Molecular action of the vitamin D hormones

Introduction
1α,25-Dihydroxyvitamin D3, also known as calcitriol,
exerts its effects in cells by both genomic and
non genomic mechanisms. These involve long-term
modulation of gene expression and short-term activation
of intracellular signalling pathways, respectively.
The genomic actions are mediated by the vitamin
D receptor (VDR) which, on binding the hormone,
interacts with the DNA to induce or inhibit new protein
synthesis. Nongenomic actions are mediated by a
membrane receptor that is distinct from the VDR. The
binding of 1α,25(OH)2D3 to the membrane receptor triggers signal transduction pathways which involve
second messengers and which modulate the genomic
actions of the hormone.
Historically, 24R,25(OH)2D3 has been considered
by many to possess little or no intrinsic activity, its
formation serving only to divert the metabolism of
25(OH)D3 away from 1α,25(OH)2D3. However, several
reports indicate that 24R,25(OH)2D3 is a functionally
independent hormone and plays a crucial
role in intramembranous and endochondral bone
formation and in the repair of bone fractures. The genomic action of 1α,25(OH)2D3 is mediated
by the VDR, which functions as a ligand-activated
transcription factor in the cells of target tissues. The
sequence of events involved in the control of gene transcription
by the VDR is (1) binding of 1α,25(OH)2D3
to the VDR in the cytosol, (2) translocation of the
hormone–receptor complex to the nucleus, (3)
binding of VDR–RXR heterodimers (RXR, retinoid
X receptor) or, less commonly, VDR homodimers
to the vitamin D response element (VDRE) in the
promoter of primary vitamin D-responding genes
and (4) recruitment of other nuclear proteins into the
transcriptional pre-initiation complex. The VDRE
functions as a transcriptional enhancer.
One must always bear in mind that an increase in
the level of mRNA is not necessarily due to increased
transcription: an increased mRNA stability will also
lead to its accumulation.
Expression and regulation of the VDR
The VDR is a protein of 53 kDa which selectively
binds 1α,25(OH)2D3 with high affi nity. The protein
is a type II member of the nuclear hormone receptor
superfamily and possesses the characteristic two zinc
fi nger motifs in the DNA binding site (see Section
6.8.5). The VDR is present in most tissues that have
been examined, including activated immune cells
such as T lymphocytes, where it plays a role in modulating
the levels of cytokines such as interleukin-2.
Bone, kidney and especially small intestine have high
levels of receptor compared to other tissues. There are
no isoforms of the VDR. The level of VDR expression
in a cell determines the magnitude of the response
evoked by 1α,25(OH)2D3. Within each target tissue,
the level of VDR is not fi xed but rather it is dynamically
regulated by multiple factors. These include
1α,25(OH)2D3, which up-regulates the amount of
receptor (homologous regulation), and other hormones
and growth factors which may cause up- or
down-regulation of receptor abundance (heterologous
regulation). The mechanisms underlying the
regulation of VDR abundance include alterations in
the rate of transcription of the VDR gene, the stability
of the VDR mRNA and post-translational events. The
protein kinase A (PKA) and protein kinase C (PKC)
signal transduction pathways interact at the level of
VDR regulation. Activation of the PKA pathway by
forskolin up-regulates VDR gene expression whereas
activation of the PKC pathway by phorbol ester downregulates
VDR gene expression (Krishnan & Feldman,
1991, 1992). There are profound tissue- and cell-specifi
c variations in VDR regulation.
Post-translational modifi cation of the VDR by
phosphorylation
The binding of 1α,25(OH)2D3 to the VDR results in
a substantial increase in phosphorylation of the receptor
(Brown & DeLuca, 1990). Two sites of serine
(Ser) phosphorylation on the human VDR have been
identifi ed, each with a different function. Ser-51 in the
zinc fi nger region is phosphorylated by PKC (Hsieh et
al., 1991), a post-translational modifi cation that inhibits
the receptor’s ability to interact with the VDRE
(Hsieh et al., 1993). Phosphorylation of Ser-208 in the
ligand-binding domain by casein kinase II enhances
the transcriptional activating capacity of the receptor,
with no effect on receptor–ligand binding, receptor
partitioning into the nucleus or association of receptor
with a VDRE (Jurutka et al., 1996). Replacement of
Ser-51 or Ser-208 with amino acids that are incapable
of being phosphorylated does not affect DNA binding
or attenuate 1α,25(OH)2D3-mediated transcriptional
activation. This demonstrates that phosphorylation of
the two serine residues is not an obligatory switch for
VDR function; rather, these modifi cations represent
both positive (casein kinase II) and negative (PKC)
modulatory mechanisms that apparently govern
receptor activity under appropriate cellular conditions.
Jurutka et al. (1996) envisaged two populations
of liganded VDR: one that is hypophosphorylated at
Ser-208, yet still active in transcriptional enhancement,
and a superactive, hyperphosphorylated form
that is even more effective at co-operatively recruiting
coactivators and/or basal transcription factors.
Desai et al. (1995) used staurosporine, an inhibitor
of PKC and related protein kinases, and okadaic
acid, an inhibitor of protein phosphatases, to investigate
the contribution of VDR phosphorylation/
dephosphorylation to vitamin D-stimulated transcription
of the rat osteocalcin gene. The results
suggested the presence of at least two functionally
distinct phosphorylation sites on the VDR. At one site,
staurosporine inhibits a phosphorylation event that is
specifi cally required for the intrinsic transactivation
function of VDR–RXR heterodimers. At the other
site, okadaic acid inhibits a post-translational dephosphorylation
event that is required for VDRE binding
of VDR-containing transcription factor complexes.
200 Vitamins: their role in the human body
Nuclear localization of the liganded VDR
The unoccupied VDR exists in equilibrium between
the cytosolic and nuclear compartments of the target
cell. Receptor–hormone binding shifts this equilibrium
to favour nuclear localization (Walters et
al., 1986). The hormone–receptor complex rapidly
translocates to the nucleus along microtubules facilitated
by specialized motor proteins. Disruption of
microtubular integrity impairs the genomic response
to 1α,25-dihydroxyvitamin D3 in human monocytes,
which clearly underlines the physiological importance
of the intracellular tubulin transport system
(Kamimura et al., 1995).
Role of ligands in VDR–RXR heterodimer binding
to DNA
Thompson et al. (1998) investigated the molecular
function of 1α,25(OH)2D3 and 9-cis retinoic acid ligands
in the binding of the VDR and RXR to mouse osteopontin
and rat osteocalcin VDREs. Effi cient binding
to either VDRE occurred as a VDR–RXR heterodimer,
not as a VDR homodimer. 1α,25-Dihydroxyvitamin
D3 dramatically enhanced heterodimer–VDRE interaction,
whereas somewhat higher concentrations of
9-cis retinoic acid inhibited this association. A possible
explanation for this inhibition is that the binding
of 9-cis retinoic acid to the RXR partner destabilizes
the heterodimer and induces RXR homodimer formation.
MacDonald et al. (1993) offered this explanation
when they demonstrated that 9-cis retinoic acid
repressed vitamin D3-induced transactivation of the
osteocalcin gene. Thus liganded RXR is diverted from
vitamin D-activated transcription toward expression
of vitamin A-dependent genes.
Thompson et al. (1998) showed that the transcriptional
response to hormone depends on the sequential
order in which the components assemble. They
proposed the existence of two alternative allosteric
pathways for VDR–RXR association and response to
ligand. (1) An unliganded VDR associates with RXR
to form an apo-heterodimer in solution. Subsequent
binding of 1α,25(OH)2D3 induces a conformational
change in the heterodimer, which results in enhanced
binding to the VDRE. The RXR partner can readily be
dissociated from the DNA-bound heterodimer by the
addition of 9-cis retinoic acid, leading to the formation
of RXR homodimers that mediate retinoid-responsive
pathways. This 9-cis retinoic acid-receptive
conformation is proposed to exist also in monomeric
RXR and in the apo-VDR–RXR. (2) When VDR
binds 1α,25(OH)2D3 before heterodimer formation,
it is postulated to acquire a conformation distinct
from that in the fi rst pathway. After heterodimerization,
the liganded VDR allosterically alters the ligandbinding
domain of its RXR partner, rendering the
RXR unable to bind its own ligand and thus making
the heterodimer resistant to 9-cis retinoic acid-elicited
dissociation.
Interaction of the VDR with basal transcription
factor TFIIB
In the formation of the pre-initiation complex on a
gene promoter, the binding of the basal transcription
factor TFIIB to the TATA-binding protein is a requisite
for the recruitment of RNA polymerase II (Section
6.5.3). VDR interacts with TFIIB through a highly
specifi c, ligand-independent, direct protein–protein
contact and enhances transcription in the manner
of an activator (Blanco et al., 1995; MacDonald et al.,
1995).