tetra acetic acid

EGTA ethylene glycol tetra acetic acid

ER estrogen receptor

EtOH ethanol

FXR famesoid X receptor

GR glucocorticoid receptor

• ms histidine

hsp90 heatshock protein of 90kDa

Hyp hypophosphatemic

IP9 inverted palindrome separated by 9 base pairs

Kb kilo base pairs

K<t dissociation constant

kDa kilo dalton

LBD ligand-binding domain

LEU leucine

LUC luciferase

LXR liver X receptor

rnRNA messenger ribonucleicacid• 8

• NCBI National Center for Bioteebnology Information

N-tenninal amino terminal

one omithine decarboxylase

Olïgo dT oligomeric deoxythymidine

PBS phosphate buffered saline

PCR polymerase chain reaction

PMSF phenylmethylsulfonyl fluoride

PPARy Peroxisome proliferator-activated receptor gamma

PR progesterone receptor

PTH parathyroid hormone

Pfu Pyrococcus furiosus

• RAR retinoic acid receptor

RT-PCR reverse transcriptase-polymerase chain reaction

RXR retinoid X receptor

RXRE retinoid X response element

S sedimentation coefficient

Td temperature of dissociation

TR thyroid receptor

VAS upstream activating sequence

UTR untranslated region

VDR vitamin D receptor

VDRE vitamin D response element

• 9

• 1• ABSTRACf

•

•

Although 24~25(OH)2D3 bas been implicated in bone development, its biological

role and mechanism of action remain controversial. In searcb for evidence of a receptor,

nuclear and cytosol exttacts were isolated from mandibles and calvaria of E 17.5 mice.

Competition and saturation analysis identified a saturable, specific and higb affinity

(~=I.lnM) 24R,25(OHhD3 binding-protein. The results of these and sucrose

sedimentation studies indicate tbat this protein is not vitamin D receptor (VDR) or

vitamin D binding protein (DBP). Tissue specificity experiments suggest that this

putative receptor is also present in liver but Dot brain.

pBDGal4-hRXRa bait was used to screen neonate and embryonal

mandiblelcalvaria cDNA libraries using the yeast two-bybrid system. PCR screening was

also perfonned using primees from the zinc-fmger region of the VDR. To date no

positive clones bave been identified. Isolation of this putative receptor will provide

valuable insight ioto the mechanism of this metabolite's role in bone development.

10

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o. RÉSUMÉ

Des travaux récents de notre laboratoire suggèrent que le métabolite de la vitamine

D, 24R,25-dihydroxyvitamin 03 (24R,25(OH)2D3), pourrait être impliqué dans le

développement osseux intramembranaire. Nous avons émis l'hyPOthèse qu'un récepteur

spécifique pour la 24R,25(OHhD3 était exprimé dans l'os intramembranaire en

développement. Des extraits nucléaires et cytosoliques de calottes craniennes et de

mandibules ont été préparés à partir d'embryons à 17.5 jours de développement. Des tests

de liaisons utilisant la 24R,25(OH)2D3 tritiée ont mis en évidence un site de liaison

nucléaire de haute affmité et spécificité (Kcs=1.1 nM). L'analyse de ces sites de liaison par

sédimentation en gradient de sucrose a pennis d'éliminer la possibilité que l'activité de

liaison soit dOe au récepteur classique de la vitamine D ou à la globuline sérique de

transport de la vitamine D. L'activité de liaison de la 24R,2S(OHhD3 rot détectée dans

l'os et le foie, mais pas dans le cerveau, suggérant une certaine restriction d'expression

tissulaire.

Une stratégie de criblage par interaction fonctionnelle chez la levure Cyeast two­

hybrid screen'), utilisant le partenaire potentiel 'Retinoid X Receptor', a été utilisée dans

l'espoir de cloner le récepteur 24R,25(OH)2D3. La librairie criblée fOt produite à partir

d'os intramembranaire fétal. Une stratégie basée sur l'homologie de séquence probable

entre le récepteur pour la 24R,25(OH)2D3 et le récepteur classique de la vitamine D et

utilisant l'amplification en chaîne ('polymerase chain reaction'; PCR) a aussi été utilisée.

Ces tentatives sont restées infructueuses jusqu'à maintenant.

Il

•

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Le clonage d'un récepteur spécifique à la 24R,2S(OH)ZD3 pennettrait d'approfondir

de façon marquée notre compréhension du mécanisme d'action de la vitamine D et des

mécanismes moléculaires contrôlant le développement osseux.

12

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m. INTRODUCTION

A. The Vitamjn D Endocrine System:

Despite its name, vitamin 0 is not a vitamin at a1l, but rather, a hormone. Both

plants and animais are able to syntbesize vitamin 0 during exposure to sunlight. In

humans, what is not formed endogenously in the skin is obtained through the diet from a

variety of plants, dairy products, eggs and 6sh. Vitamin 02 (also referred to as

ergocaiciferol) is the form of the vitamin that is produced in plants, whereas vitamin D3

(cholecaiciferol) is that which is syntbesized by vertebrates. Vitamin~ is unsaturated at

carbon centers Cn and C23 and bas an extra methyl group at C24 (1). Although this form

has been shown to contribute significantly to the vitamin 0 functions of humans and other

vertebrates (2, 3) and undergoes similar metabolic pathways to form a hormonally active

metabolite (4), for the pUl'POse ofthis paper vitamin 03 is the focus.

Figure 1 illustrates a simplified schematic of vitamin D metabolism. Penetration of

sunlight's ultra violet B photons through the skin allows photolysis of a cholesterol-like

precursor called 7-dehydrocholesterol (also termed pro-vitamin D3) present in

mammalian skin, converting it into pre-vitamin 03 (5). UPOn thermoisomerization, this

becomes the more stable secosteroid, vitamin D3 that then enters the circulation where it

binds to the vitamin D binding protein (DBP) (6).

The vitamin D binding protein is the principal carrier of vitamin DJ and its major

metabolites. Il is a serum protein that circulates in high amounts (5 x lO~ M) compared

to 25(OH)D3, its major ligand (5-12 x IO·IM) (7). Although the ligand binding domain of

DBP has highest affinity for 2S(OH)DJ (the most abundant circulating vitamin 0

13

• • •II n Il lt l1 n Il :t II II Il 20

24,25(OH)2D3

ri Il It

HO

Kidney 25(OH)Df 24R-hydroxylase" , OH

HO

25(OH)D3

~

37 oC.....

HO

Liver D3-25 hydroxylase l "pre-D3

Kidney 25(OH)Df 1a-hydroxylase

" " · · JI'

4 Â'THO'-'V, 1a,25(OH)2D

OH 3

~7-dehydrocholesterol

Figure 1: Simplified schcmatic of"itamin Dmetabolism. Vitamin DJ, synthesized by the skin or obtained throllgh the diet, is transportedby DBP to the liver where it becomes hydroxylated to 25(01-l)D

3• After transport to the kidney the Illetabolite is further hydroxylatcd to

1a.,25(OH)2DJ' the active form of vitamin DJ• Alternativcly, 24-hydroxylation in the kidney Icatls to the production of 24,25(OH)2DJwhosc function has yct to be dctcrmined.

•

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metabolite), it is also able to bind other metabolites, including 24R,2S(OHhD3 (but not

24S,25(OH)2l>J, its synthetic cpimer) and la,25(OH)2D3 (8). Several conflicting reports

exist on the relative order ofbinding affinity for sterols by DBP. A couple of reports state

the order is thought to he: 25(OH)D3 = 24R,25(OH)2D3 > la,25(0H)203 > vitamin 03 (7,

9), while others suggest it is: 25(0H)03 > 24R,25(OH)2D3 > la,25(OHhl>J (10, Il), and

still a third shows it could he 24R,25(0H)03 > 25(08)D3> la,25(0H)2D3 (12). Sorne of

these variations may he due to the source of DBP used and/or the conditions in which the

experiments were performed.

Vitamin 03, associated with OBP, gets transported to the liver, at which point the

vitamin D 25-hydroxylase enzyme (25-0Hase) adds a hydroxyl group to C25 (figure 1) (5,

6). This is the initial step in vitamin D activation and leads to the production of

25(0H)D3, the most abundant, but still inactive, form of vitamin D3. The liver production

of 25(0H)D3 does not seem to he stringently regulated and mainly depends on substrate

concentration (1). This inactive metabolite is then carried to the kidney, again by DBP,

where it can follow one of two pathways (figure 1). It can become hydroxylated at either

the la-position by the renal enzyme, 25-hydroxyvitamin D la-hydroxylase (la-OHase),

or al the 24-position by the 25-hydroxyvitamin D 24-hydroxylase (24-0Hase) (5, 6). In

the former situation, the resulting metabolite is la,25(OH)2D3 (calcitriol), the biologically

active form of vitamin D3. The circulating concentration of 1a,25(OH)203 is about 1000­

fold less than 25(OH)03 al 5-15 x 10-H M (13). The latter pathway leads to the formation

of 24R,25-dihydroxyvitamin D3 (24R,25(OHhD3), the most abundant dihydroxylated

metabolite in blood (-6 x lO-~ (14). Countless other side chain oxidation pathways

14

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lead to the formation of over 40 other vitamin I>J Metabolites, Most of which, however,

are considered non-functional and eatabolic in nature.

The primary role of la,2S(OHhD3 is to regulate calcium and phosphoms

homeostasis. It does this in three ways: 1) by increasing intestinal calcium and

phosphorus absorption, 2) by inducing mobilization of calcium from bone whenreq~

in synergy with parathyroid hormone (PfH) and 3) by increasing renaI reabsorption of

calcium and phosphoros, aIso in conjonction with PTH (15).

1a,25(OH}2D3 and 24R,25(OH)2D3 production are reciprocally regulated. When

calcium is needed, 1a,25(OH)2D3 synthesis is enhanced, while the 24-0Hase

hydroxylation pathway is suppressed. Conversely when calcium levels are adequate,

synthesis shifts to 24R,2S(OH)2D3. This increase in 1a,2S(OH)2D3 synthesis upon

hypocalcemia is secondary to increased PTH and is due to an induction of the 1a-OHase

enzyme. Thus there appears to he a calcium-mediated feedback system regulating the

la-OHase enzyme whicb prevents sustained levels of 1a,25(OH)2D3 that could lead to

hypercalcemia (16). 1a,2S(OH)2D3 production is aIso reguiated by plasma phosphate

levels. As plasma phosphate level decreases, production shifts from 24R,25(OHhD3 to

increased la,25(OH)2D3 synthesis. In addition, high levels of la,2S(OHhD3 act to

decrease 1a-OHase activity and stimulate 24-0Hase activity (1), further regulating

Metabolite levels.

The major site of 24-bydroxylation is in the kidney (1). The cytochrome P450

enzyme, 25-hydroxyvitamin D-24-hydroxylase, is able to hydroxylate both 2S(OH)D3

and la,25(OH)2D3. Contrary to la-OHase, it is positively regulated by la,2S(OH)2D3

15

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and negatively regulated by PI'H (17). It seems tbat the 24-hydroxylation of 25(08)1»

and 1(1,25(0H)203 is the first step in a metabolic pathway to inactivate these Metabolites

and halt their fonctions. However, although the major site of expression of 24-0Hase is

in the kidney, its expression in osteoblasts and other tissues indicates it May serve to

sYDthesize active Metabolites (such as 24R,25(OH)203) that could play an important role

in bone mineralization (18). The possible fonctions of 24R,25(OH)203 are discussed in a

later section.

B. Bone Bistogenesis:

It is weil known that 1a,25(08)203 is vital for normal bone formation, as it is an

important regulator of osteoblastic differentiation and function. Abnormalities along the

pathway of the vitamin D endocrine system leads to osteomalacia and rickets. In

addition, a significant amount of evidence exists which suggests that 24R,25(OH)2D3 also

plays an important biological role in bone development. A brief overview of bone

histogenesis will allow for a better understanding of these raIes.

There are three main cell types in the skeleton: (a) chondrocytes, the cartilage

fonning cells which differentiate from eartier multipotential mesenchymal progenitor

(stem) cells; (h) osteoblasts, the bone forming cells which aIso differentiate from

mesenchymal progenitor ceUs and deposit a specialized matrix that undergoes

mineraiization (see below); and (c) osteoclasts, the bone resorbing ceUs which are large,

multinucleated cells derived from baematoPOietic stem cells. Two distinct tyPes of bone

histogenesis exist: intramembranous and endocbondral ossification, the major difference

being the presence or absence of a cartilagjnous phase (19, 20).

16

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Endochondral ossification, which occurs in long bones and the growth plate,

involves the formation of a cartilaginous matrix befoIe conversion ioto bone. Essentially,

mesenchymal ceUs differentiate into prechondroblasts and !hen chondroblasts. These

cells secrete a cartilaginous matrix into which they embed tbemselves and become

chondrocytes. During the development of this avascular, embryonic cartilage a ring of

woven bone is formed through the process of intramembraneous ossification (see below)

in the future midshaft area. Angiogenesis then allows blood vessels to invade this

calcified woven bone, bringing along osteoclast precursors. The osteoclasts begin to

destroy the calcified cartilage matrix as weil as excavate the hematopoietic bone marrow

cavity, while osteoblasts are recroited to replace the cartilage witb bone matrve At the

ends of long bones, the cartilage serves as a growtb plate to aIIow longitudinal growth.

The growth plate chondrocytes form columns beginnjng with resting cartilage cells,

followed by proliferating chondrocytes that become progressively larger, hypertrophie,

and eventually die. Their cartilaginous matrix becomes mineralized just below the

growth plate hypertrophie zone. Eventually the calcified cartilage is partially resorbed by

osteoclasts, and osteoblasts (that differentiated from the mesenchymal cens brought in by

the invading blood vessels) begin to deposit woven bone on top of the cartilaginous

remnants. This immature woven bone and calcified eartilage is further remodeled lower

in the growth plate, eventually becoming replaced by mature lameUar bone (19,20).

Intramembranous bone formation, which occurs in the fiat bones (such as the

calvari~ mandible, and clavicle) as well as the outer cortex of the long bones

(periosteum), also begins as a cluster of mesenchymal cells. However, in this case they

do not differentiate into ehondrocytes, but rather undergo proliferation and differentiation

17

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directIy into osteoblasts. The proliferating precursors secrete an organic mabix, called

osteoi<L that contains coUagen fibers embedded within it. Once differentiated, osteoblasts

begin to form an immature woven bone by dePOsiting crystals of calcium phosphate on

and around the coUagen fibers in random orientation. EventuaUy the woven bone

becomes invaded by blood vessels, remodeled and replaced by mature, lameUar bane (19,

20).

c. Nuclear Hormone Receptors:

As early as the 1970s steroid actions were shown to he mediated by SPeCifie high

affinity receptor proteins. It was believed that the binding of hormone to its receptor

induced an allosteric change that enabled the hormone-receptor complex to bind to high

affmity sites in chromatin and modulate transcription. However, it wasn't until 1985 that

the nrst nuclear hormone receptor was isolated (glucocorticoid receptor, GR) (21). Sïnce

then receptors have been identified for alI the known fat-soluble, nuclear hormones.

Furthermore, an endless number of so-called orphan receptors have been identified for

which no known ligands have been found, bringing the total number of members of this

nuclear receptor superfamily to over ISO (22). Of these, only about 50 are mammalian

receptors, the rest coming from C. elegans and other species. The majority of these

orphan receptors were discovered by using low stringency hybridization screening, while

still others were found by using more modem molecular cloning techniques, sucb as the

yeast two-hybrid.

Nuclear hormone receptors are Iigand-modulated transcription factors that mediate

responses to steroids, retinoids, and thYroid hormones. They are modular proteins

18

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•

consisting of five distinct domains" each with separate fonctions (figure 2) (22" 23). The

N-tenninal (NB) and the C-terminal (F) domains are highly variable, as is the hinge (D)

region, which allows Cree rotation of the ligand-binding and DNA-binding domains. This

hinge region has also been implieated in heatshock protein 90 (hsp90) interaction as well

as nuclear localization (24). The N-terminal domain is involved in transcriptional

activation (AF-l domain) (in sorne receptors) and promoter selection (24). AlI nuclear

hormone receptors have a DNA-binding (C) domain (DBD) consisting of two highly

conserved zinc-fmgers. This region is responsible for localizing the receptor to specific

DNA sequences (called hormone response elements) on target genes (22). In addition to

protein-DNA interactions this domain also alIows protein-protein interactions (25).

Finally, the ligand-binding (E) domain (LBD) of these receptors, aIso highly conserved, is

responsible for ligand recognition which ensures specificity and selectivity of the

physiologic response. This region aIso encodes the dimerization properties of the

receptors (possessing homo- and/or hetero- dimerization interfaces). Furthermore,

hormone-dependent transcriptional activation (AF-2 domain) and/or repressor functions

are also encoded in this domain (23). Essentially, the LBD is a molecular switch that, in

most cases, tums the receptor on to a transeriptionally active state upon ligand binding

(22). The mechanism for this involves complicated interactions with various co­

activators and co-repressors. In simplest terms, co-repressors interact with the unliganded

receptors, resulting in inhibition of basal transcriptionaI activity. Binding of hormone

causes the AF-2 domain to change confonnation, which subsequently leads to the

dissociation of the co-repressors, recJUitment of co-activator proteins, and transcriptional

activation of the receptor (23).

19

•

"....-__....A_I...8 ........;;;,C__~-D...... E FN < i'iIIIft..'_. c

Staroid Receplora

Dimeric 0rphM Aeceptora

tIryroId hotmoI..."."... RA

'.2S-lOHJ7~

~ttoItâ

~

NGf1.8 (CE8-1') '1ELP 1 SF-1 (Rz-F1) '1,

ri i

lIonomeric Orphan Aeceptors

glu(C1 COificoitI

""'.,&01"',,, 'fi

PR7"...........a'.n....e,."

AXA (wp) ~RACOUP/.... '1HNF.... .,

•

•Figure 2: Nuclear hormone receptor superfamily structure and classification. Ail nuclearhonnone receptors have an N-tenninal region (AIB), a DNA-binding domain (C), a hingeregion (0), a ligand-binding domain (E), and aC-terminal region (F). The superfamily isdivided into four classes depending on their dimerization and DNA-binding properties asdescribed in the text. From: Mangelsdorf, DJ., et al. 1995. Cell. 83: 835-839.

•

•

•

The nuclear bormone receptor superfamily is divided into four classes depending on

their dimerization and DNA-binding propertics (figure 2) (22). Class 1 receptors are the

steroid bormone receptors. These fonction as ligand induced bomodimers and bind to

DNA-response elements consisting of inverted repeats. Class fi receptors fonction as

obligate RXR heterodimers and usuaUy bind to directly repeated DNA balf-sites. This

group contains the rest of the known ligand-dependent receptors. Most orpban receptors

belong to either class m or class IV. Class mconsists of the dimeric orphan receptors,

which bind as homodimers to direct repeats. Finally, class IV monomeric orphan

receptors bind to DNA response elements as monomers (22).

The actions of la,25(OHhD3 are mediated by a bigh affinity, highly specifie

receptor, called the vitamin D receptor (VDR). VDR is a elass fi member of the nuelear

hormone receptor superfamily, and thus is able to beterodimerize with RXR. Figure 3

depicts the mechanisms by whicb la,25(OH)2D3, VDR and RXR act to modulate

transcription. When la,25(OH)2D3 enters its target celI, it binds to the unliganded VDR

in the nucleus. The reœptornigand complex heterodimerizes with RXR, conferring upon

the receptor an increased affmity for specific DNA sequences located upstream of the

target genes (Le. in the target gene promoters). Tbese are refened to as vitamin D

response elements (VDRE). This interaction leads to the modulation of transcriptional

activity of target genes responsible for carrying out the pbysiologjcal functions of

A typical VDRE consists of two directly repeated hexanucleotide sequences

separated by three base pairs (DR3). However, DR4 and DR6 type VDREs also exis~ as

do inverted palindromic arrangements of two core binding motifs spaced by nine20

•9-cis retinoic acid (.6)

+1i •

t..U-'ra"s ftlinol0' -\ ~ .... ,("11,011

t1

DietarySources

Sunlight

~coon

.U·trllns retin.ld~hyde

~Cll()

• ll-trllns retinoic uid (8)~C()()fl

t

~4

if ...--110

RXRE

-- .~VDRE t.::::/

ŒI"---c1-.I~~~

1,2S(OH)2D3 (e)

~OH +-4_

.& kidneyHO OH

/t!

l~1: Target1\ Cell\ Nucleus

•

•Figure 3: VDR-mediated transcription. In the presence of la,25(OH)..,D., RXR- )

heterodimerizes with the bound VDR, fonning a transcriptionally active complex on theVDRE. 9-cis-retinoic acid may promote RXR homodimer fonnation, shifting occupiedRXR to RXREs. From: Whitfield, GK., et al. 1995. J. Nutr. 125: 16905-16945.

•

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Ducleotides (IP9) (25). 1be general consensus sequence of the core binding motifs of

Most members of the supcrfamily is RGGTCA (R= A or 0), although there is a high level

of divergence from this consensus in VDREs of various promoters.

Figure 3 also shows the effect of 9-cis retinoic acid (the RXR ligand) on VDRIRXR

heterodimerization. For some RXR heterodimers (termed I&pennissive"), the presence of

RXR ligand acts to potentiate the activation action of the partner ligand. However, this

seems Dot to he the case with the VDR, where, in the presence of 9-cis retinoic acid, RXR

May preferentiaUy homodimerize and bind to RXR. response elements (RXREs), thereby

activating different target genes. This may indirectly destabilize the VDRIRXR

heterodimer formation.

The VDR is present in ail tissues that exhibit a respoDse to la,25(OH)2D3

(including intestine, kidney, parathyroid glands, and bane). Its sedimentation coefficient

is 3.2 S in human and rat, and 3.7 S in chicken (26). Ils abundance (as measured by

la,25(OH)2D3 binding capacity of extraets) in Most la,25(OHhD3 target tissues is fairly

low, between 10 - 100 fmol/mg proteÎD, but can he as high as 1pmollmg protein in certain

tissue extracts (26). This could indicate that those ceUs with a higher VDR content are

more responsive to la,2S(OHhD3, than those with lower VDR expression. However, it

is also likely to involve differences in the ability to intemalize and metabolize the ligand

and/or the availability of partner proteins needed for gene activation (26). Finally,

although highly specifie for la,2S(OH)2D3 (Kct =1-2 x lO-I~, VDR does bind to other

vitamin D metabolites (including 24R,25(OHhD3) with a 500-1000 fold lower affinity

(27,28).

21

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It is important to note here that in addition to its genomïc actions, la,25(OHhI>] is

also thought to carry out a nomber of non-genomic activities in various tissues (reviewed

in 28a). These include transcaltaehia (movement of calcium from the intestinal lumen to

the bloodstream), as weil as rapid changes in membrane Iipid turnover and prostaglandin

production. These actions are proposed to he mediated by the interaction of

la,25(08)2D3 with a œil surface (membrane) receptor (VDRmcm) that is distinct from the

nuclear receptor, VDRnuc (28b). Though it bas Dot yet been studied, it is wonhwbile to

keep in mind the possibility that some of the actions of 24~25(0H)203May a1so he

mediated by non-genomic mecbanisIDS. Interestingly, a putative candidate for a

24R,25(OH)203 membrane receptor has already been identified (Il).

The role of 1a,25(OH)2D3 in intestinal calcium and phosphorus absorption,

mobilization of calcium from bone, and renal reabsorption of calcium and phosphoros is

weil established and widely accepted. On the other band, the physiological mIe of

24R,25(OH12D3 remains a controversial topie. 24R,25(OH)2DJ is the Most abundant

dihydroxylated Metabolite of vitamin 03, yet for years scientists bave believed that it is

simply a catabolite of 25(0H)03 whose production is meant only to regulate

la,25(OH)2D3 levels. This line of thought stemmed, in part, from experiments using

analogs of vitamin D that had been tluorinated on C24 (thereby preventing hydroxylation

at this position). When this analog (24,24-ditluoro-25-hydroxyvitamin 03) was used as

the sole source of vitamin D, no effects different from those obtained wben using

25(OH)D3 alone were observed. This was taken to indicate that metabolites of vitamin D

22

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•

that are hydroxylated al position 24 serve no biological function, other than as a

mechanism to inactivate 2S(OH)D3 (29, 30). However, the first evidence contradicting

this hypothesis and suggesting that other vitamin 0 metabolites migbt have biologjcal

importance came from experiments sbowing that administration of la,2S(OH)2D3 alone

was unable to produce the same biologjcal effects as those obtained wben vitamin 03 was

given (31). In these eXPeriments, bens raised solely on la,25(OH)2D3 produced fertile

eggs that were unable to batcb. In contrast, bens raised OD a combination of

la,25(OHhD3 and 24R,2S(OH)2DJ produced eggs able to batcb equally effectively as

those of bens given vitamin D3. These results suggested a bioiogical role for

24R,25(OH)20J tbat bad never been sunnised.

Since then growing evidence bas been obtained for physiological funetions of

24R,25(OH)20J. One of the fust in vivo experiments that suggested a role of

24R,25(OH)2D3 in embryonic development came from Sunde et al. in 1978 (32). These

investigators observed that the impaired development of vitamin D-deficient cbick

embryos could he rescued by treatment of the eggs with vitamin D3 itself, but not

completely by treatment with la,25(OH)2D3. This suggested that other vitamin D

metabolites were necessary for normal chick embryogenesis.

A significant number of reports iodicate that growth plate cartilage is a target organ

for 24R,25(OH)2D3 (reviewed in 33). It was sbown that injected radiolabeled

24R,25(OH)2DJ was preferentially taken up by growtb plate cartilage. In the same set of

in vivo experiments, when radiolabeled 25(08)D3 was injected ioto vitamin D-replete

rats an accumulation of labeled 24R,25(OHhD3 in growtb plate cartilage but not in

articular cartilage was discovered. Moreover, Corvol et al. (34) demonstrated that very

23

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•

low concentrations (10-13 M - 10-10 M) of 24R,25(OHhOJ stimulated proteoglycan

synthesis in cultured rabbit growth plate chondrocytes.

Other lines of evidence suggest a mIe of 24R,2S(OHhD3 in regulation of bone

growth, development and repaire As far back as 1978, Omoy et al. (35) discovered that

24R,25(OH12D] was necessary for the bone healing process of racbitic cbicks. Birds

treated with 24R,2S(OH)2D3 alone grew as weIl as those treated with vitamin D3 but were

unable to maintain calcium homeostasis. Conversely, those treated with la,25(OHhD3

alone could maintain nonnal plasma calcium and phosphorus levels but still were unable

to prevent the racbitic changes in bones. Only birds treated with both dihydroxylated

metabolites were normal in both bone and mineral bomeostasis, indicating that both

metabolites are required but perform distinct functions. Another group of investigators

examined the effects of 24R,25(OH)2D3 on bone fracture healing (36, 37). Firstly, it was

detennined that circulating levels of 24R,25(OH)2D3 are elevated in chicks during tibial

fracture bealing. This is due to an increase in the renal 24-0Hase activity and suggests

that 24R,25(OH)2D3 is involved in the early process of fracture repair (37). Secondly,

Seo et al. (36) aIso demonstrated that a normal physiological concentration (10-9 M) of

24R,25(OH)2D3 is necessary and sufficient for normal bone growth and integrity, and is

essentiaI, in combination with la,2S(OHhD3, for the fracture healing process in chicks.

This confmned in vitro studies by Schwartz et al. (38) based on organ culture of mice

fetallong bones, suggesting a raie for 24R,25(OH)2D3 (at physiological concentrations)

in the growth of fetaI mice bones as shown by increased diaphyseal length, periosteal

bone area, and hydroxyproline content.

24

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Using a different approach to examine the putative physiological role of

24R,25(OHhD3 our own laboratory took advantage of the modem teehnology of

homologous recombination to ultimately create mice lacking the 24-0Hase gene (20, 39).

A null mutation in the 24-0Hase gene was created, thereby eliminating the source of

vitamin D3 Metabolites hydroxylated al the 24-position in animaIs homozygous for this

mutation. Nonnal, fertile animaIs heterozygous for the mutation were obtained and

crossed to produce homozygous mutants. These are barn with the expected Mendelian

frequency, though about one-half die before weaning. The reason for this perinatal

lethality has not yet been determined but a possible explanation could he an inability of

these pups to maintain minerai homeostasis. The homozygous pups are unable to

eliminate la,2S(OHhD3, leading to its accumulation and thus MaY die from

hyPercalcemia. The surviving fllSt-generation homozygous mutants are fertile and appear

normal in minerai homeostasis, macroscopic anatomy of liver, spleen, kidney, and 8Ot, as

weIl as in bone histology. This is surmised to he due to the fact that the mother May

provide adequate 24R,25(OH)2~ to the pup during development through the placenta.

However, when homozygous mutants are bom of homozygous mothers, bone

development becomes impaired in the fonn of a mineralization defect. Accumulation of

unmineralized osteoid matrix at sites of intramembranous ossification, such as the

calvaria, mandible, clavicle, and exocortical (periosteal) surface of the long bones, is

evident (figure 4). Heterozygous littermates are nonnal. Though further detailed

examination needs to he done, initial observation of the growth plate seems normal,

possibly indicating that 24R,2S(OHhI>] is not important in chondrocyte maturation in

vivo.

25

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24-0Hase

-/-

Figure 4: Abnormal bone formation in 24-0Hase-deficient mice. Thehomozygous mutant shows an accumulation of osteoid and acorresponding lack of mineralized bone compared to its heterozygouslittermate. Samples shawn are mandibles from 8 day-old pupsstained with Goldner Trichrome.

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In addition to suggesting a plausible role for 24R,2S(OH)203 during

intramembranous bone development, these results support the existence of specific

putative receptors to carry out the actions of this Metabolite and indicate sites of

intramembranous ossification as a possible location for these receptors. Interestingly, it

has been observed that chick embryos from hens maintained ooly on la,25(08)203 as

their source of vitamin D3 also presented with abnormal mandibles (40). Admittedly, it

could be argued that these effects may be due to the lack of la,25(08)203 catabolism

instead of a requirement for 24R,2S(OH)203. This possibility is currently being

addressed by crossing the 24-0Hase mutant mice with vitamin D receptor (VDR)

mutants. If the observed effects are due ta an excess of la,2S(OH)2D3 acting on the

VDR, then the 24-0HaselVDR double mutant mice should no longer demonstrate the

abnormal intramembranous bone phenotype (39).

Phannacological activity of 24R,2S(OH)2D3 has also been established by various

groups. High doses of 24R,2S(OH)203 have been shown to increase bone volume and

decrease bone resorption in vivo in rats (41), rabbits (42), and ovariectomized dogs (43,

44). Furthermore, pharmacological doses of 24R,25(OH)2OJ produced dose-dependent

effects in promoting bone formation without causing excessive bone resorption in

hyPOphosphatemic (Hyp) mice (a model for familial X-linked hypophosphatemic rickets

in humans). This was in contrast to the effects of equivalent doses of la,25(OH)2D3 (45,

46).

A large number of these slodies have aIso suggested that 24R,25(OH)2D3 and

la,2S(OH)2D3 exert independent effects on various tissues. For example, it bas been

shown that radiolabeled 24R,2S(0H)203 localized to epiphyseaI chondroblasts while26

•

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1~25(OH)2D3 concentrated in diaphyseal booe cells, suggesting that each of tbese

metabolites bas its specifie site of action in developing bone (47). FurthermoIe,

experiments indicate that rat costochoodral cartilage chondrocytes isolated from the

resting zone respond to 24R,25{OHhD3, whereas those from the growth zone respond to

1~25(OH)2D3 (48-50). These responses included protein synthesis, cell proliferation,

plasma membrane and matrix vesicle synthesis, and phospholipid metabolism. Aloog

these same lines, Schwartz et al. discovered that treatmeot of resting zone chondrocytes

with 24R,25(OHhD3 induces their differeotiation and maturation iota growth zone

chondrocytes tbat are lcx,2S(OH)2D3 responsive (51). This suggests that 24R,25(OH)2D3

plays a role in cartilage development. 24R,25(OH)2D3 has also been sbown to stimulate

the activity of the brain isozyme of creatine kinase (CKB) in cultured chick limb-bud

cartilage ceUs, whereas 1~25(OHhD3 does not (52). Moreover, in vivo experiments

demonstrated that CKB aetivity is stimulated by 24R,25(OH)2D3 in the epiphyses of rat

long banes, but by lcx,25(OH)2D3 in the diaphyses of these bones, again suggesting

separate sites of action for the two dihydroxylated metabolites (53). A [mal indication of

the distinct effects of 24R,25(OHhD3 and la,25(OH)2D3 are found in a report by Yamato

et al. (54). These investigators sbowed that 24R,25(OH)2D3 antagonizes the stimulating

effect of la,25(OH)2D3 on the fonnation and fonction of osteoclastic cells.

Although strongly suggestive, the above observations are not unequivocal evidence,

and thus final proof of the different effects of 24R,25(OH)2D3 and1a,25(OH)2D3 will

depend upon the demonstration of two receptors.

27

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The presence of hypotbetical 24~25(OH)21>J receptors in growth plate

chondrocytes (55) and epiphysis of rat bone (47, 56, 57) has been suggested by severa!

groups, mostly using autoradiographic and binding saturation slodies. Other slodies have

provided support for the existence of 24R,25(OHhI>J receptors in chick embryo limb-bud

cultures (58) and in the parathyroid gland of chicks (59). These reports are somewhat

outdated and not much follow up has occurred. However, a recent slody by Kato et al.

e11) indicates the quest for the receptor bas not been given up by all, and provides

evidence for a membrane receptor specifie for 24R,2S(OHhD3 in a fracture-healing eallus

of chick tibiae. A1though all these studies are persuasive, they are also merely

insinuative, and thus, the isolation of the receptor has remained elusive to date. We

provide here, evidence of a putative 24R,25(OH)2D3 receptor in intramembranous bone of

the developing mouse embryo and describe attempted efforts for its isolation.

28

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IV. HYPOTBESIS

Previous experiments have indicated that a receptor for 24R,25-dibydroxyvitamin

0 3 does exist (56, 58, 59). However, these studies are outdated and have not been

followed up, and so the topic remains controversial. Moreover, tbese studies were

perfonned in long bones (56), parathyroid gland (59) and chondrocytes (58), tissues that

appear normal in the 24-0Hase knockout mice (20). Tbus it is possible that the receptor

bas never been cloned because the wrong tissue was heing examined. The abnonnal

ossification of the mandible and calvaria (and other sites of intramembranous

ossification) in the 24-0Hase knockout mice leads us to propose that a likely site of

expression for the putative receptor would be in these tissues. Furthermore, former

attempts to clone the receptor were often performed on postnatal tissue (56, 59). Since

ossification starts at E14.5, and since it is unknown whether expression of this putative

receptor would be turned off during or after development, it seems more rational to look

at embryonic tissue. Finally, by analogy with the vitamin 0 receptor that binds

1~25(OH)2D3, and by the fact that the structures of other RXR heterodimer ligands are

similar to that of 24R,25(OHhD3, we hypotbesize that the putative receptor will be a class

II member of the nuclear hormone receptor superfamily. It is worthwhile to note that the

retinoic acid system bas more than one Metabolite (all-trans and 9.-cis retinoic acid), eacb

of whicb binds its own nuclear hormone receptor (i.e. RAR, RXR). In contrast, the

estrogen system bas two distinct receptors (ERa and ERP) binding the SarDe Metabolite.

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Therefore it is not unreasonable to hypothesize that the vitamin D system could similarly

have more than one receptor for its metabolites.

Thus, the hypothesis propose<! is that a receptor for 24R,2S(OH)203 exists in

embryonic mandible and calvaria tissue, and that this receptor is a class n member of the

nuclear hormone receptOf superfamily, heterodimerizing with RXRa.

30

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v. OBJEcrIVES AND RATiONALE

The goals of tbis project were tirst ta provide evidence for the existence of a unique

receptor for 24R,2S-dihydroxyvitamin D3 and second, ta attempt ta clone il. This would

further our understanding of the molecular mecbanisms of 24R,2S(0H)21» action and its

function in bone development. The fICSt objective was to look for 24R,2S(OHh03

binding in mouse mandible and calvaria tissue. The rationale behind using these tissues

cornes from the observation that bone development is impaired at sites of

intramembranous ossification (including mandible and calvaria) in mice lacking the 24­

OHase enzyme (and hence deficient in 24R,25(OH)203). This was done by isolating

nuclear and cytosol extraets from 17.5 day-old mouse embryo mandibles and calvaria and

performing saturation analysis on them using eH]-24R,2S(OHhD3.

The next objective was to characterize this binding with respect ta its specificity for

24R,2S(OHhD3 versus other vitamin 03 metabolites. Competition assays were carried

out using 10-200 foid excess of various cold metabolites, including 24R,2S(0H)2D3,

245,25(0H)2D3 (the unnatural, inactive epimer), la,2S(OHhD3, and 25(OH)03 as

competitors for binding.

Thirdly, it was necessary to rule out the possibility of the vitamin D receptor (VDR)

or the vitamin D binding protein (DBP) accounting for the observed binding. The VDR

was ruled out on the basis tbat la,25(OH)2D3 did not compete for binding with the eH]­

24R,25(0H)2D3 (opposite to wbat would have occurred if the binding protein was the

VDR). Sucrose gradient sedimentation experiments were used to evaluate the possibility

of DBP as the binding protein by comparing proftles elicited by DBP and the nuclear

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extraet upon addition of actin and DNase I. The basis for this was the observation that

DBP fonns a complex with actin and DNase 1, which cao be visualized as a shift in

sedimentation peak.

Lastly, with enough evidence supporting the existence of a receptor in the tissue,

the next objective was to try to clone it. This was attempted by using the yeast two­

hybrid system since il" bad already been successfully employed in our lab and had also

been used by others to identify novel nuclear hormone receptors (60-62). The rationale

for using retinoid X receptor (RXR) as bait was based on functional homology of the

putative receptor with the VDR, which was known to function as a RXR heterodimer. In

addition, the ligands for other class II receptors are structurally similar to

24R,25(OHhD3• Mandible and calvaria tissue was selected for the cDNA libraries to he

screened for the reasons discussed above. The PCR-based screening was employed as an

alternate strategy to isolate the receptor since the highly conserved zinc-fmger region of

the DNA-binding domain of the VDR provided a good region for a 3'primer to allow

amplification of inserts containing a similar motif. Another advantage to this technique is

that it did not require the receptor to he an RXR heterodimer, thereby increasing our

chances of finding the clone.

32

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VI. MATERIALS AND METRODS

A. Preparation of Nuelear and Cytosoi Extracts:

Bone (mandible and calvaria), brain, and liver nuclear and cytosol extracts were

isolated using a protocol adapted from Roy et al. 1991 (63). Briefly, bone (O.55g), liver

(2.0g) and brain (l.Og) tissues from 17.5 day-old C57B16 mouse embryos were dissected

and frozen in liquid nitrogen. The tissue was homogenized in a 40ml Dounee manuaI

tissue grinder (Wheaton "200", Minville, NJ) containing 2 ml/gram tissue of NEI buffer

(250mM sucrose, 15mM Tris-HCI [pH 7.9], l40mM NaCI, 2mM EDTA, O.5mM EGTA,

O.15mM spermine, O.5mM spermidine, ImM dithiothreitol [DTI], 0.4mM

phenylmethylsulfonyl fluoride [pMSF), 25mM KCl and 2mM MgCh) (63) using pestle

B. Nonidet P-40 (Sigma Chemical, St Louis, MO) was added to a fmal concentration of

0.5% and the homogenate dounced 5 more strokes with pestle B. The homogenate was

centrifuged in a Sorvall RC-3 centrifuge using the HL-S rotor (Sorvall, Wilmington, DE)

at 2.2K (-lOOOg), 4°C for S minutes. The supematant containing the crude cytosol

extract was flash frozen in 200J.1l a1iquots and stored at -SO°C. The nuclei (pellets) were

washed in 5 ml of NEl buffer containing 0.5% Nonidet P-40 and recentrifuged as before.

This wash supematant was later assayed for protein content and then discarded. The

nuclei were then Iysed in 2 packed cell volumes (PCV) of NE2 buffer (NE1 buffer

containing 3SOmM Kel) (63) containing 0.5% Nonidet P-40, 1110 total volume 4M KCI,

and 11500 total volume O.SM sodium bisulfite (Sigma Chemicals, St. Louis, MO) for 5

minutes at 4°C. After douncing for 20 strokes in a 7ml dounce using pestle A, the

homogenate was transferred to a Sm! Ultra-elear ultracentrifuge tube (Beckman

33

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Instruments, Palo Alto, CA) and centrifuged al 24K for 1 hour (4°C) using a Beckman

LB-M ultracentrifuge with the SWS5Ti rotor (Beckman Instruments, Palo Alto, CA).

For brain and liver tissues, the supematant was too viscous to pellet, thus it was

subsequentIy sonicated twice for 5 seconds using an MSE 500 Watt ultrasonic

disintegrator (Measuring and Scientific Equipment Ltd., Sussex, Eng), then recentrifuged

at 35K for 1 hour. The supematant was dialyzed using a O.S-3.0ml Slide-A-Lyzer

cassette (pierce, Rockford lllinois) for 1 houc al 4°C in 500m1s of Dingam's Buffer D

(20mM Hepes [pH 7.9], l00mM KCl, 0.2mM EDTA, O.SmM DIT, O.5mM PMSF, 20%

glycerol). The dialyzed nuclear extract was recovered and spun in a I.Sml

microcentrifuge tube at 13000rpm (4°C) for 10 minutes in an IEC Centra MP4R

microcentrifuge (International Equipment Company, Needham Hts, MA). The

supematant was flash frozen in SOJ1l aliquots and stored at -SO°C. A small amount of

nuclear and cytosol extract was used to determine protein concentration using the Bio-

Rad Protein Assay kit (Bio-Rad Laboratories (Canada) Ltd., Mississauga, Ont.).

B. Crude Extracts:

Crude extraets were isolated from mandible and calvaria of 24-0Hase homozygous

mutant and heterozygous mutant mouse embryos. Tissues were collected at embryonic

day 17.5 (E17.5) as follows: 0.3g of tissue was isolated, placed directIy in liquid nitrogen

and stored at -sooe until ready for extraction. The tissue was crushed in an autoclaved

mortar and pestIe on dry ice until it was a powder. Liquid nitrogen was added to the

mortar, the powder transferred to a 7ml dounce on ice and the liquid nitrogen allowed to

evaporate off. ImL of NE2 buffer containing O.SfI, Nonidet P-40, 1110 total volume of34

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4M KCl, and 1/500 total volume of NaBisulfite was added and the mixture was dounced

severa! tilDes with pestle B until viscous. It was then transferred to an ultraelear

centrifuge tube on ice and sonicated twice for 5 seconds. The homogenate was then spun

at 24K for 1 hour at 4°C. The cmde extraet supematant was dialyzed, spun, aliquoted and

quantified as above.

C. Competition Assays:

Mandiblelcalvaria or liver, nuclear (25~g protein) or cytosol (65~g protein) extraets

(in lOO~ of NEl buffer) were incubated with InM (nuclear) or O.25nM (cytosol) eH]­24R,25(OH)2D3 (Amersham Pharmacia Biotech, Baie d'Urfe, QC) (in S~ 95% ethanol).

This was done both in the presence and absence of increasing amounts (10-200 molar fold

excess) of cold Metabolites. Non-radioactive 24R,25(08)2D3, 24S,25(OH)2D3 (the

unnatural, biologjcally inactive epimer (64, 65», 1«,25(08)2D3. or 25(0H)D3 (in SJ.1l of

95% ethanol) were used as the competitors for the eHl-24R,2S(OH)2D3 putative receptor

binding interaction. In control assays, either O.5Jlg of Gc-Globulin (Sigma Chemicals, St.

Louis, MO) with O.2Smglml BSA (66) in the same reaction volume, or 50flg of Bovine

Globulin Cohn Fraction IV (Sigma Chemicals, St. Louis, MO) in 320J.Ü fmal volume, was

used as a source of DBP. Each binding reaction was performed in duplicate.

In the ligand binding assays for determining tissue specificity and for all cmde

extraet experiments, 25~g of relevant extract was incubated with 1nM eH]­24R,2S(OH)2D3 in the presence and absence of 2S-fold molar excess of unlabeled

24R,2S(OH)2D3.

35

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The incubation tubes (I.Sml microcentrifuge tubes), containing extraet or DBP,

labeled ligand and cold competitor, were vortexed to start the binding reaction and

incubated at room temperature for 4S minutes (67). The reaction was terminated by

transferring the tubes to ice and immediately adding 400IJl of hydroxyapatite slurry (67).

The tubes were vortexed and left on ice for IS minutes with vortexing every S minutes.

The samples were then centrifuged at 13000rpm for 3 to 4 seconds in the microfuge. The

hydroxyapatite pellets were washed tbree times with SOOJ.ü of 10mM Tris/HCI-o.S%

Triton X-lOO [pH 7.5] by vortexing and then centrifuging as above. The final washed

hydroxyapatite pellets were extraeted twicc with 9OOJJ.l 2: 1 methanol:chloroform to

ensure complete removal of radioactive ligand (67). The extraction solvent was then

transferred to scintillation vials and dried under a stream of nitrogen. Econotluor-2

scintillation fluid (packard, Meriden, CI') was added to the vials and the samples counted

for 5 minutes at 57% counting efficiency using a TRI-CARB liquid scintillation analyzer

(Canberra Packard Canada, Mississauga, ON). Competition curves were then obtained

using Prism's Non-linear Regression One Site Competition equation (GraphPad Prism

Version 2.0, San Diego, CA). From the ligand binding experiments to determine tissue

specificity, maximum binding in each tissue was calculated (in fmol) and graphed as a bar

graph using Prism.

D. Saturation Analysis Experiments:

The saturation analyses were carried out using a similar method as described for the

competition assays. SJ.lg of mandiblelcalvaria (or liver) nuclear extract were incubated

with eH]-24R,2S(OHhD3 over the range of O.OS - 6.4nM, in the presence and absence of

36

•

•

•

2S-fold excess cold 24R.25(OHhD3 in a final reaction volume of 110J,Ll. AlI reactioos

were perfonned in duplicate. Separation of bound and unbound ligand was earried out

with BioGel HTPGeI hydroxyapatite (BiO-Rad Laboratories, Hercules, CA) as in the

competition assays (67). Bound ligand was counted as above, where total binding

represents bound eH]-24R,2S(OH)2D3 in the absence of non-Iabeled hormone and oon­

specifie binding represents bound eH]-24R,2S(OH)2D3 in the presence of excess non-

labeled hormone. Specifie binding was ealeulated by subtracting the non-specifie from

the total binding. The saturation curve was obtained using Prism's Non-linear Regression

One Site Binding (Hyperbola) equation.

E. Sucrose Gradient Sedimentation Experiments:

2SJ1g of liver nuelear extrael (in lOSJ1l of NEt buffer) or IJlg DBP (in lOSIJ,1 of

PBS) was incubated with toM of eHl-24R,2S(OH)2D3 (in SJ.Ll 95% EtOH) for 4S minutes

at room lemperalUre. As per modifications in the protocol of Van Baelen, et al. (68), in

tubes where actin was ta he added, 2J.lg (DBP experiments) or 501J.g (nuelear extraet

experiments) of actin from bovine muscle (Sigma, St. Louis, MO), dissolved in Hepes

buffer (SmM HePes [pH 1.5], O.lmM CaCh, O.lmM NaN3, O.2mM ATP) was used. An

equal volume of bePes buffer alone was added to tubes wbere actin was Dot ineluded. The

actin (or Hepes buffer) was added to the tubes following the 45-mînute incubation and

incubated for another 15 minutes al room temperature.

In tubes where DNase 1 was to he added, the DNase 1 from bovine pancreas

(Boehringer Mannheim, Laval, Canada) was dissolved in O.IM KeI. Once the 15

minutes incubation of actin was completed, 16J.lg (DBP experiments) or 400J,Lg (nuelear37

•

•

•

extract experiments) of DNase 1 was added to the desired tubes containing actin and

incubated for a final 15 minutes at room temperature. To the tubes not containing DNase

1. O.IM KCI was added instead. In aU tubes water made up the final reaction volume to

220J.Ù.

In the anti-actin antibody experlments, 1Of.ù of a 2001J.glml stock of anti-actin

antibody in PBS (Santa Cruz Biotechnology, Santa Cruz, CA) was added to the

incubation mixture containing nuclear extraet or DBP and actin. Finally, 5.5J,11 of 4M KCl

and water was added to the tubes to obtain a fmal volume of 22otJ.l.

Linear 5-25% sucrose density gradients (4.2ml) in 0.3M KCl-TED (O.3M KCl,

10mM Tris, 1.5mM EDTA, ImM dithiothreitol, pH 7.4) (11) were prepared using a

peristaltic pump, in 5m! ultraelear centrifuge tubes that bad been coated for 1 bour with

5% BSA and dried for 1 bour. FoUowing the incubations, aU 220fJ.l of sample was loaded

onto the sucrose gradient with a pipet and tben centrifuged at 240000g (45000rpm) for 20

hours at 4°C (68). Fractions of 4 drops were tben collected into scintillation vials from

the bottom of the tube using a peristaltic pump and counted as in the competition assays.

F. mRNA Extraction:

Mandibles and calvaria (0.59g) from twenty-four 17.5 day old mouse embryos were

extracted and immediately placed into a sterile 50ml screw cap conical tube containing

4ml GTC Extraction Buffer (4M guanidine tbiocyanate, 25mM sodium citrate [pH 7.1])

and 1641Jl of p-Mercaptoethanol. mRNA was isolated from this fresh tissue by foUowing

38

•

•

•

the large-seale protocol for mRNA isolation from tissue samples of the PolyATtraet

System 1000 kit from Promega (Promega, Madison, WI).

G. Two-Hybrid cDNA Library Construction:

Stratagene's HybriZAP-2.1 Two-Hybrid eDNA synthesis kit (Stratagene, La Jalla,

CA) was used ta eonstnlct a cDNA library for ycast two-bybrid screening. FoUowing

Stratagene's protocol, SlJ.g of the isolated mandiblelcalvaria poly(AtmRNA was used to

synthesize the fust strand cDNA, labeled with [a_32P]dCfP (800 Ci/mmol). eDNA was

size fractionated using a Sepharose CL-2B spin eolumn and the fust two fractions were

used separately to continue the procedure. The Gigapack III Gold Packaging Extraet

(Stratagene, La Jalla, CA) was used to package the two fractions, the primary libraries

were amplified, and the secondary libraries in-vivo mass excised, as per the instnletion

manual for the kit. The exeised libraries were amplified following Stratagene's protocol

and the DNA isolated by a1kaline lysis.

H. DNA·Binding Domain Vector Construction:

Stratagene's pBD-Gal4 pbagemid vector (lOJ1g) was digested with EcoRI and Pstl.

The ligand-binding domainlhinge region of buman RXRa was obtained for the bait insert

by cutting the full-Iength clone with EcoRI and Pst 1. The insert was tben subcloned iota

the digested vector and the new bait plasmid was sequeneed to ensure it was in the correct

reading frame.

39

•

•

•

1. Yeast Two-Hybrkl Screening

The prepared two-hybrid libraries were screened foUowing the Library

Transformation and Screening Protocols in Clonteeh's Matehmaker GaI4 Two-Hybrid

user manual (CLONTECH Laboratories, Palo Alto, CA) with the following

modifications: YRG-2 (Stratagene, La Jalla, CA) was the yeast host strain used for the

simultaneous co-transformation of SOOJ.t.g of library plasmid and Img of bait plasmid.

24~S(OHhD3 (lO-6M) was added to ~e transformed yeastjust before plating onto 10-

20 SD 3- plates containing S-lOmM 3-AT. For sorne screeos, the ms3 Ujump-start"

protocol was followed as per the Clontech user manual. HIS3+ clones were assayed for

Il-Gal activity using the colony-lift ~-galactosidase fliter assay (Clontech) and positive

clones grown for 1-2 days in LEU" media. The yeast plasmid DNA was isolated using

Stratagene's Isolation of Plasmid DNA From Yeast protocol (Stratagene, La Jolla, CA),

transfonned ioto XL-l Blue Mlf cells by electroporation and plated on LBIAmp plates.

Insert DNA was isolated from the colonies by alkaline lysis and sequenced using the

ThermoSequenase radiolabeled tenninator cycle sequencing kit (Amersham Phannacia

Biotech, Baie d'Urfe, QC). The sequence Was resolved on a 6% acrylamide gel (19:1 Bis

to acrylamide), analysed by EditSeq program (DNASTAR IDc, Madison, WI) and

compared to known sequences using a BLAST search of the NCBI (National Center for

Biotecbnology Information) data bank.

J. PCR ScreeniDg of the Yeast Two-Hybrid Libraries:

PCR was used as an aItemate method of screening the yeast two-hybrid libraries.

PCR reactions (SOJÙ) were set up containing 1~ of library DNA (sOOng/JÙ), S~ lOx

40

•

•

•

cloned Pfu DNA polymerase reaction buffer (Stratagene, La JoUa, CA), 4OIJ.l 820, and

IJ.Ù eacb of IOmM dNTPs, S'and 3' primers (SOpmlJ1l) and Pfu polymerase enzyme

(Stratagene, La Jolla, CA). As a negative control no DNA was added to the tube, and as a

positive control IIJl (-IOOng) of a plasmid containing a full-Iength buman vitamin D

receptor (VDR) insert was added instead of library DNA.

Analysis of the full-Iength sequence of VDR showed that it has the highest

sequence similarity with FXR (Famesoid X Receptor) and LXR (Liver X Receptor),

especially in the zinc-finger domaine Based on this, an internaI 3' primer was chosen in

an area of tbis domain where there is only a 1 bp difference between VDR and FXR and a

2 bp difference between VDR and LXR out of the 21 bp length of the primer. Thus this

primer consisted of 21 bases of the 3' end of the zinc-finger region of mVDR (S'> AAT

GTC CAC GCA GCG TIT GAG <3') (Td=70.10C). The 5' primer was either 21 bases

of the 5' end of the zinc-linger region of mVDR (S'> TGT GOA GTG TGT GOA GAC

CGA <3') (Td=68.5°C) or 21 bases of the pADGal4 vector upstream of the c10ned inserts

(5) GAT ACC CCA CCA AAC CCA AAA <3') (Td=67.8).

The peR reactions were placed in a Hybaid OmnïGene (InterSciences, Markham,

Ontario). The screening by PCR was done as follows: After a 2 min denaturation at

95°C, the cycling parameters were: 95°C for 30 s,65°, 62°, or 59°C for 1 min, and 72°C

for 1 min, for a total of 30 cycles. A finaI extension of 10 min at 72°C was added. The

PCR products were directly anaIyzed by gel electrophoresis through a 1.2% agarose gel.

Bands of interest (<300bp when using both VDR primers, and up to 3kb when using the

41

•

•

•

pADGal4 5' primer) were isolated from the gel and the DNA extraeted usmg the

QlAquick Gel Extraction Kit (Qiagen Inc., Mississauga, Ont).

The DNA of severa! bands was subcloned into TOPIO ceUs using the TOPO TA

Cloning System (Invitrogen, Carlsbad, CA). Before perfonning the TOPO Cloning and

the One Shot Transformation Reactions, the addition of 3' A-overbangs post­

amplification was perfonned as per the protocol appendix. White colonies (containing

insert) of each PCR reaction product were cultured ovemight in LB medium containing

lOOlJ,glJ.1l ampicillin and the miniprep DNA isolated using alkaline lysis. This was then

digested with Eco RI and run on a 1.2% gel. A couple of IDÏnipreps of each band were

selccted for DNA sequencing using the ThennoSequenase radiolabeled terminator cycle

sequencing kit (Amersham Pharmacia Biotech, Baie d'Urfe, QC). The sequence

reactions were loaded on a 6% acrylamide gel and the DNA sequences obtained were

subjected to a National Center for Biotechnology Information BLAST search to compare

with known sequences.

42

•

•

•

vu. RESULTS

A. Ligand Saturation Analysis of Nuclear Extrads:

The search for a nuclear receptor which binds specifically to eH]-24R,2S(OH)2D3

was based upon, among other studies, the observation that mice lacking the 24-0Hase

enzyme suffer from abnonnal development of bones formed by intramembraneous

ossification (39). Since this phenotype was observed in the mandible and calvari~ these

tissues were isolated from 17.5 day-old CS7B 16 mouse embryos, homogenized, and

purified nuclear and cytosol extracts were obtained. Saturation analysis using tritiated

24R,25(OH)2D3 over the range of O.OS-6.4nM was carried out on the nuclear extraet to

test for the presence of saturable ligand binding to a protein present in the extract. Figure

5 shows the saturation curve obtained from this experiment. As is evident in the figure,

saturable binding of eH]-24R,2S(OH)2D3 was observed in the mandiblelcalvaria nuclear

extraet. This binding is of high affmity (Kci =1.1 ::t 0.14nM) and has a Bmax of 1.79 ::t

0.08 pmoVmg proteine An identical saturation analysis experiment on liver nUclear extract

was later carried out and also demonstrated saturable binding, with a Kct =0.44 ± 0.07 DM

and a Bmax of 2.34 ±0.11 pmollmg protein (data not shown).

B. Competition Analysis of Nuclear and Cytosol Extracts and of OBP:

In arder to elucidate the ligand specificity of the nuclear extract protein binding

24R,2S(OH12D3, competition analysis experiments were conducted on both cytosol and

nuclear fractions using related vitamin D Metabolites as competitors of eH]­24R,2S(OH12D3 binding. These Don-radioactive, competitor Metabolites were: the

43

•

2000

76543

Bmax 1.79 ± O.OSpmoVmg protKd 1.1nM ±O.1nM

21O-T=---~---r-----r-----""------'r----"""-----'

o

500

al 1500c=ac_:aE.!:! e- 1000I-f/)

•Figure 5: Saturation analysis of [3H]-24R25(OH)2D3 binding bymandible/calvaria nuclear extract. Aliquots of nuclear extract (5J.1g

protein) were incubated with [3Hl-24R25(OH)2D3 over the range ofO.05-S.4nM, in the presence or absence of 25-fold excess ofnonlabeled hormone for 45min at room temperature. Specifiebinding was calculated as outlined in Materials and Methods.

•

•

•

•

synthetic epimer 24S,2S(OHhI>], la,2S(OHhDJ, and 25(OH)~, in addition to unlabeled

24R,25(OHhD3. Figures 6a and b show the competition curves for eHl-24R,2S(OH)2D3

binding in mandiblelcalvaria nuclear and cytosol extraets, respectively. In figure 6a, it is

evident that non-radioactive 24R,2S(OH)2D3 competes Most effectively with the labeled

ligand for binding to the nuclear protein and thus has the highest affinity for the binding

sites compared to the other vitamin D3 metabolites. At only a 2S-fold molar excess

([competitorl =2S DM) of cold 24R,2S(OHhD3, the specific binding of labeled ligand to

the protein drops to 6%, compared to the 33-38% specific binding seen with competition

by the same excess of either 24S,2S(OHhD3 or 25(0H)03. These latter two Metabolites

were equal in tbeir ability to displace the eHl-24R,2S(OHhD3 from the binding protein.

Finally, la,25(OH)2D3 competes only minimally for binding, even at 200-fold molar

excess ([competitor] = 200nM). This specificity of the 24R,2S(OHhD3 binding protein in

the nuclear extraet suggests that it is not the vitamin D receptor (VDR) since

1a,25(OHh03 would have had the highest affmity for the VDR (27, 28, 69), which is

clearly not the situation.

As seen in figure 6b, similar trends appear in the cytosol extraet, though in aU cases,

there is somewhat less competition tbroughout the curves (e.g. at So-fold molar excess of

unlabeled 24R,2S(OH)203 specific binding is less than 5% in the nuclear extraet whereas

it is 29% in the cytosol extraet). Finally, nuclear and cytosol extraets were a1so isolated

from the livers of the same 17.5 day-old embryos and competition assays perfonned on

these. Ligand binding was observed, and the competition assays elicited a similar profùe

to those of the bane extraets (figure 6c).

44

•100

6

75mc:cc:a • 24R2503u

i 50 0 24825036 1a25D3

(1)25(OH)03

~A

0

25

•00.0 0.5 1.0 1.5 2.0 2.5

log (competitor]

Figure 6a: Specificity of [ 3Hl-24R25(OH)2D3 binding tomandible/calvaria nuclear extract. 251J,g of nuclear extract protein was

incubated for 45min at room temperature with 1nM eHl-24R25(OH)203and various concentrations (1o-200nM) of unlabeled vitamin 03 analogs.

•

•

Cft 75c:acliu;; 50"ü8.fi) •fi. 24R25D325 •

0 2482503• --6-1a25D3• 25(OH)03

a0.0 0.5 1.0 1.5 2.0

log [competitor]

Figure 6b: Specificity of [ 3H]-24R25(OH)2D3 binding tomandible/calvaria cytosol extract. 65J.1Q of cytosol extract proteinwas incubated for 45min at room temperature with O.25nM

[3H]-24R25(OH)2D3 and various concentratoins (2.5-50nM) ofunlabeled vitamin 03 analogs.

•

•100r---"2r'"----..A..-_

en 75c:ac 24R2503:s •() 0 24S2503;;: 50 1Œ25D3·ü 6

B- .6 25OHD3en';R.0

25•o-l------'T-----~----=:::;:=~I-------,0.0 0.5 1.0 1.5 2.0

log [competitor]

Figure 6e: Specificity of [3Hl-24R25(OH)203 binding to Iivernuclear extract. 251J,9 of nuclear extract protein was incubated for

45min at room temperature with 1nM [3H]-24R25(OH)203 and variousconcentrations (2.5-50nM) of unlabeled vitamin 03 analogs.

•

•

•

•

ln order to determine if there was a difference in the amount of binding seen in

heterozygous versus homozygous 24-0Hase knockout mice, crode extraets were obtained

from the mandible and calvaria of bo~ as explained in Methods. Traditional ligand

binding assays, using 25J1g of extraet, tnM of eHl-24R,2S(OH)2D3, and the presence or

absence of 25-fold excess cold 24R,2S(OH)2OJ, were Performed on these extracts.

Specific binding was calculated in fmollmg protein and compared between both

genotyPes. The heterozygous mouse embryo crode extract bound 748 ± Il fmol

ligand/mg protein while the homozygous extraet bound less than half of that at 336 ± 9

fmolligand/mg protein (data not shown).

It is weU known that 24R,2S(OH12D3 can bind to other proteins (e.g. VDR and

OBP) (7, 69). DBP (vitamin D binding protein) acts as a carrier protein for 2S(OH)D3 in

the circulation (7). It is possible that DBP could bave been present in the nuclear extract

due to contamination from blood, though this is unlikely since the nuclei were washed

weIl before lysing. Nevenheless, it was necessary to mie out the POssibility that the

protein in the extraet that was binding the ligand was DBP. Thus competition assays

were conducted using DBP as the binding protein, and the same metabolites as

competitors, with the intention of comparing the competition profIles of the two proteins.

lnitially, Bovine Globulin Cohn Fraction IV (Sigma Chemicals, St Louis, MO),

known to bind 24R,2S(0H)2D3 specifically, was used as a source of DBP to carry out the

experiment. Figure 7a shows that the order of competition in this case was different from

that obtained when using the nuclear extraet. Instead of a noticeably more effective

competition by 24R,25(OH)2D3 as seen with the extraet, when using DBP

24R,25(OH)2D3 and 25(0H)D3 competed equally with the labeled ligand, followed by

4S

•

• 24 5 3

o 24S25034 la25D3A 25(OH)D3

25

100r- ~

•O.....l...----r"------~-----__r_-----____,

0.0 0.5 1.0

log [competitor]

1.5

Figure 78: Specificity of [3H]-24R25(OH)2D3 binding to bovineGlobulin Cohn Fraction IV (Sigma) as a source of DBP. 50J,lQ ofDBP was incubated for 45min at room temperature with O.OSnM

eH]-24R25(OH)203 and various concentrations (O.S·17nM) ofvitamin 03 analogs.

•

•

en 75c:gc:s •u

i 50

en • 24R250afi!. 0 245250a

25 6 1a25D3

• 25(OH)Da• 01.0 1.5 2.0

log [competitor]

Figure 7b: Specificity of [ 3H]-24R25(OH)2Da binding toGe Globulin (Sigma) as a source of DBP. 0.51-19 of OBPwas incubated for 45min at room temperature with O.25nM[3H]-24R25(OH)20a and various concentrations (6.25­1OOnM) of unlabeled vitamin Da analogs.

•

•

•

•

24S,25(01{h03 and finaUy la,25(0H)20J. The OBP competition profile obtained here

agrees with one reported arder of binding affinities for sterols by DBP, which is:

25(OH)DJ =24R,25(0H)203 > 1«,25(08)203 (7, 9). This suggests that OBP and the

protein in the nuclear extraet are not the same protein sincc they elicit a different

competition profile.

Other investigators (7, 10, 68) have used Sigma's Gc-Globulin (Sigma Chemicals,

St. Louis, MO) as a source of DBP. In arder to verify their results we repeated the

competition experiments using this agent. However, as cao he seen in figure Th, very

little competition was obtained even when increasing to 400-fold excess. Although the

findings of the fmt experiment suggested that the protein in the nuclear extract binding

the ligand was not DBP, since reports on the order of sterol affinities for DBP are

conflicting (7, 9, 10, 12), as were our own observations, more convincing evidence was

necessary to determine whether or not OBP was binding the 24R,2S(OHhD3 in the

mandiblelcalvaria nucIear extraets.

c. Sucrose Gradient Sedimentation 01 Nuclear Extracts:

To further ensure that the binding observed in the extracts was not due to

contamination by DBP present in semm, an approach was taken that exploits the

interactive propenies of DBP, actin and DNase 1 (7, 68). Using a modified version of

Van Baelen' s approach (68), a sedimentation profile of DBP in a sucrose gradient system

was obtained. Figure 8a shows that when DBP a10ne was incubated with eH]­24R,25(OHhD3 the labeled DBP elution peaked al fraction 18. Upon addition of actin,

this peak shifted to fraction Il. This indicates that the actin bound to the DBP (68),

46

•

•

S-u

1500

1000

500

-M-OBP--0- OBP+Actin-e- OBP+Actin+DNase1

403020

fraction number

10o-l-i~~~~~~~~~..,....-r-"""""""""'I"""""-.--r"~--r-1~I""""T"'""'f"""'T"-'f"""'T"-~

o

Figure 8a: Complex formation between DBP, actin and DNase 1. 11J,g of

DBP (Gc Globulin, Sigma) was incubated with 1nM eHl-24R25(OH)2D3 for45 min at room temperature. The 220).11 samples were layered onto 50/0-250/0sucrase gradients prepared in 0.3M KCI-TED butter and spun @ 240000gfor 20h. Fractions were collected using a peristaltic pump. Fraction 1 =bottom of the tube. DBP (1IJ,g), Actin (2IJ,g), DNase 1(16IJ,g) .

•

•1200

4030100~;"";";-'T'"""'f"-r-.,....,.........-P"""'T"""'P-"I""--""""'_---"--"'-T"""T"~r""'"r'""T""""I'-T""""I""""T'~t""""T".......-r--T""""""'''''--'''''''''

o

1000 ~NUC

~NUC+Aetin

800 ~ NUC+DNase1

S. 600u

400

• 200

Figure Sb: Sucrose gradient sedimentation of liver nuclear extract after

labeling with 1nM [3H]-24R25(OH)2D3' NUC (25Jlg), Actin (SOJlg), DNase 1(400Jlg). Abbreviations: NUC; liver nuclear extract.

•

•

•

•

making it heavier 50 that il migrated furtber down the tube during the spin and was eluted

in an earlier fraction when drops were collected from the bottom of the tube. Also in

accordance with Van Baelen's observations, the eH]-24R,2S(OHhDJ-DBP peak was

further shifted to an earlier fraction towards the bottom of the tube (fraction 8) with the

addition of both actin and DNase 1, indicating DBP formed a trimer with tbese

compounds (68).

To compare the sedimentation profile of the nuclear extract with that of DBP, the

experiment was repeated onder the same conditions, using 2SJ1g of liver nuclear extraet

instead of DBP. Sïnce previous competition and saturation experiments with liver

nuclear extract showed tbe same propertîes as the mandiblelcalvaria extract, which was in

short supply, liver extraet was used in these experiments. When the nuclear extraet alone

was incubated with eH]-24R,2S(OH)2DJ the bound protein elution peaked at fraction 12

(figure Sb). This eH]-24R,2S(OH)21>]-nuclear protein peak did not shift towards an

earlier fraction upon addition of exogenous actin alone or actin and ONase 1 (figure 8b).

These results show that the protein in the nuclear extraet that is binding eH]­

24R,25(OH)20J does not complex with actin or with DNase 1 and suggest that the nuclear

24R,25(OH)2D) binding activity is not due to OBP.

To provide evidence against the possibility that the binding activity was due to DBP

complexed with tissue-derived actin (11), another sedimentation experiment was

perfonned using anti-actin antibodies. This would determine if tissue-derived actin was

present in the sample or not. Anti-actin antibodies would bind to actin present in the

sample and the eH]-24R,2S(OHhD3-nuclear protein-actin peak would shift to an earlier

47

•

•

•

fraction. If no actin was binding the nuclear protein in the sample then no shift would he

seen.

Figure 9a is a conttol experiment using DBP and exogenously added actin to ensure

correct conditions for the anti-actin antibodics. DBP atone elutes neac fraction 18. The

addition of anti-actin antibody to the DBP alone does not cause a shift in the elution~

as expected. When actin and DBP are present in the tube, the elution peak is shifted to

the left (fraction 15). Finally, wben DBP, actin and anti-actin antibody are incubated

together, the elution peak shifts further to the left to fraction Il. This indicates that the

antibody is binding to the DBP/actin dîmer, making the complex heavier so that it elutes

in an earlier fraction.

Next, the experiment was repeated using the nuclear extract. Figure 9b shows that

the elution of the protein in the nuclear extraet alone, when incubated with eH]­24R,25(OH)2D3, peaks at fraction 12. When anti-actin antibody is added to the nuclear

extract the elution peak does not shift to an earlier fraction, suggesting there was no actin

hound to the nuclear proteine This was perfonned in duplicate. This provides convincing

evidence that the nuclear protein binding eHl-24R,25(OH)2D3 is not DBP complexed

with endogenous actin.

D. Tissue Speclficlty:

In order to test for tissue specificity of the eH]-24R,25(OH)2D3 binding protein

observed in the mandiblelcalvaria nuclear extraets, ligand binding assays were also

Performed on isolated liver and brain nuclear extraets of the same mouse embryos, using

25J1g protein and loM labeled ligand. The resulls, summarized in figure 10, indicate that

48

•1750

•

1500

1250

i 1000

u750

500

250

~DBP

~DBP+ab

-+- DBP+actin~ DBP+actin+ab

403020

fraction number

10o~_"~i:t:~~~,....-r-"'l~--r-T"""T""""'I"-r-T''''''''''''''''''''''''~::!~~~

o

Figure 9a: Complex formation between DBP, actin and anti-actin

antibody. OBP was incubated with 1nM [3H]-24R25(OH)2D3 for 45min atroom temperature. The final 220J,ll samples were layered onto 5-25%sucrase gradients prepared in O.3M KCI-TED buffer and spun @240000g for 20h. Fractions were collected using a peristaltic pump.Fraction 1 = bottom of the tube. DBP (1IJ.Q). actin (2 IJ.g). ab (2 J.lg).Abbreviations: ab; anti-actin antibody.

•

•

504020 30

fraction number

10O-+-r...........~.,...,.....,...,..."I""""'I"""I""""I"~ .......... "I""""'I".,........~ .......................r--r-r""'T""'r"""I"""............,........~..........r--r-....................'Y""'T"""I

o

200

1000

800 ~nuc

-<>- nuc+ab-4- nuc+ab (2)

600

S-u

400

•

Figure 9b: Sucrose gradient sedimentation profile of 25J.lQ liver nuclearextract incubated with 1nM [3H]-24R25(OH)2D3 and anti-actin antibody toensure the absence of tissue-derived actin in the extract. Duplicates wereperformed with the antibody. NUC (25J.1Q), ab (2f.1g). Abbreviations: NUC;Iiver nuclear extract, ab; anti-actin antibody.

•

•

12.5

10.0

'0C~0,g 7.5

CI):::::'"

Q 0LI) EN:e,a:::~ 5.0~%

CI)

• 2.5

0.0....1.---bone liver brain

•

Figure 10: Tissue specificity of the protein binding eH]-24A25(OH)2D3 in

nuclear extracts. Maximum fmol of [3H]-24A25(OH)2D3 bound by 25f.1Q ofnuclear extract protein from 17.5 day old mouse embryo bone, liver, andbrain.

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there is very little binding in brain extraets (60.0 ± 2.0 fmol/mg nuclear protein),

compared with that of bone (304 ± 16 fmoVmg nuclear protein). Furthermore, liver

produced a maximal binding of (432 ± IOfmoVmg nuclear protein), even greater than that

of bone. This data suggests some degree of tissue specificity of the protein expression.

E. Yeast Two-Hybrid Screening:

The yeast two-hybrid strategy for cloning the putative 24~2S(OH)2D3 receptor was

based on the premise that since 24R,2S(OH)203 and la,2S(OH)2D3 are functionally and

structurally related ligands, the putative 24R,25(OH)203 receptor may he of the same

class as the vitamin D receptor (VDR) that binds la,25(OH)2D3. As a member of the

Class fi nuclear hormone receptors, VDR heterodimerizes with the retinoid X receptor

(RXR) (70-73). On this basis we constructed a bait protein consisting of the hRXRa

ligand-binding domain fused in frame with the GAL4 DNA-binding domain in the pBD­

GAL4 yeast expression vector. As mentioned previously, this ligand-binding domain

(LBD) of the receptor contains the dimerization interface. This bait was used to screen

two cDNA libraries fused to the GAlA activation domain in the pAD-GAIA vector: a one

week-old mouse calvaria library, and a 17.S-day-old embryonal mouse mandiblelcalvaria

library. After verification of the size of the inserts in the libraries it was determined that

the average insert size was between SOObp - 1.Skh, though inserts ranging to 5kb were

obtained.

The yeast two-hybrid screens of the post-natal library repeatedly pulled out a

-1.7kb insert whicb turned out to be the mouse peroxisome proliferator-activated receptor

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gamma (pPARy). This is anather member of the class n nuclear bormone receptors and

is known ta interact with RXR (74). This finding demonstrated that the tools and the

methodology used were functional. In addition, non-specific artifacts~ such as mouse

osteopontin~ collagen~ osteonectin and ribosomal proteins were also pulled out of bath the

post-natallibrary and initial screenings of the embryonic library. These false-positives

are one of the major drawbacks of the yeast two-bybrid system. As of yet~ no potential

positive clones bave been identified. Continued screening of the embryonic library is

underway.

F. PCR ScreeniDg of the Yeast Two-Bybrld Libraries:

The rationale for using a PCR-based strategy for attempting to clone the putative

24R,25(OH)2D3 receptor is based on the fact tbat the DNA-binding domains of nuclear

hormone receptors are highly conserved and contain two zinc-fmger motifs (22). Thus by

PCR-amplifying the cDNA libraries using primers from the zinc-fmger region of the

VDR and lower annealing temperatures, il sbould he possible ta amplify fragments in the

library containing similar zinc-finger regions. In addition ta potentially identifying such

transcripts, this strategy may also pull out the VDR since the primers were generated

from its sequence. The PCR screens, performed onder the parameters outlined in

Materials and Methods section~ produced four bands. Deposition of their sequences in

the NCBI <1ata bank ÏDdicated that they were artifacts. Further screens altering the PCR

parameters and/or cbanging the primers will continue to he carried out.

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VOL DISCUSSION

Many previous studies bave indicated a role for 24R.2S(OHhD3 in bone function

(35-39,45-47, 51, 54, 64, 75, 76). Still others have suggested the existence of receptors

for this vitamin D Metabolite (56, 58, 59). However, the evidence to date bas been

inconclusive and therefore the exact biological role and the mecbanism of action of

24R,25(OH)2DJ have not yet been elucidated. The purpose of this study was to provide

evidence for the presence of, and try to isolate, a putative receptor for 24R,2S­

dihydroxyvitamin D3 in developing bone.

Recent evidence in our own laboratory bas demonstrated that mice lacking the 24­

OHase enzyme (and consequently, vitamin D metabolites hydroxylated at the 24­

position) suffer from abnonnal development of bones formed by intramembraneous

ossification, such as the mandible, calvaria, and clavicle (figure 4) (20, 39). These results

support the notion that 24R,25(OHhD3 does bave an important fonction in bane, that

receptors mediating its function could exist, and that a probable sile of their expression

would he intramembraneous bane.

Based on these observations, we bave isolated nuclear and cytosol exttacts from

mandiblelcalvaria tissue of 17.5 day-old CS7B16 mouse embryos in an attempt to identify

binding of 24R,2S(OHhD] to a protein in this tissue using ligand binding studies.

A. Ligand Saturation ADalysis of Nudear Extracts:

Saturation analysis is used to determine if, with a given amount of protein and the

addition of increasing amounts of ligand, binding of ligand saturates as all the available

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receptor molecules become occupied. This experiment demonstrated the presence of

saturable binding of eHl-24R925(OHhD3 to a protein in the nuclear extraet. This high

affinity binding <Kd = 1.1 ± O.14nM) strongly supports the notion that there is a

24R,25(OH)2D3 receptorlbinding protein in the nuclear extraet and that the number of

these receptors available for ligand binding is finite and abundant (Bmu = 1.79pmoVmg

protein). These values compare with the~ (1.79nM) and Bmax (2.79pmoVmg protein) of

a putative cytoplasmic 24~25(OH)2D3 receptor described by Somjen et al. (56), but not

with those (1Cd = 15.3nM, Bmax = 43.9 fInol/mg) of a putative membrane receptor

described by Kato et al. (11). This could suggest that different fractions (i.e. nuclear,

cytosol, membrane) or possibly even different tissues, may contain unique

24R,25(OH)2D3 receptors for separate functions. Altematively, variations observed in the

abundance of the receptor could suggest that certain tissues (with higher Bmu) are more

highly responsive to 24R92S(OH)2D3 than those with lower levels ofexpression (26).

The circulating concentration of 24R,2S(OH)2D3 is reported to he hetween loM

(chick) (37) and 6nM (human) (14). This is in the same order of magnitude as the Kct of

the putative receptor (1.1nM) and MaY not cause a problem. However, it could be argued

that, with a circulating 24R,25(OHhOJ concentration of as mucb as 6nM and a receptor

with a dissociation constant of ooly 1.1nM, all available receptors could he occupied all

of the lime and there would he no on/off mecbanism. One possible explanation is that

sorne of the circulating 24R,2S(OH)2D3 is bound to DBP and is thus not available to bind

the putative 24R,25(OH)2OJ receptor. Another possibility could he that 24R925(OH)2D3

is acting via an intraerine mecbanism, wberebya specific ceU produces 24R,2S(OH)2D3

which binds the receptor within the same celle In sucb a case, the receptor would not he

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exposed to the higher circulating levels of 24R,2S(OHh~. There is evidence of such an

intraerine action of la,25(OHh~with the VDR in a chick myelomonocytic œil line

(77). As weU, there are reports of similar intraerine and autocrine functions for other

nuclear honnone receptors including the androgen receptor, AR (78-80) and the

progesterone receptor, PR (81, 82). Ooly once it is possible to determine if the putative

receptor localizes to specific ceU types known to produce 24R,2S(0H)2DJ will it he

feasible to test this hypothesis.

B. Competition Analysis of Nuclear and Cytosol Extracts and of DBP:

It is known that the vitamin D receptor (VDR) binds lo.,2S(0H)2D3 with highest

affinity and exquisite specificity. However, with 500-1000 fold lower binding affinity, it

can also bind other vitamin D Metabolites, including 24R,2S(OH)2DJ (27, 28). Moreover,

it is also well documented that plasma vitamin D binding protein (DBP), which circulates

in the blood at very high titers (5 x 10-6 M), is also capable of binding 24R,25(OH)2DJ (7,

9, 10, 12). Therefore it remains crucial to determine the ligand specificity of this

24R,25(OH)2D3 receptorlbinding protein and to eliminate the possibility that the binding

seen in the saturation experiments was due to either the VDR or DBP.

In order to do this, ligand competition assays using structurally related vitamin D

Metabolites as competitors were carried out. Assays performed on nuclear and cytosoI

extracts exhibited similar competition profiles, though competition was comparatively

less in cytosol than in nuclear extraets. This does not necessarily signify that there is

another unique receptor for 24R,2S(OHhDJ in cytosol, but could conftrm the notion that

nuclear hormone receptors are found in the cytosol as weU as in the nucleus (83, 84).

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Altematively, the lower level of competition seen in the cytosol could be due to the

presence of DBP since the cytosol fraction did not have washes, incœasing the possibility

of contamination with blood. The contaminating DBP could he binding some of the cold

metabolites, theœby leaving less available to compete for binding to the protein.

Another possible explanation for the differing levels of competition between

nuclear and cytosol fractions involves complex formation of nuclear honnone receptors

with accessory factors in the cytoplasm. It is reported that Many nuclear hormone

receptors (e.g. GR, PR, ER, but not TR, RXR or VDR) are found in the cytoplasm

complexed to accessory proteins (83-87). Tbese bave been characterized as 9 S

complexes consisting of the receptor bound with a 90kDa beat shock protein, hsp90

(and/or other accessory factors) and are mostly observed in cytosol fractions (83, 88).

This is in contrast to the 4 S receptor in the nucleus. However, there are significant

differences between receptors in this family with respect to: 1) their ability to bind hsp90

and/or other proteins, 2) where this complex is found (for some receptors (e.g. AR and

ER) the 9 S receptor complex is thought to he mainly in the nucleus in intact cells,

leaking out into the cytoplasm only upon celllysis), and 3) its effect on the affmity of the

receptor for its ligand (84, 88). Tberefore, it remains possible that the lower affinity of

the putative 24R,25(OH)2D3 binding protein for the ligand seen in the cytosol extract

(figure 6b) could he due to the receptor heing complexed to hsp90 or other accessory

factors. To determine if the cytosol protein is complexed to other factors sucrose

sedimentation experiments cao he carried out on both the mandiblelca1varia cytosol and

nuclear extraets. The profl1es elicited from these can men he compared. A sbift of the

peak in the cytosol extract to an earlier fraction (i.e. towards the bottom of the tube)

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compared with that of the nuclear extraet would indieate that the cytosol binding protein

was complexed to other factors, making it beavier.

In both the cytosol and nuclear fractioDS, the putative receptor clearly demonstrated

highest binding affmity for 24~25(OH)2D3 (figures 6a and b). An important result

obtained from these experiments was tbat the putative receptor showed little competition

from 1a,25(OH)2D3. This indicates the receptor had a low aftmity for la,25(OHhD3. If

the protein in the extraet was the vitamin D receptor, one would expect la,25(OHhD3 to

compete most effectively for the binding sites, compared to the other metabolites. As this

is clearly not the case, these results therefore provide unequivocal evidence that the

protein binding 24R,25(OHhD3 in the extracts is not VDR. Identical competition assays

and saturation analyses were carried out using the liver nuclear extraet as weil (figure 6c).

Since these elicited similar proftles as the bone extract we concluded that the two tissues

contained the same receptor and therefore could he used interchangeably.

Vitamin D binding protein (DBP), which is known to bind circulating 25(OH)DJ, is

present in high amounts in serum (7). Though contamination of the nuclear extract is

unlikely because of the washes performed in its isolation, it remained important to rule

out the possibility of DBP being responsible for the 24R,2S(OH)2D3 binding seen in this

extract. An attempt was made to do this by carrying out similar competition assays using

DBP instead of extract with the intention of comparing the two binding profiles. In the

frnt of these experiments, using bovine globulin Cohn fraction IV as the source of DBP,

the DBP did not appear to distinguish between 24R,2S(OH)2D3 and 2S(OH)D3, whereas

the nuclear extraet protein had a distinctly higher affinity for 24R,2S(OH)2D3. This was

in agreement with some published reports on DBP (7, 9) and suggests that DBP and the

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nuclear protein are not one and the same. However, as mentioned in Resolts, a suitable

competition assay could not he obtained when using Gc-Globulin as the source of DBP

(figure Th), even at 400-fold excess of cold metabolite. This could bave been because too

little of the protein wu used or too much labeled ligand prevented competition by the

cold metabolites. Since this conttoversy prevented elimination of the plssibility of DBP

involvemen4 more convincing evidence from alternative experiments was necessary.

c. Sucrose Gradient Sedimentation of Nudear Extracts:

The strategy used to determine the presence or absence of DBP in the nuclear

extract was based upon observations tirst described by Van Baelen, et al. (68). The

investigators used sucrose gradient sedimentation to detennine that DBP, actin and

DNase 1 can fonn a trimeric complex that cao he viewed as a shift in the elution peak in

sedimentation experiments. Based on this we compared sedimentation proftles elicited

from DBP with those obtained from the nuclear extraets that we prepared.

The sedimentation constants for DBP, DBP + actin, and DBP + actin + DNase 1 are

4.1 S, 5.8 S, and 7.0 S respectively (68, 89). Since the nuclear protein elutes al a peak

between those of DBP a10ne and DBP + aclin, and assuming a linear relationship, an

estimated sedimentation value of s.s S can he given for the nuclear binding proteine A

more accurate value could he assigned by using known standards in the sante tube as the

sample. This would eliminate error due to slight variations in the gradients as well as in

the number of fractions collected. Again it remains possible that this value is actually that

of the receptor complexed to an accessory factor aod not the value of the receptor alone.

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The shifts that occur to the DBP peak upon addition of actin alone or actin + DNase

1 (figure Sa) are not seen with the nuclear extraet peak upon tbese same additions (figure

Sb), suggesting tbat the protein responsible for 24R,2S(OHh1>J binding in the nuclear

extract is not DBP. Only one doubt remained. In a similar experiment by Kato et al. (11)

it was shown that the reason for the lack of shift upon addition of external actin was the

presence of tissue-derived actin already hound to the protein in the sample (which

therefore was confirmed to he DBP). In addition, as is evident in figure Sb, the peak

obtained upon the addition of DNase 1 and actin to the nuclear extraet was not as clean as

the others (probably due to a disturbance of the sucrose gradient during the experiment).

For these reasons, a final experiment was performed in order to be absolutely sure that

DBP was not responsible for the ligand binding seen in the nuclear extraets.

Exploiting the same principles of complex formation, anti-actin antibody was added

to the nuclear extraet tube and layered on a sucrose gradient to try to detect the presence

of tissue-derived actin. In this experiment, if a shift of the eHl-24R,2S(OH)2D3-nuclear

extraet peak towards an earlier fraction was seeo, compared to that of ouclear extract

alone, it would be an indication that tissue-derived actin was hound to the protein in the

ouclear extraet. This would then strongly suggest that the protein binding the ligand was

DBP. However, as shown in figure 9b, wc did not observe a shift of the peak. This

therefore indicates tbat the lack of shift seen upon addition of actin to the nuclear extraet

iD the fust set of sedimentation experiments was not due to the presence of tissue-derived

actin, which further supports the evidence that the 24R,2S(OH)21>J-binding protein in the

nuclear extraet is not DBP.

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Absolute, unequivocal praof of this cao only he obtained by using nuclear extraets

Crom DBP knockout mice ta do the ligand binding experiments (90). If the nuclear

extract of these mice no longer binds the eHl-24R,2S(OHhD3 this would he evidence

that, in the normal mice, the binding was due to DBP and not an independent receptor for

24R,25(OHhD3. On the other hand, the presence of binding in the DBP knockout mice

would provide unequivocal evidence that DBP is not a factor. These experiments are

currently being arranged, using the DBP knockout mice from Dr. Nancy Cooke (90).

Taken together, these ligand binding assays strongiy support the existence of a

specific receptorlbinding protein for 24R,25(OH)2D3 in developing intramembraneous

bone. Furthennore, we have proven that this putative receptor is different from the

vitamin D receptor (VDR) and seems not to he the vitamin D binding protein (CBP).

D. Tissue Specificity:

Since the phenotype observed in the 24-0Hase knockout mice occurred in bone

(specifically in those formed by intramembraneous ossification) this suggested a possible

site of receptor expression. However, an interesting question to pose was that of tissue

specificity. In arder to detennine ü this putative receptor was specifie to bone or ü it was

also present in other tissues, nuelear and cytosol extracts were obtained from the livers

and brains of the same 17.5 day-old mouse embryos. Ligand binding assays were tben

carried out to detennine if 24R,25(0H)2D3 binding could he detected in either of these

tissues. Using identieal amounts of nuclear extraet protein (2SJ,lg), identical

•concentrations of labeled ligand (toM) and 2S-fold molar excess of unlabeled

24R,25(0H)2D3 (2SnM), ligand hound (in finol) was calculated and compared between

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tissues. It is impottant to note tbat these maximum ligand binding values do not represent

the number of binding sites present (as the SIDa values from the saturation analyses do)

since these experiments are carried out in sub-saturating conditions.

As is evident in figure 10. brain tissue at this stage of development did not show

much binding in comparison to bone, indicating it is unlikely to he a site of expression of

the putative receptor. A literature searcb failed to find reports wbere fonction of

24R,25(OHhD3 bas been described in the brain. However, the level of binding seen in

liver was even greater than in bone. suggesting that there is high expression of the

putative receptor in tbis tissue. This observation is of particular interest since the

presence of putative 24R,25(OHhD3 receptors in the liver could suggest another possible

site of action for 24R,25(OHhD3. The livers of the 24-0Hase knockout mice bave never

been histologically examin~ nor bas the cause of death been detennined since it is

unlikely that these mice are dying from their bone abnonnalities. It is possible that a lack

of 24R,25(OH)2D3 during development could lead to abnormalities in the liver. possibly

severe enough to cause death. Livers from tbese mice are currently heing barvested and

examined by histological techniques for any possible abnormalities to detennine if this is

the case.

Anotber area of interest that would warrant looking into, would he sites of

endochondral ossification. Past reports have indicated endochondral bone as a target for

24R,25(OH)2D3 fonction and a candidate for receptors (35. 36. 38, 56, 64, 75). However,

this contradicts the evidence obtained in our laboratory, which shows tbat endocbondral

bone formation appears nonnal in the 24-0Hase mockout mice (20, 39). In addition.

many of tbese former experiments used long bones (such as tibiae and femur) which have

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an exocortical surface (periosteum) that is fonned by intramembraneous ossification (20)

and could affect the results. Ligand binding assays on nuclear extraets isolated from

growth plates at the same stage of development could address this controversy.

E. Yeast Two-Hybrid ScreeniDg:

The above ligand binding studies have provided strong evidence that a

receptorlbinding protein for 24~2S(OH)2D3 is expressed in developing

intramembraneous bone. The task remained to clone it. The yeast two-hybrid system

was chosen for this purpose because there is documented use of this strategy for pulling

out new nuclear hormone receptors (60-62). In addition, the system has been used with

success in our own laboratory and many of the tools (the kits, the reagents, the yeast etc.)

were already available. Furthermore, a one-week-old mouse calvaria osteoblast cDNA

library fused to the Gal4 activation domain in Stratagene's pAD-Gal4 vector was already

constructed. This library was used for initial yeast two-hybrid screening using the

hRXRa(LBD)-pBDGal4 bait.

This strategy repeatedly pulled out a -1.7kb insert encoding for the mouse

peroxisome proliferator-activated receptor gamma (mPPARy). PPARyis a known class n

member of the nuclear hormone receptor superfamily that heterodimerizes with RXRa

(74). Thus, this was an encouraging result as it indicated that the sttategy and tools heing

employed were functional. However, due to the unusually high nombers of this insert

pulled out over several screens, it was suspected that during amplification of the excised

library a misrepresentation of inserts bad occurred leading to its over-representation.

Unfortunately, re-excision and re-amplification of the library faile<! to correct this

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problem and mPPARy was pulled out severa! more tilDes. This strategy eould also he

expected to identify other known RXR-interacting partners present in bane (sueh as VDR,

RXR or RAR). RXR was not isolated since it ooly homodimerizes in the presence of its

own ligand and these experiments were performed using 24R,25(0H)2D3, not 9-cis

retinoic acid as the ligand. Neither VDR Dor RAR were identified in the screens,

suggesting that their representation was low in the screened libraries. In addition to

ppARy, non-specifie artifacts sueh as mouse collagen, osteopontin, and ribosomal

proteins were pulled out. Sueh '-ralse-positives" are a common problem with this system

and certain proteins seem to he pieked up more often than others (e.g. ribosomal proteins,

collagen-related proteins, heat shock proteins, mitochondrial proteins etc.) (91). This

could he because these proteins may have surfaces (such a large hydrophobie surfaces)

that have low affinity for Many different proteins, alIowing them to form complexes

strong enough to he detected in the yeast two-hybrid system (92). These false positives

remain one of the system's major drawbacks.

The bone phenotyPe of the 24-0Hase knockout mice was evident during embryonic

development (20, 39). In addition, it is unknown if the expression of this putative receptor

would he tumed off during development, preventing its c1oning. On this basis, it was

decided to conslIUct another eDNA library from mRNA of the mandible and ealvaria of

17.5 day-old mouse embryos, since allligand binding assays were perfonned al this age.

Screening of this library has so far ooly isolated other non-specifie proteins that seem to

he sticking to the bait.

A Jack of isolation of a potential clone al this stage does not negate the existence of

a receptor. There are several possible explanatioDs as to why this method May not he

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picking up the desired clone (or other known clones besides ppARy, such as VDR or

RAR). The fust is that Many nuclear hormone receptors have very long 3' untranslated

regions (3' UTR's). Stratagene's Two-Hybrid cDNA sYDthesis kit uses an oligo (dT)

primer to convert the poly(A)+mRNA ioto the first-strand cDNA. If the receptor has a

long 3'UTR it is possible that the reverse transcriptase enzyme was interropted during the

reverse transcription before it reached the region of the gene encoding the dimerization

domain. If this is the case, either no fusion protein is created or it MaY not include the

necessary residues for an interaction with the bait (93). Thus, even though the mRNA

transcript for the putative receptor may have been present, it would not be detected in the

yeast two-hybrid library. The isolation of ppARy, which contains a long 3' UTR, as weil

as the presence of long inserts in our libraries are good indications that this may not he a

problem in our case. Other possibilities are that the gene for the putative receptor is not

present in the correct reading frame in the GAlA-AD library or tbat the hybrid proteins

are not stable in the yeast. preventing detection of an interaction (93). Finally, it remains

possible that the receptor is not a class II nuclear hormone receptor and doesn't interact

with RXR. in which case the yeast two-hybrid strategy using RXR as a bait would not

pull out the clone.

An interesting question to address was that of possible upregulation of the receptor

in mutant mice homozygous for the 24-0Hase gene knockoUL If, in resPOnse to a lack of

its ligand. the putative receptor becomes upregulated (in a similar fashion to some

enzyme/substrate reactions), then it would he wortbwhile to constnlct a new bybrid

library from these homozygous mutant mice (witb a higher receptor representation) in

order to increase our chances of cloning it. Firstly, in order to detennine if the receptor is

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upregulated in a situation where there is a Jack of ligan~ cmde extraets were obtained

from mandible and calvaria of both the homozygous and heterozygous mutant mice.

Ligand binding assays were perfonned on bath as a measure of receptor levels. Binding

of the labeled ligand (in fmollmg protein) in the homozygous extract was less than balf of

that obtained from the heterozygous extraet (336 ± 9 finol/mg protein vs. 748 ± Il

finol/mg protein). Since this is the case, it would oot he useful to construct a library from

these knockout mice, however, these results MaY suggest that a Jack of 24R.2S(OH)2D3

could lead to downregulation of ils receptor. Interestiogly, Mahonen et al. (94) have

shown that 1a,2S(OHhD3 induces the expression of VDR mRNA and protein in bone

cells. Thus it seeOlS reasonable to suggest that 24R,2S(OH)203 may have a regulatory

fonction on its own receptor, much in the same way as 1a,2S(0H)2D3 does with its

receptor.

F. PCR Screening of the Yeast Two-Bybrid Libraries:

In addition to the yeast two-hybrid strategy, we have employed another approach to

clone the putative receptor. One such method uses PCR to take advantage of the fact that

nuclear hormone receptors have highly conserved DNA-binding domains containing two

zinc-fmgers. On the basis of functional homoJogy with the VDR, it foUows that the

putative receptor for 24R,2S(OH)2D3 would have highest sequence homology in this

region with the VDR. Thus by using a downstream primer from the zinc-linger region of

the VDR and an upstteam primer from the pAD-Gal4 vector, it should he possible to

amplify inserts containing a similar zinc-fmger domain and anything upstream of il in the

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insert. Although three different annealing temperatures were used, the four bands that

were isolated using tbis method tumed out to he artifacts.

These results do not invalidate the procedure, however. Good primers are keyand

optimal conditions must he determined in order to ensure that the primers do not stick to

everything, yet are still able to pick up inserts with sequences that vary sligbdy from the

primers. Setting the annealing temperature depends upon the similarity of the primer to

the insert to he amplified, which in our case is UnknOWD. The greater the similarity

between the primers and the putative receptor, the cIoser the annealing temperature

should he set to the Td of the primers. In order to decrease the likelihood of picking up

artifacts the annealing temperature can be increased to 66- 68°C. However, this must he

done on the assomption that the putative receptor sequence in the primer area will he

identical to or only a few base pairs different from that of the VDR primer being used. It

is also possible to decrease the annealing temperature helow 59°C to try to pick up the

receptor under the assumption that its similarity to the VDR will he further away than that

of FXR or LXR. Another alteration that cao he made is to vary the concentration of salt

in the buffer and then alter the annealing temperatures, since salt concentration is known

to play a factor in primer annealing. Altematively, in order to reduce the number of

artifacts that are produced in this procedure the numher of cycles could he decreased from

30 to 25, since increasing the nomber of cycles augments the likelihood of amplifying

non-specifie targets.

As is the case with the yeast two-hybrid system, fallure to identify the putative

receptor using this strategy does not indieate the receptor is not in the tissue we are

looking at. This is because the strategy requires that the DNA-binding domain containing

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the zinc-linger regioo of the putative receptor was reverse transeribed in the library, not a

guarantee, especially if the receptor bas a long 3'UTR. Non-representative amplification

of the libraries could a1so bias the results of this type of screeo. A possible solution

would he to do RT-PCR on the mRNA rather tban the cDNA libraries. Thus, continued

pursuit using this and other strategies will he ongoing.

G. Alternative Strategies to Clone the Z4R,2S(OR)JI)3 Receptor:

ln the event that the putative receptor is not cloned using the yeast two-hybrid or

PCR screening strategies, alternative methods could he developed to attempt cloning.

One option is to use the strategy that was employed to isolate the majority of the orphan

receptors before the yeast two-bybrid system evolved. This involves constructing a

degenerate oligonucleotide probe corresponding to the most higbly conserved region of

the nuclear hormone receptor family, the DNA-binding domain (DBD) (95). This could

then he used to screen a mandiblelcalvaria cDNA library onder low stringency conditions

(96). The advantage to using such a procedure is that it does not require the putative

receptor to he a class fi member interacting with RXR. However, since there is no way of

increasing the specificity of the screen towards the desired clone tbis sttategy would most

likely identify other nuclear hormone receptors found in bone such as VDR, R.XR, RAR,

or ER.

Another possible strategy involves a modification of the yeast one-hybrid system.

The classical yeast one-hybrid system was devel0Ped as a method to identify and clone

genes encoding for proteins that reco8Dize a specific DNA sequence (97). The system

bas two basic components: 1) an expression library of hybrid proteins constmcted by

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fusing protein coding sequences to the Gal4 transeriptional activation domain, and 2) a

UAS-less lacZ reporter gene containing multiple copies of the binding site of interest

within its promoter region. Wben transfonned into yeast, hybrid proteins that recognize

this binding site will induce expression of the reporter gene and tum the cell blue in a p­

galactosidase assay. This occurs due to the hybrid protein's dual ability to bind the

promoter region and activate transcription. This type of classical one-hybrid system

would not work in our case due to the fact that we do not know the sequence of the

response element to which the putative receptor would bind.

A modification of this system bas been developed to screen for possible ligands for

orphan receptors. In this strategy, the ligand-binding domain (or portions thereof) of an

orphan receptor is fused to the Gal4 DNA-binding domain and transfonned into a

mammalian cellline containing a lacZ reporter gene with a Gal4 UAS. Putative ligands

are added to the ceUs which are then assayed for activation of the reporter gene. The

Gal4 DBD of the fusion protein would bind the Gal4 UAS and, in the event ofbinding by

a tnle ligand, the AF-2 domain of the receptor would adopt a confonnation that would

allow the teeruitment of coactivators and activation of the reporter gene. Clones are then

identified by their blue color upon addition of X-Gal.

In our case, a sunHar protocol could he used for the purposes of isolating a receptor

for a known ligand. In this procedure, a new mandiblelcalvaria cDNA library would he

constnlcted fused with the Gal4 DNA-binding domain in a mammalian expression vector.

This library would he ttansformed into mammalian ceUs and the cells exposed to

24R,2S(OH)2D3. The Gal4 DDD cao interact with the GAIA upstream activating

sequence (UAS) located upstream of an integrated GALl-lacZ gene. If one of the cDNA

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inserts fused to the GaI4 DBD encodes the putative receptor, then upon addition of

24R,25(OH)2D3 to the transformed cells, a conformational change would occur within the

AF-2 domain of the receptor which, together with the Gal4 DBD would activate

transcription of the GALl-lacZ gene (98). These colonies could then he recognized by

their blue color upon incubation with X-gal. In the absence of 24R,25(OHhD3 or if the

insert does not encode the putative receptor then transcription would not he activated and

the ceUs would not tum blue. A possible drawback to this system is that if the library

insert does not encode the correct portion of the LBD of the receptor, the method would

not pick it up. In addition, other activators could he cloned if they contain an activation

domain that does not require its ligand to he active. On the other hand, this strategy does

not rely on the hypothesis that the putative receptor interacts with RXR and is aIso fairly

straightforward to cany out.

Previous studies indicate that 24R,25(OHhD3 induces the activity of the brain

isoform of creatine kinase (CKB) in cultured chick-embryo limb-bud cartilage ceUs (52)

and in the epiphyses of rat tibiae (53), whereas la,25(OH)2D3 does not. FUIthermore, it

was shown that this increase in activity was due to an increased rate of synthesis of CKB,

not just an increase in the activity of the enzyme that was already present. In addition,

other reports indicate 24R,2S(OH)2D3 induces omithine decarboxylase (ODe) activity in

the epiphyses of rat long bones (64). These results suggest that the CKB and OOC

promoters may contain a 24R,25(OH)2D3 response element. In light of tbis, another

strategy for isolating the putative 24R,25(OH)2D3 receptor arises.

We have obtained plasmids containing various lengths of the ODe promoter from

Dr. Andrew Butler (99) as weil as plasmids containing various lengths of the CKB

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promoter from Dr Micbael Ritebie (100, 10l). Tbese promoters would he subcloned

behind a lacZ reporter gene. In addition, an expression library driven by a mammalian

promoter (ex. CMV) would he constructed from the EI7.S mouse mandiblelcalvaria

rnRNA. The expression library and one of the promoter constructs would then he

transfected together into CV-1 cells (or any other mammalian cellline that does not sbow

background) and exposed ta 24R,2S(OH)2D3. If the library contained a cDNA insert tbat

encoded for the putative receptor the ligand would bind to il, the receptorRigand complex

(heterodimerized with RXR, if necessary) would bind to the 24R,2S(OHhD3 response

element in the CKB or OOC promoter and drive the expression of the lacZ gene. These

blue clones would he picked, the plasmid DNA isolated, and the insert amplified by PCR

using primers from the library expression vector. A possible drawback to this strategy is

that the insert would bave to encode for bath the DBD and the LBD of the receptor in

order for transcriptional activation to he possible.

An alternative to using the expression library in the strategy would he ta transfect

the promoter constructs into ceUs that are known to respond to 24R,25(OH)2D3 on the

assumption that if the ceUs respond to the ligand then they May contain a receptor for it.

In this situation, it would he necessary to transfect only the promoter plasmid and add

ligand. Past studies have sbown that kidney ceU cultures of one-week old rats respond to

24R,25(OH)2D3 and not 1a,25(OH)2D3 but lose this responsiveness as they approacb 4

weeks, at wbich time they become only responsive ta la,25(OHhD3 (102, 103). In

addition, it has been reported that this response profùe parallels the profl1e of the binding

activity of the respective ligands, suggesting the presence of receptors. Other studies

have indicated that cbick mesencbymal cells (52), rat epipbyseal chondrocytes (58), and

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cultured growth plate cbondrocytes (34) are also œsponsive to 24R,2S(OHhI>]. If there

was a receptor for the ligand in any of tbese cells, then upon transfection of the promoter

plasmid into, for example, one-week old rat kidney ceUs and addition of 24R,2S(OHhIl],

a ligandlreceptor complex would form (with RXR if necessary), bind to the response

element in the promoter and drive the reporter gene. Progressive deletion of the promoter

would then lead to the identification of the sequence of the response element, whicb could

then he used to isolate the receptor using the classical yeast one-hybrid strategy as

described above.

The above strategies provide a wide may of possibilities to approach the task of

isolating the receptor from a variety of angles. With the curreot technology and the

increasing evideoce supporting the existence of a 24R,2S(OHhD3 receptor, it should just

he a matter of time hefore it is cloned. Once it has beeo cloned it will he necessary to

completely sequence il, to characterize it for binding specificity using other vitamin D

metabolites, and to identify a putative DNA binding site. 115 pattern of expression will

need to he studied, and i15 ttanseriptional regulatory function elucidated in order to

progress towards a full understanding of the mechanisms of 24R,2S(OH)2D3 action.

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IX. SUMMARY AND CONCLUSIONS

Traditionally, it has been believed that 24R,25-dihydroxyvitamin D3, the most

abundant dihydroxylated vitamin D metabolite circulating in the blood, does not have a

unique physiological role but rather is simply the initial Metabolite in vitamin D

catabolism. However, previous studies have suggested that a fonction for

24R,25(OHhD2 does exist, and still others indieated the existence of specifie receptors

for this metabolite. Most recently, our own laboratory has demonstrated that mice

Iaeking the 24-0Hase enzyme responsible for converting 25(08)D3 to 24R,25(OH)2D3,

suffer from abnormal development of bones formed by intramembraneous ossification,

such as the mandible and ealvaria. These results support the notion that receptors

mediating the action of 24R,2S(OH)2D3 do exist, and also identify the probable sites of

their expression as being intramembraneous bone.

Nuclear and cytosol extraets bave been isolated from the mandible and calvaria of

17.5 day-old mouse embryos. Saturation analysis on these have identified saturable

binding of eHl-24R,25(OH)2D3 to a protein in the extracts. This binding is of high

affinity <Kcs = 1.1nM) and has a Bmu of 2.79pmoUmg proteine Competition assays

showed that this binding protein was specifie for 24R,25(OH)2D3 and was not the

Vitamin D receptor (VDR). Furthermore, sucrose gradient sedimentation assays were

perfonned to eliminate the possibility tbat this protein was the vitamin D binding protein

(DBP), which is a1so known to bind 24R,25(08)2D3. Finally, tissue specifieity

experiments earried out on Uver, brain and bone nuclear extraets revealed that this

putative receptor was also present in liver but not in brain.

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Two strategies have been employed to isolate the receptor cDNA. Firstly, a hybrid

protein consisting of the GAIA DNA-binding domain fused to the ligand-binding domain

of buman RXRa was used to screen a one-week-old mouse calvaria cDNA library and a

E17.5 mouse mandible/calvaria cDNA library, both fused to the GAIA activation domain.

This strategy bas so far pulled out mouse peroxisome proliferator-activated receptor

(mPPARy) from the neonate library. Continued screening of the embryonallibrary will

follow. Secondly, a 5' primer from the library vector and a 3' primer located within the

highly conserved zinc-finger region of the VDR have been used to screen the embryonal

library by PCR for inserts containing a similar motif. To date no positive clones have

been isolated.

The substantial evidence presented here for the existence of a putative nuclear

receptor for 24R,2S(OHhDJ indicates that continued efforts to isolate the receptor, both

by these methods and by altemate strategies, should result in its successful cloning. The

isolation of such a receptor would provide valuable insigbt into the mechanism of action

of the vitamin D metabolite and its involvement in bone development.

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Xe ORIGINAL CONTRIBUTION TO KNOWLEDGE

The answer to the question of whetber 24R,25-dihydroxyvitamin OJ is an active

vitamin D3 metabolite or an inactive eatabolite bas long been sought. The bypotbesis tbat

there may exist a receptor for this metaboüte bas been the basis of many studies in the

past two decades. This is the flI'St report to show specifie binding of 24R,2S­

dihydroxyvitamin 03 in intramembranous bene during embryonic development. It is also

the flCSt time anti-actin antibodies have been used in sucrose gradient sedimentation

experiments to show that observed ligand binding was not due to DBP complexed witb

endogenous actin. Furthermore, the mRNA that we have isolated from embryonic mouse

mandible and calvaria as weIl as the cONA libraries that we have generated are the fmt

of their kind and provide strong tools with which furtber eloning attempts cao he carried

out.

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