Bone Vol. 17, No. 2, Supplement
August 1995:33S-38S
ELSEVIER
New Understanding of the Molecular Mechanism of
Receptor-Mediated Genomic Actions of the
Vitamin D Hormone
M. R. H A U S S L E R , P. W. J U R U T K A , J.-C. H S I E H , P. D. T H O M P S O N , S. H. S E L Z N I C K , C. A. H A U S S L E R ,
and G. K. W H I T F I E L D
Department of Biochemistry, College of Medicine, The University of Arizona, Tucson, AZ, USA
of the receptor as a DNA-bound active heterodimer of
liganded hVDR and unoccupied RXR. (Bone 17:33S-38S;
The nuclear vitamin D receptor (VDR) binds the 1,25dihydroxyvitamin D 3 [I,25(OH)2D3] hormone with high aff'mity and elicits its actions to regulate gene expression in
target cells by binding to vitamin D-responsive elements
(VDREs). VDREs in positively controlled genes such as osteocalcin, osteopontin, [~3-integrin, and vitamin D-24-OHase
are direct hexanucleotide repeats with a spacer of three nucleotides. The VDR associates with these VDREs with the
greatest affinity as a heterodimer with one of the family of
retinoid X receptors (RXRs). VDR consists of an N-terminal
zinc finger domain that determines DNA binding, a "hinge"
segment and a C-terminal hormone binding domain which
also contains two conserved regions that engage in heterodimerization with an RXR on the VDRE. The role of the
1,25(OH)2D 3 ligand in transcriptional activation by the
VDR-RXR heterodimer is to alter the conformation of the
hormone-binding domain of VDR to facilitate strong dimerization with RXR, which results in ligand-enhanced association with the VDRE. Thus RXR is recruited into a heterocomplex by liganded VDR. The natural ligand for the RXR
coreceptor, 9-cis retinoic acid, suppresses both VDR-RXR
binding to the VDRE and 1,25(OH)2D3-stimulated transcription, indicating that 9-cis retinoic acid diverts RXR away
from being the silent partner of VDR to instead form RXR
homodimers. Recent data reveal that after binding RXR, a
subsequent target for VDR in the vitamin D signal transduction cascade is basal transcription factor liB (TFIIB). VDR
can be shown to bind directly to TFIIB, in vitro, and synergizes with it in transcriptional control by 1,25(OH)2D 3 in
transfected cells, thus unveiling a molecular mechanism
whereby 1,25(OH)2D 3 activates the transcription machine.
Finally, natural mutations in hVDR that confer 1,25(OH)2D 3
resistance in a number of patients have been characterized.
The mutations fall into three categories: (i) DNA binding/
nuclear localization; (ii) hormone binding; and (iii) RXR heterodimerization. These natural mutations are consistent with
the structure/function analysis of hVDR via biochemical and
molecular biological approaches and confirm the basic model
1995)
Key Words: Vitamin D receptor; Vitamin D-responsive element; Retinoid X receptors; Transcriptional control; 1,25Dihydroxyvitamin D3; Hereditary hypocalcemic vitamin D-resistant rickets.
Introduction
As depicted in Figure 1, vitamin D obtained from diet or photoactivation in the skin is metabolized to its active form, 1,25dihydroxyvitamin D 3 [I,25(OH)2D3], by sequential hydroxylations in the liver and kidney. The 1,25(OH)2D 3 hormone is a
member of the subfamily of steroid hormone lipophilic ligands
that enters the nucleus directly and binds to a DNA-associated
aporeceptor. 8 Similar to the vitamin D receptor (VDR), the thyroid hormone (TR) and retinoic acid receptors (RAR) are localized in the nucleus even in the unoccupied state. VDR is a
phosphoprotein which undergoes additional phosphorylation
upon 1,25(OH)2D 3 binding. 8 Human VDR (hVDR) is phosphorylated by protein kinase C at serine-51, in a basic nuclear localization domain between the two zinc fingers in the DNA-binding
region, and by casein kinase II (CKII) at serine-208, a residue
near the N-terminus of the hormone-binding domain which is
potentially involved in positive modulation of transactivation. 7
The association of 1,25(OH)2D 3 with VDR elicits the recruitment of a retinoid X receptor (RXR), forming a VDR-RXR
heterodimer with high affinity for a number of vitamin D-responsive elements (VDREs) which lie in the regulatory regions
of 1,25(OH)2D3-controlled genes. 17 Altered expression of these
genes (Figure 1) results in target cell modification and the ensemble of biologic effects of vitamin D including bone remodeling, intestinal Ca" ÷ and PO43- absorption, PTH suppression,
and catabolism of the 1,25(OH)2D 3 hormone by 25(OH)vitamin
D-24-hydroxylase (24-OHase). As detailed in what follows, Figure 1 also illustrates that the novel vitamin A metabolite which
serves as a natural ligand for RXR, namely 9-cis retinoic acid
(9-cis RA), negatively modulates 1,25(OH)2D3-dependent transcription by diverting the RXR silent partner away from its complex with VDR. The opposing effects of 1,25(OH)2D 3 and 9-cis
RA could have profound effects on the cell differentiation and
mineral homeostatic functions of vitamin D, 16 but this requires further elucidation in defined cell culture and with in vivo
systems.
Address for correspondence and reprints: Dr. Mark R. Haussler, Department of Biochemistry, College of Medicine, The University of Arizona, Tucson, AZ 85724, USA.
© 1995 by Elsevier Science Inc.
33S
8756-3282/95/$9.50
SSDI 8756-3282(95)00205-R
34S
M.R. Haussler et al.
Structure/function of the vitamin D receptor
Liver
Vitamin D Constitutive ) 25tOH~D~
~ i .~
Bone Vol. 17, No. 2, Supplement
August 1995:33S-38S
Kidney
." 1,25(OH)2D3
....
~"PTH/1 25
Hormone
Hegulalea ~ Ca/P64
Target Cell
uc,eu
o,c
<
\
+
~r hnRNA ~
:riptional Control J
Osteoblast function (Osteocaicin 1"; Osteopontin 1"; Collagen J,)~
Macrophage/Osteociast differentiation (c-myc J,; ~3 Integrin 1 " ) )
Intestinal Ca/PO4 absorption (CaBP28k 1"; CaBPgk 1`)
Renal vitamin D catabolism (24-OHase 1')
Parathyroid hormone synthesis (PTH ,I,)
Immunomodulation (IL-2 J,)/Cell Differentiation
L
(
/
J
VDREs Identify Primary Vitamin D Target Genes
Table 1 lists the VDREs in 1,25(OH)2D3-controlled genes that
have thus far been definitively identified by combinations of
promoter-reporter construct transfections, mutagenesis, and in
vitro VDR binding. Positive VDREs consist of hexanucleotide
direct repeats spaced by three nucleotides (DR3s) with the half
element being similar but not identical to the A G G T C A estrogen-responsive half-element. 26 Recent data from studies of
VDR-RXR heterodimers on a DR3 element ~2 reveal that the
RXR partner lies on the 5' half-element. This orientation of
heterodimers has been observed as well with TR and RAR, 29
which form RXR-containing heterodimers on positively controlled DR4 and DR5 elements, respectively. Thus, in all cases
of positive control by the VDR/TR/RAR subfamily characterized
to date, the RXR partner lies on the 5' half-element. Viewing the
3' VDR half-element in Table 1 (top portion), it is evident that
the guanine in the second position is absolutely conserved and
that there are two variants of the VDRE half-element. The first
type, characteristic of rat and human osteocalcin, ~s'28 avian 133
integfin, 2 and the proximal 24-OHase 22 VDRE, possesses pu-
Table 1. Selected natural vitamin D responsive elements
Gene controlled
Type of
regulation
5'-half
element
"Spacer"
3'-half
element
+
+
+
+
GGGTGA
GGGTGA
GGTTCA
GGTTCA
ATG
ACG
CGA
GCG
AGGACA
GGGGCA
GGTTCA
GGTGCG
+
+
AGGTGA
GAGGCA
GTG
GAA
AGGGCG
GGGAGA
Rat osteocalcin
Human osteocalcin
Mouse osteopontin
Rat 24-OHase-distal
Rat 24-OHaseproximal
Avian 133 integrin
Positive VDRE
consensus
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
Human PTH
Avian PTH
Rat bone
sialoprotein
.
.
+
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
GGGTCA
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
GTG
.
.
.
.
.
.
.
.
.
.
.
.
GGGGCA
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
-
GGTTCA
GGGTCA
AAG
GGA
CAGACA
GGGTGT
-
AGGGTT
TAT
AGGTCA a
~Overlaps an inverted TATA box
"
mRNA
,j,,
Figure 1. Overview of vitamin D metabolism and action to regulate transcription in target cells.
ProteinsBi°active
~1~
Altered
Cell
Functions
/ /
J
fines (primarily guanine) in the third and fourth positions. In
contrast, the mouse osteopontin 21 and distal 24-OHase 3° VDREs
contain, for the most part, thymine bases in these positions. In
spite of these differences, a consensus-positive VDRE can be
tentatively assigned and is listed in Table I. It is important to
note that all natural VDREs identified to date that function as
strong enhancers of transcription have the core DR3 motif, and
that all bind the RXR-VDR heterodimer. While alternative
VDRE motifs are not excluded, they will likely turn out to be
weaker elements which may require additional adjacent ciselements that bind cooperating accessory transacting factors.
Several negative VDREs have also been characterized (Table 1).
For example, the rat bone sialoprotein VDRE overlaps an inverted TATA box in the basal promoter, illuminating occlusion
of TATA-binding protein as one mechanism for negative regulation. 14 Avian 13 and human 5 PTH VDREs differ in that the
former is quite similar to a positive VDRE, whereas the latter has
a very weak 3' half-element that could affect VDR conformation
or even bind a repressor partner, with VDR being relegated to the
transcriptionally inactive 5' half-element. It is probable that negative regulation by VDR will have a number of mechanisms,
some perhaps not even involving DNA binding, but instead resulting from a type of transcription factor squelching.
What is the Molecular Role of 1,25(OH)2D3?
From published VDR DNA-binding experiments carried out by
gel mobility band retardation assays, it is clear that when physiologic quantities of VDR are utilized, the receptor only binds
efficiently to DR3 VDREs when purified RXRs or suitable nuclear extract sources of RXRs are supplied. 2'13'17As"E2,2s However, there has been some confusion and controversy over
whether the 1,25(OH)2D 3 hormone is required for such binding. 17.~s,22,24,2s One problem in interpretation of gel mobility
shift assays is that often vast excesses of expressed VDR/RXR
are included in the reaction and only the minuscule fraction that
actually binds to the labeled VDRE probe is visualized. Thus,
even in the absence of 1,25(OH)2D3, a small fraction of VDR
will exist in the active conformation and produce a positive band
shift. The role of 1,25(OH)2D 3 is probably to shift the conformational equilibrium to favor the active species, much like an
allosteric activator stimulates enzyme activity. A second problem is that nonphysiologic concentrations of salt (~< 0.1 mol/L
KCI) have been used in some studies, allowing the formation of
Bone Vol. 17, No. 2, Supplement
August 1995:33S-38S
M.R. Haussler et al.
Structure/function of the vitamin D receptor
relatively low affinity protein-DNA complexes. In reactions that
best mimic physiologic conditions, namely 0.15-0.2 mol/L
KC117'22'28 and limiting concentrations of VDR/RXR, the
1,25(OH)2D 3 hormone can be shown to be required for heterodimeric VDR-RXR binding to DR3 VDREs.
The model depicted in Figure 2 illustrates our concept of
VDR-DNA binding, the essence of which is that there are two
distinct dimerizing forms of RXR-VDR, with the 1,25(OH)2D 3
hormone eliciting a transformation of the weakly associated beterodimer into a more stable complex. This "sliding hinge"
model includes several hypotheses not previously put forth. We
propose (Figure 2, top) that in the absence of 1,25(OH)2D 3 (resting state), a significant fraction of VDRs exist as preheterodimerized species bound weakly to DNA. Weak dimerization
is, by analogy with RXR-TR and RXR-RAR, 29 proposed to
result from contact between the D-box in the second zinc finger
of RXR and the first zinc finger or prefinger region of VDR. This
weakly dimerized form is configured such that it can slide along
DNA but is unable to penetrate deeply enough into the major
groove of DNA to recognize and bind to DR3 VDREs (Figure 2,
top). By traversing in the plane of DNA, the VDR-RXR heterodimer is positioned within reach of its target sites and is thus
poised to accept ligands. This model therefore represents an
interesting analogy to the plasma membrane dimeric receptors
for several growth factors that lie in the plasma membrane awaiting circulating ligands. It should be noted that, as detailed below, VDR-RXR heterocomplexes can also form in solution (presumably in the nucleoplasm?) in a process accelerated by
1,25(OH)2D 3. Therefore, all activation events need not occur on
DNA per se.
Upon 1,25(OH)2D 3 binding (Figure 2, center), we propose
Resting State
35S
that a strong dimerization interface is exposed in the hormonebinding domain of VDR. Heterodimerization then conformationally alters or " h i n g e s " the RXR-VDR complex such that the
zinc finger regions of each receptor now can not only penetrate
into the major groove of DNA to contact fully the half elements,
but also are configured such that they correctly discriminate the
spacing of a DR3, as opposed to DR4s or DR5s that contain
similar half-elements. We can therefore summarize the role of
the 1,25(OH)2D 3 hormone as follows: the primary function of
1,25(OH)2D 3 is to activate allosterically the RXR-VDR heterocomplex and allow it to specifically recognize the VDRE.
Although others assert that the ligand for RXR, 9-cis RA,
either synergizes with 1,25(OH)2D 3 in activating transcription in
insect cells 3 or has no significant effect in mammalian cells, 6
w e 17 and two additional groups 4"t2 have clearly shown that 9-cis
RA exerts a negative influence on 1,25(OH)2D 3 actions. Accordingly, 9-cis RA prevents RXR-VDR from full association with
DR3 VDREs, 4"17 whereas 9-cis RA and a more specific RXR
ligand partially repress 1,25(OH)2D3-stimulated transcription in
transfected cells, t 2. ~7 This effect of 9-cis RA binding to the VDR
silent partner is depicted in the lower portion of Figure 2. If
added or present prior to 1,25(OH)2D 3, 9-cis RA is a potent
suppressor of RXR-VDR binding to the VDRE, acting by diverting RXR from forming heterodimeric RXR complexes. 4']7
This diversionary action by 9-cis RA does not absolutely require
the presence of an RXRE (Thompson and Haussler, unpublished
data), but as illustrated in Figure 2 (bottom), RXR homodimer
binding to the DR 1 RXRE can then occur. Most importantly, and
likely explaining the conflicting data in experiments involving
coaddition of 1,25(OH)2D 3 and 9-cis RA to gel mobility shift
and transfected cell transcription reactions, we (Thompson and
Haussler, unpublished data) have observed that if 1,25(OH)2D 3
initially occupies VDR to facilitate RXR-VDR heterodimers,
this species is relatively resistant to 9-cis RA binding and inhibition (Figure 2, lower right). Therefore, as in classic physiologic systems of opposing hormones, the state of activation will
be dictated by the individual concentrations and order of appearance of the 1,25(OH)2D 3 and 9-cis RA nuclear ligands.
Structure/Function of Human VDR and Naturally
Occurring Mutations
_
Activated State x~,25(OH)2D 3
~
p
x~,25(OH)2D3
P
VDREI
D,
VDRE2
Partially Repressed State
9-cis RA
•
RXRE
D'
P
VDREI
P
9-cis RA
P
P
VDRE2
Figure 2. Sliding hinge model for 1,25(OH)2D3-VDR binding to the
VDRE and its diversion by 9-cis RA.
Illustrated in Figure 3 is a schematic map of human VDR. In
addition to their classic roles in DNA binding and hormone binding, respectively, the N-terminal and C-terminal domains of
hVDR subserve other functions. For example, the zinc finger
region contains two nuclear localization signals, one mapping to
the T-box region just C-terminal to the second finger ]5 and a
second residing in an area of five basic amino acids between the
two fingers. 9 Heterodimerization with RXR is a property of the
C-terminal 1,25(OH)2D3-binding domain and the relevant portion of VDR appears to consist of at least a bipartite region
encompassing a conserved sequence known as E123 as well as
several heptad repeats within a leucine zipperlike domain, particularly the fourth and ninth heptads. 2° Until X-ray crystallographic data are available for the actual tertiary structure of VDR
and its contacts with RXR and the VDRE, models of VDR will
by necessity be derived from biochemical and artificial mutagenesis studies. However, a number of natural mutations in hVDR
have been characterized that produce the autosomal recessive
disorder of hereditary hypocalcemic vitamin D-resistant rickets
(HHVDRR) (reviewed in refs. 16 and 25). These mutations are
pictured in Figure 3 and their characterization provides
36S
M.R. Haussler et al.
Structure/function of the vitamin D receptor
Bone Vol. 17, No. 2, Supplement
August 1995:33S-38S
DNA Binding Domain
Hormone Binding (E) Domain
~,_~.,,~.~
H3+Nq
I
(~)
\
1/21
\
[
tr
Localization
_ H35Q
~
~
E1
R274L
R73Q
HeptadRepeats
I314S
Y295stop/
]l
c~ ~
L
t I i-T---
Q152stop
~
I
Transcriptional
Activation
Heterodimerization Domains
,
[
N u c l e ation"
r
G33D
, Hing_......~e ,
I
R73stop '
Hormone /|
1
Binding
Mutations I
j9 t
427
R391C
P a t i e n t #2)
(p
NovelMutation
M~
Novel
affecting
predon
predominantlyRXR
hetero
heterodimerization
K45E
r F47I R50Q
DNABindingandNuclearLocalizationMutations
Figure 3. Schematic structure/function map of hVDR and summary of natural mutations that cause the hereditary hypocalcemic vitamin D-resistant
rickets phenotype. An encircled P indicates a site of phosphorylation. Patient No. 2 (R391C) refers to the study of Selznick et al. 27
strong independent verification of the present model for VDR.
The first and most abundant class of mutations resides in the zinc
finger region and affects DNA binding and nuclear localization.
Interestingly, these altered amino acids are for the most part in
residues highly conserved in the steroid receptor superfamily and
fall into three subcategories: (i) the tip of the first zinc finger
which may constitute a weak dimerization interface with the
D-box of the RXR partner on DNA29; (ii) the " k n u c k l e " region
just C-terminal of finger 1 which comprises an a-helix in the
estrogen receptor (ER) that recognizes the ERE half-element26;
and (iii) the a-helical region(s) on the C-terminal side of finger
2 which make contacts with the DNA phosphate backbone in
the ER-ERE structure. 26 A second class of naturally mutated
VDRs are those possessing missense or nonsense (stop) codons
that preclude 1,25(OH)2D 3 hormone binding via alteration or
truncation, respectively, of the C-terminal domain (Figure 3).
Finally, we have recently discovered a novel mutation (R391C)
in an HHVDRR patient which compromises primarily RXR heterodimerization. 27 This latter substitution in hVDR appears to
establish yet a third class of mutant VDRs and unequivocally
demonstrates that RXR-VDR heterodimerization is obligatory to
prevent the HHVDRR phenotype. The natural mutations identified in the lower portion of Figure 3 therefore support both the
schematic structure/function of hVDR pictured in the top portion
of this figure and the model for vitamin D action depicted in
Figure 2. The paramount conclusion from the characterization
of natural mutants of hVDR is that, to function biologically,
VDR must localize to the nucleus and bind to three entities:
1,25(OH)2D3, an RXR isoform, and DNA.
How Does VDR Regulate the Transcription Machine?
In the basal state of DNA transcription, the TATA-box binding
protein (TFIID) and its associated factors (TAFs) are bound to
the TATA box at approximately position - 20 in the 5' region of
controlled genes, but the frequency of transcriptional initiations
is very low because the RNA polymerase II-basal transcription
factor liB (TFIIB) enzyme complex is not stably associated with
TFIID-TAFs. The recruitment of the TFIIB-RNA polymerase II
complex appears to be the rate-limiting step in preinitiation complex formation, and is stimulated dramatically when a transacting factor or factors bind to upstream enhancers. In a process
involving DNA looping, transactivators are thought to attract
TFIIB and also interact with TAFs, forming a stable preinitiation
complex that executes repeated rounds of productive transcription. Recent data indicate that the activation function in the hormone-binding domain of the estrogen receptor, AF-2, associates
specifically with a TAF known as TAFn30 ~1 and that the ER
binds to TFIIB in vitro, l° In collaboration with the groups of
Ozato at the National Institutes of Health and Tsai and O'Malley
at the Baylor College of Medicine, we have observed that hVDR
specifically associates with hTFIIB, l In that work, Blanco et al.
showed that VDR binds to a TFIIB-glutathione S-transferase
fusion protein linked to glutathione-laden beads.l It was also
observed that both TRI3 and R A R a , but not RXR, interact with
hTFIIB. These results indicate that the RXR silent partner is not
actively engaged in TFIIB contact, but that the ligand-binding
partners in the VDR/TR/RAR subfamily provide a hard-wired
connection to the assembly and enhancement of the transcription
machine. Moreover, simultaneous independent data obtained by
MacDonald et al. 19 using the powerful yeast two-hybrid system
to detect protein-protein interactions also revealed that hVDR
binds efficiently to TFIIB. MacDonald et al. 19 further exploited
the yeast two-hybrid system to prove that, although hVDR and
RXR interact, no homodimeric association occurs for hVDR
alone, providing conclusive evidence against the existence of
VDR homodimers in biologically relevant settings. Utilizing fusion protein technology, they also showed that VDR interacts
directly with RXR to form a heterodimer in solution and not just
on DNA; this process was enhanced eightfold by the presence
Bone Vol. 17, No. 2, Supplement
August 1995:33S-38S
o f 1,25(OH)2D 3 h o r m o n e . 19 B e c a u s e h V D R - T F I I B association
is not d e p e n d e n t o n the 1 , 2 5 ( O H ) 2 D 3 ligand, 1"~9 the role o f
1,25(OH)2D 3 c a n n o w be further r e s o l v e d to an early participation in c o n f o r m i n g V D R s u c h that it attracts R X R followed by
the targeting o f the resulting R X R - V D R b e t e r o d i m e r to V D R E s
(see Figure 2). A l t h o u g h we h a v e yet to e x a m i n e w h e t h e r the
p r e s e n c e o f R X R further facilitates V D R - T F I I B association, it is
probable that the s i m p l e p o s i t i o n i n g o f a certain d o m a i n or dom a i n s o f h V D R i m m e d i a t e l y u p s t r e a m o f target structural g e n e s
constitutes the direct physical link to T F I I B a n d transcriptional
e n h a n c e m e n t by 1,25(OH)2D 3.
B l a n c o et al. ~ h a v e also reported functional studies w h i c h for
the first time s h o w the interaction o f T F I I B with a n y m e m b e r o f
the steroid receptor s u p e r f a m i l y in l i g a n d - d e p e n d e n t activation
o f transcription in intact cells. In pluripotent P19 m o u s e e m b r y onal c a r c i n o m a cells, transfection o f h V D R or hTFIIB alone
p r o d u c e d no better t h a n a t w o f o l d i n d u c t i o n o f V D R E - l u c i f e r a s e
reporter e x p r e s s i o n b y 1 , 2 5 ( O H ) 2 D 3. H o w e v e r , w h e n transfected together, h V D R a n d h T F I I B m e d i a t e d a synergistic transcriptional
response of approximately
30-fold when
1,25(OH)2D 3 w a s added, a n d this effect w a s absolutely dependent on the p r e s e n c e o f the V D R E in the luciferase construct. It
s h o u l d be n o t e d that the V D R - T F I I B positive cooperation appears to be cell-specific b e c a u s e similar e x p e r i m e n t s in contacti n h i b i t e d N I H / 3 T 3 S w i s s m o u s e e m b r y o c e l l s r e s u l t e d in
hTFIIB-elicited s q u e l c h i n g o f transcription. Therefore, in m o r e
differentiated cells, p e r h a p s i n c l u d i n g osteoblasts or fibroblasts,
a c c e s s o r y coactivators m a y be p r e s e n t to m o d u l a t e TFIIB or
bridge b e t w e e n V D R a n d TFIIB. In c o n c l u s i o n , V D R and TFIIB
are h y p o t h e s i z e d to exist in a m u l t i - s u b u n i t transcription c o m plex w h i c h also c o n t a i n s T A F s and/or coactivators that m a y be
p r o m o t e r - or t i s s u e - s p e c i f i c . F u r t h e r c h a r a c t e r i z a t i o n o f this
c o m p l e x will require the d i s c o v e r y o f cell type a n d promoterspecific c o m p o n e n t s via transfection a n d b i o c h e m i c a l interaction
studies. U l t i m a t e l y an in vitro transcription s y s t e m m u s t be dev i s e d that utilizes d e f i n e d c o m p o n e n t s to faithfully replicate
1,25(OH)2D3-stimulated g e n e e x p r e s s i o n .
Conclusion and Perspectives
T h e p r e c e d i n g description o f the m o l e c u l a r m e c h a n i s m w h e r e b y
the v i t a m i n D h o r m o n e controls g e n e e x p r e s s i o n is as yet i n c o m plete. To a d v a n c e our u n d e r s t a n d i n g , a physical characterization
o f the structure o f V D R via X - r a y c r y s t a l l o g r a p h y is required.
F u r t h e r m o r e , to c o m p r e h e n d the g e n o m i c action o f v i t a m i n D in
c a l c i u m h o m e o s t a t i c a n d o t h e r target cells it will be n e c e s s a r y to
elucidate the detailed i n v o l v e m e n t o f various R X R i s o f o r m s ,
specific T A F s , a n d n o v e l coactivators that m i g h t i n f l u e n c e the
regulation o f different v i t a m i n D - c o n t r o l l e d promoters. Additional d e v e l o p m e n t a l a n d biologic insights into the v i t a m i n D
endocrine s y s t e m will no d o u b t arise w h e n a V D R g e n e k n o c k o u t
is created in m i c e . All o f this i n f o r m a t i o n m a y assist in determ i n i n g if the n o n g e n o m i c actions o f v i t a m i n D c o m p o u n d s that
have b e e n o b s e r v e d in vitro are biologically relevant a n d perhaps
will aid in e v a l u a t i n g the potential role for V D R in the pathop h y s i o l o g y o f osteoporosis.
Acknowledgments: This work was supported by NIH Grant Nos.
DK33351 and AR15781 to M.R.H. and DK40372 to G.K.W.
References
1. Blanco, J. C. G., Wang, I.-M., Tsai, S. Y., Tsai, M.-J., O'Malley, B. W.,
Jurutka, P. W., Haussler, M. R., and Ozato, K. Transcription factor TFIIB
M . R . Haussler et al.
Structure/function of the vitamin D receptor
37S
and the vitamin D receptor cooperatively activate ligand-dependent transcription. Proc Natl Acad Sci USA 92:1535-1539; 1995.
2. Can, X., Ross, F. P., Zhang, L., MacDonald, P. N., Chappel, J., and Teitelbantu, S. L. Cloning of the promoter for the avian integrin [33subunit gene and
its regulation by 1,25-dihydroxyvitamin D3. J Biol Chem 268:27371-27380;
1993.
3. Carlberg, C., Bendik, I., Wyss, A., Meier, E., Sturzenbecker, L. J., Grippo,
J. F., and Hunziker, W. Two nuclear signalling pathways for vitamin D.
Nature 361:657-660; 1993.
4. Cheskis, B. and Freedman, L. P. Ligand modulates the conversion of DNAbound vitamin D3 receptor (VDR) homodimers into VDR-retinoid X receptor
heterodimers. Mol Cell Biol 14:3329-3338; 1994.
5. DeMay, M. B., Kieman, M. S., DeLuca, H. F., and Kronenberg, H. M.
Sequences in the human parathyroid hormone gene that bind the 1,25dihydroxyvitamin D3 receptor and mediate transcriptional repression in response to 1,25-dihydroxyvitamin D3. Proc Natl Acad Sci USA 89:8097-8101;
1992.
6. Ferrara, J., McCuaig, K., Hendy, G. N., Uskokovic, M., and White, J. H.
Highly potent transcriptional activation by 16-ene derivatives of 1,25dihydroxyvitamin D3. J Biol Chem 269:2971-2981; 1994.
7. Hanssler, M. R., Jurtuka, P. W., Hsieh, J.-C., Thompson, P. D., Selznick,
S. H., Haussler, C. A., and Whitfield, G. K. Receptor mediated genomic
actions of 1,25(OH)2D3: Modulation by phosphorylation. Norman, A. W.,
Bouillon, R., and Thomasset, M., Eds. Vitamin D: A Pluripotent Steroid
Hormone: Structural Studies, Molecular Endocrinology and Clinical Applications. Berlin: Walter de Gruyter; 1994; 209-216.
8. Haussler, M. R., Mangelsdorf, D. J., Komm, B. S., Terpening, C. M., Yamaoka, K., Allegreno, E. A., Baker, A. R., Shine, J., McDonnell, D. P.,
Hughes, M., Weigel, N. L., O'Malley, B. W., and Pike, J. W. Molecular
biology of the vitamin D hormone. Recent Prog Horm Res 44:263-305; 1988.
9. Hsieh, J.-C., Jurutka, P. W., Nakajima, S., Galligan, M. A., Haussler,
C. A., Shimizu, Y., Shimizu, N., Whitfield, G. K., and Hanssler, M. R.
Phosphorylation of the human vitamin D receptor by protein kinase C: Biochemical and functional evaluation of the serine 51 recognition site. J Biol
Chem 268:15118-15126; 1993.
10. Ing, N. H., Beckman, J. M., Tsai, S. Y., Tsal, M.-J., and O'Malley, B. W.
Members of the steroid hormone receptor superfamily interact with TFIIB
(S300-1I). i Biol Chem 267:17617-17623; 1992.
11. Jacq, X., Brou, C., Lutz, Y., Davidson, I., Chambon, P., and Tora, L.
Human TAFu30 is present in a distinct TFIID complex and is required for
transcriptional activation by the estrogen receptor. Cell 79:107-117; 1994.
12. Jin, C. H. and Pike, J. W. DNA binding site and coregulator requirements for
1,25-dihydroxyvitumin D3-dependent activation [abstract]. J Bone Min Res
9(suppl. I):S160; 1994.
13. Koszewski, N. J., Lapuz, M. H., Russell, J., and Malluche, H. H. Vitamin D
receptor interactions with positive and negative DNA response elements: An
interference footprint comparison [abstract]. J Bone Min Res 9(suppl. 1):$290;
1994.
14. Li, J. J. and Sodek, J. Cloning and characterization of the rat bone sialoprotein
gene promoter. Biochem J 289:625-629; 1993.
15. Luo, Z., and M~ienpii~i,P. H. A peptide C-terminal to the second Zn-finger of
human vitamin D receptor is able to specify nuclear localization. Norman,
A. W., Bouillon, R., and Tbomasset, M., Eds. Vitamin D: A Pluripotent
Steroid Hormone: Structural Studies, Molecular Endocrinology and Clinical
Applications. Berlin: Walter de Gruyter; 1994; 249-250.
16. MacDonald, P. N., Dowd, D. R., and Haussler, M. R. New insight into the
structure and functions of the vitamin D receptor. Sem Nephrol 14:101-118;
1994.
17. MacDonald, P. N., Dowd, D. R., Nakajima, S., Galligan, M. A., Reeder,
M. C., Haussler, C. A., Ozato, K., and Haussler, M. R. Retinoid X receptors
stimulate and 9-cis retinoic acid inhibits 1,25-dihydroxyvitamin D3-activated
expression of the rat osteocalcin gene. Mol Cell Biol 13:5907-5917; 1993.
18. MacDonald, P. N., Hanssler, C. A., Terpnning, C. M., Galligan, M. A.,
Reeder, M. C., Whiffield, G. K., and Haussler, M. R. Baculovirus-mediated
expression of the human vitamin D receptor: Functional characterization, vitamin D response element interactions, and evidence for a receptor auxiliary
factor. J Biol Chem 266:18808-18813; 1991.
19. MacDonald, P. N., Sherman, D. R., Dowd, D. R., Jefcoat, S. C., Jr., and
DeLisle, R. K. The vitamin D receptor interacts with general transcription
factor IIB. J Biol Chem 270:4748~.752; 1995.
20. Nakajima, S., Hsieh, J.-C., MacDonald, P. N., Galligan, M. A., Haussler,
C. A., Whitfield, G. K., and Hanssler, M. R. The C-terminal region of the
38S
M . R . H a u s s l e r et al.
Structure/function o f the vitamin D receptor
vitaruin D receptor is essential to form a complex with a receptor auxiliary
factor required for high affinity binding to the vitamin D responsive element.
Mol Endocrinol 8:159-172; 1994.
21. Noda, M., Vogel, R. L., Craig, A. M., Prahl, J., DeLuca, H. F., and Denhardt, D. T. Identification of a DNA sequence responsible for binding of the
1,25~lihydroxyvitamin D 3 receptor and 1,25-dihydroxyvitamin D 3 enhancement of mouse secreted phosphoprotein 1 (Spp-I or osteopontin) gene expression. Proc Natl Acad Sci USA 87:9995-9999; 1990.
Bone Vol. 17, No. 2, Supplement
August 1995:33S-38S
26.
27.
22. Ohyama, Y., Ozono, K., Uchida, M., Shinki, T., Kato, S., Suda, T., Yamamoto, O., Noshiro, M., and Kato, Y. Identification of a vitamin D-responsive element in the 5' flanking region of the rat 25-hydroxyvitamin D 3 24hydroxylase gene. J Biol Chem 269:10545-10550; 1994.
28.
23. Rosen, E. D., Beninghof, E. G., and Koenig, R. J. Dimerization interfaces of
thyroid hormone, retinoic acid, vitamin D, and retinoid X receptors. J Biol
Chem 268:11534-11541; 1993.
29.
24. Ross, T. K., Darwish, H. M., Moss, V. E., and DeLuca, H. F. Vitamin
D-influenced gene expression via a ligand-independent, receptor-DNA complex intermediate. Proc Natl Acad Sci USA 90:9257-9260; 1993.
30.
25. Rut, A. R., Hewison, M. Kristjansson, K., Luisi, B., Hughes, M. R., and
O'Riordan, J. L. H. Two mutations causing vitamin D resistant rickets: Mod-
elling on the basis of steroid hormone receptor DNA-binding domain crystal
structures. Clin Endocrinol 41:581-590; 1994.
Schwabe, J. W. R., Chapman, L., Finch, J. T., and Rhodes, D. The crystal
structure of the estrogen receptor DNA-binding domain bound to DNA: How
receptors discriminate between their response elements. Cell 75:567-578;
1993.
Selznick, S. H., Nakajima, S., Hsieh, J.-C., Haussler, C. A., Hanssler,
M. R., and Whitfield, G. K. Vitamin D receptors from two patients with
hereditary resistance to 1,25-dihydroxyvitamin D 3 contain point mutations in
the conserved heptad repeat region of the hormone binding domain [abstract].
J Bone Min Res 8:(suppl. 1):S138; 1993.
Sone, T., Kerner, S., and Pike, J. W. Vitamin D receptor interaction with
specific DNA: Association as a 1,25-dihydroxyvitamin D3-modulated heterodimer. J Biol Chem 266:23296--23305; 1991.
Zecbel, C., Shen, X.-Q., Chen, J.-Y., Chen, Z.-P., Chambon, P., and Gronemeyer, H. The dimerization interfaces formed between the DNA binding domains of RXR, RAR and TR determine the binding specificity and polarity of
the full-length receptors to direct repeats. EMBO J 13:1425-1433; 1994.
Zierold, C., Darwish, H. M., and DeLuca, H. F. Identification of a vitamin
D-responsive element in the rat calcidiol (25-dihydroxyvitamin D3) 24hydroxylase gene. Proc Nail Acad Sci USA 91:900-902; 1994.