C H A P T E R
23
Target Genes: Bone Proteins
Gerald J. Atkins 1, David M. Findlay 1, Paul H. Anderson 2,
Howard A. Morris 2
1
Bone Cell Biology Group, Discipline of Orthopaedics and Trauma, University of Adelaide, Adelaide,
South Australia, Australia, 2 Chemical Pathology, SA Pathology, Adelaide, South Australia, Australia;
School of Pharmacy and Medical Sciences, University of South Australia, Adelaide, South Australia, Australia
VITAMIN D AND SKELETAL
HOMEOSTASIS
Genome-wide analyses indicate vitamin D, through
its active metabolite 1,25-dihydroxyvitamin D3 (1,25
(OH)2D3) and the vitamin D receptor (VDR), has a poten-
tial to regulate the expression of some 3000 genes [1].
However, current evidence indicates that the strongest
phenotype exhibited by vitamin-D-deficient humans or
animals relates to impaired skeletal health. The first
scientific reports in the early 1900s that rickets was
due to vitamin D3 deficiency placed vitamin D as central
to the regulation of calcium and phosphate homeostasis.
In this context, it appears that the most critical and most
widely studied endocrine role of vitamin D is its contri-
bution to the maintenance of plasma calcium and phos-
phate at physiological levels through regulation of
intestinal absorption of dietary calcium and phosphate.
A central question, however, is whether vitamin D
acts directly on bone tissue to modulate bone mineral
homeostasis and bone strength. This question has been
difficult to answer conclusively due in part to the direct
actions of vitamin D on plasma calcium and phosphate
levels, which indirectly affect bone mineralization and
structure. One direct action that has been clearly demon-
strated is the ability of plasma 1,25(OH)2D3, at least at
supraphysiological levels, to stimulate bone resorption
by the activation of osteoclasts [2].
The effects of vitamin D deficiency on bone in vivo
can apparently be largely corrected by increasing die-
tary calcium and phosphate [3,4], suggesting that
vitamin D is not an absolute requirement for optimal
bone health. Indeed, the fact that the osteomalacic
Vitamin D, Third Edition DOI: 10.1016/B978-0-12-381978-9.10023-X
phenotype observed in the vitamin D receptor gene
knockout (VdrKO) mouse can be rescued by feeding
a diet containing high levels of calcium and phosphate
has led some to conclude that VDR-mediated activity
in bone is essentially redundant [5,6]. Others have sug-
gested that actions of VDR in bone may in fact impair
mineralization [7,8]. These conclusions are, however,
difficult to reconcile against an accumulating large
body of evidence indicating that vitamin D activity in
bone is critical for bone cell differentiation and optimal
mineral status [9e12]. In this chapter, we examine the
evidence for direct effects of vitamin D on the bone as
a tissue, and its actions on the constituent cells of
bone, in particular bone-matrix-forming osteoblasts,
osteocytes, and bone-resorbing osteoclasts.
There is now evidence that each of the major bone
cells is capable of producing 1,25(OH)2D3 from the
25-hydroxyvitamin D3 (25(OH)D3) precursor, and that
this activity is likely to account for the skeletal effects
of circulating 25(OH)D3 (see Fig. 23.1). On the weight
of this evidence, we have proposed that bone is an
intracrine organ of vitamin D metabolism [13]. The
actions of 1,25(OH)2D3 are mediated ultimately by
direct effects on individual vitamin-D-responsive
genes. However, the effects of vitamin D on bone tissue
as a whole are not yet fully understood but are likely
due to a combination of direct effects via VDREs, down-
stream effects of the induced gene expression and
effects at specific stages of bone cell proliferation and
differentiation. The effects of 1,25(OH)2D3 on important
transcription factors, which in turn govern a down-
stream program of gene expression, will also be
considered.
411 Copyright Ó 2011 Elsevier Inc. All rights reserved.
412 23. TARGET GENES: BONE PROTEINS

The 1a-hydroxylation of 25(OH)D3 to 1,25(OH)2D3 in

UVb bone cells was reported almost three decades ago
Skin [23e25] and yet only recently has evidence arisen to
7-dehydrocholesterol suggest that locally produced 1,25(OH)2D3 in osteo-
Vitamin D3 blasts plays a role in osteoblast differentiation and
mineralization and in the regulation of osteoclastogene-
Liver
sis and osteoclast activity [9e12,26,27]. We and others
25D have characterized Cyp27b1 promoter activity and
Cyp27b1 mRNA expression in bone tissue associated
bone cells with trabecular bone and growth plate [13,16,28] (see
25D Fig. 23.2). Cyp27b1 mRNA levels were significantly
higher in rat fetal bone than in adult bone and were
Bone shown by in situ hybridization to be present largely in
CYP27B1 growth plate chondrocytes and osteoblasts [29]. We
have demonstrated the age-related regulation of
1,25D Cyp27b1 mRNA expression in rat bone, which is also
increased in the presence of high dietary calcium levels
and is positively associated with bone mineralization
VD
R +1 [20,30]. Similar findings of a relationship between circu-
lating 25(OH)2D levels and osteoid thickness have been
VDRE
reported in two separate clinical studies [31,32]. Further-
more, the expression of bone Cyp27B1 mRNA is distinct
osteoblasts - proliferation from the regulation of renal Cyp27B1 mRNA [33]. The
- differentiation
- mineralization levels of Cyp24 mRNA in bone, a gene exquisitely regu-
osteoclasts - development lated by 1,25(OH)2D3, are coupled with the levels of
- activity bone Cyp27b21 mRNA expression and independent of
osteocytes - function changes to circulating levels of 1,25(OH)2D3, suggesting
that the local bone production of 1,25(OH)2D3 is respon-
FIGURE 23.1 A major source of active 1,25(OH)2D3 to regulate sible for regulating CYP24 activity [20,34].
bone processes of turnover and mineral homeostasis is now consid-
ered to be the bone itself. The action of the 1a-hydroxylase enzyme
(CYP27B1) in bone cells including osteoblasts, osteoclasts, and osteo-
cytes, gives rise to the active form of vitamin D, 1,25(OH)2D3. 1,25
(OH)2D3 binds to the VDR in the nucleus of target cells whereby it can
alter gene transcription of vitamin-D-responsive genes, including
those involved in essential bone cell activities, such as bone formation
and resorption.

EXOGENOUS AND ENDOGENOUS

SOURCES OF 1,25(OH)2D3
Circulating 1,25(OH)2D3, under nonpathological
conditions, is derived by the actions of the renal 25
hydroxyvitamin D 1a-hydroxylase (CYP27B1) enzyme,
which is highly expressed under certain conditions in
the proximal tubular epithelial cells of the kidney
[14e16]. CYP27B1 is also expressed in a wide range of
extrarenal tissues including bone. Except in the cases
of sarcoidosis [17] and placentation [18], extrarenal FIGURE 23.2 Bioluminescence grayscale heat-map showing
production of 1,25(OH)2D3 does not appear to con- regions of Cyp27b1 promoter activity in a femur (A) of the 1501 bp
tribute significantly to circulating levels [19,20]. Thus, Cyp27b1 promoter-Luciferase reporter transgenic mouse. The darker
local or cell-specific production of 1,25(OH)2D3 in bone intensities of gray (B) represent greater activity of the CYP27B1
promoter. The overlaid grayscale heat-map with the femur image
and other tissues has been generally postulated to act (C) demonstrates greater Cyp27b1 promoter activity in proximal and
in an autocrine or paracrine manner to regulate param- distal regions of the bone consistent with growth plate and trabecular
eters of cell growth and differentiation [21,22]. bone structures.

III. MINERAL AND BONE HOMEOSTASIS

 EXOGENOUS AND ENDOGENOUS SOURCES OF 1,25(OH)2D3
In human osteoblasts, CYP27B1 mRNA expression
and conversion of 25(OH)D3 into 1,25(OH)2D3 in
transformed osteoblasts were demonstrated by Van
Driel and colleagues. By using the pan inhibitor keto-
conazole, they showed the reliance for this effect on
cytochrome P450 activity [9]. We have also shown
the expression of CYP27B1 mRNA in human primary
osteoblasts isolated from adult femoral bone samples,
and human osteosarcoma cell lines, and that 1,25
(OH)2D3 can be produced from 25(OH)D3 at physio-
logical concentrations in these cells [12,35]. Using
RNAi gene silencing, the synthesis of 1,25(OH)2D3
and the expression of osteocalcin, osteopontin, RANKL,
and CYP24 mRNA in response to 25(OH)D3, were
all dependent on CYP27B1 activity [12,35]. Thus,
autocrine 1,25(OH)2D3 synthesis and activity is a
common feature of human osteoblastic cells. We also
showed that treatment with physiological levels of
25(OH)D3 inhibited cell proliferation and stimulated
osteoblast differentiation, increasing the degree of
matrix mineralization by human osteoblasts [12]. 25
(OH)D3 mainly circulates in vivo bound to the
vitamin-D-binding protein, DBP, and this interaction
is thought to exclude 25(OH)D3 from entering cells
by passive diffusion [36]. The DBP-25(OH)D3 complex
is reabsorbed specifically by renal tubules via the
expression of two receptors for DBP, cubilin and
megalin [37,38]. In further support of 25(OH)D3
metabolism representing a physiologically important
pathway in osteoblasts, human osteoblastic cells
have been shown to express both cubilin and megalin
[9,35].
Many studies have shown an effect of 1,25(OH)2D3
on osteoclast formation, focusing on indirect effects
via the osteoblast. Certainly, these effects are impor-
tant, as indicated by studies using Vdr-null osteoblasts
[39]. However, we have recently demonstrated that
human peripheral blood mononuclear-cell-derived
osteoclasts convert 25(OH)D3 into 1,25(OH)2D3 [27].
As will be discussed further below, conversion of 25
(OH)D3 to 1,25(OH)2D3 in osteoclasts dose-dependently
inhibited the resorptive activity, with a maximal effect
(~30% inhibition) seen at 25(OH)D3 levels >50 nmol/l.
These data suggest that 25(OH)D3 metabolism in cells
of the osteoclast lineage optimizes osteoclastogenesis
and regulates the resorptive behavior of mature osteo-
clasts. Cells of the monocyte/macrophage lineage have
been shown to express Cyp27b1 mRNA and convert
25(OH)D3 into 1,25(OH)2D3 [40,41]. Bone marrow
macrophages also convert 25(OH)D3 into either 1,25
(OH)2D3 or 24,25(OH)2D3. Reichel and coworkers [42]
demonstrated that, upon exposure to recombinant
human interferon-gamma (IFN-g), bone-marrow-derived
macrophages initially synthesized 1,25(OH)2D3 from
25(OH)D3.
III. MINERAL AND BONE HOMEOSTASIS
413
Growth plate chondrocytes also express Cyp27b1
mRNA and display 1a-hydroxylase activity [43,44].
Evidence suggests that 1,25(OH)2D3-VDR signaling is
an important pathway for chondrocyte support of osteo-
clastogenesis during bone remodeling at the growth
plate [45]. More recently, gene deletion and transgenic
mouse models of CYP27B1 activity in chondrocytes
demonstrated that locally produced 1,25(OH)2D3 in
these cells regulated RANKL-mediated osteoclastogene-
sis, endochondral ossification, and chondrocyte devel-
opment in vivo [46].
Intriguing preliminary data have been obtained sug-
gesting that the locally produced 1,25(OH)2D3 in bone
tissue exerts a differential response from that derived
from the circulation. Thus in a rodent model, when
circulating levels of 25(OH)D3 are below 80 nmol/L,
bone loss is detectable as a result of increased bone
resorption, increased osteoclastogenesis, and increased
Rankl expression, driven by elevated circulating 1,25
(OH)2D3 levels [47]. On the other hand, when 25(OH)
D3 levels are adequate and metabolized by bone cells
to 1,25(OH)2D3 then Rankl expression is minimized.
Furthermore there is clearly a differential response to
induction of Cyp24 when osteoblastic cells are incubated
with 1,25(OH)2D3 or 25(OH)D3 in vitro. Cells incubated
with 25(OH)D3 at levels of approximately 100 nmol/L
induce vitamin-D-responsive genes such as osteocalcin
and osteopontin in a CYP27B1-dependent manner.
Cyp24 is not significantly induced until levels of 25
(OH)D3 reach 400 nmol/L. However, when 1,25
(OH)2D3 is incubated with osteoblastic cells, Cyp24 is
the first gene to be induced at the lowest levels of 1,25
(OH)2D3 [11]. The mechanism of such differential effects
is unknown at this time; however it could be related to
the ability of vitamin D, especially when locally metab-
olized, to induce osteoblast maturation. It has been
well established that early or pre-osteoblast-like cells
respond to vitamin D in a different manner from mature
osteoblast or pre-osteocytic-like cells, as discussed in
more detail later in this chapter.
In summary, bone cells express the vitamin D meta-
bolic enzyme CYP27B1 and have the capability of con-
verting 25(OH)D3 into 1,25(OH)2D3, to elicit various
effects central to bone remodeling, including bone
resorption, bone formation, and mineralization. The
potential and known effects of 25(OH)D3 metabolism
during bone remodeling are depicted in Figure 23.3.
Further innovative studies are required to identify and
characterize these autocrine/paracrine networks of
vitamin D metabolism and activity in the in vivo bone
microenvironment to establish the relative roles of
endogenous and exogenous sources of 1,25(OH)2D3.
Such studies have the potential to establish a new para-
digm for nutritional requirements for vitamin D for
optimal skeletal health.
414 23. TARGET GENES: BONE PROTEINS

OSTEOCLASTOGENESIS BONE FORMATION

Stromal OB preOC Mature OC Immature OB Mature OB Mature OCy

RANKL ICAM attachment ( v 3) RANKL OCN

OPG RANK RANK OPG BSP-1 DMP1
M-CSF NFATc1 CTR proliferation differentiation
OC formation TRAcP differentiation FGF23
CATK
C
CA
ATK
OC
O C resorption
M-CSF
LFA RANK
RESORPTION
ICAM RANKL 25D 1,25D
LACUNA
25D 1,25D

25D 1,25D

OSTEOID 25D
1,25D
MINERALIZED
BONE

Time / Bone Remodeling

FIGURE 23.3 The potential effects of 25(OH)D3 metabolism by bone cells. This cartoon depicts the sequence of cellular events in bone
remodeling and the potential role of metabolism of 25(OH)D3 into 1,25(OH)2D3 in osteoblasts (OB) and osteocytes (OCy), as well as in osteoclast
(OC) lineage cells. Stromal osteoblasts support osteoclast differentiation from immature OC precursors (preOC), which form bone-resorbing,
mature OC. The resorbed site is then populated by immature OB, which proliferate and differentiate into mature OB. These synthesize an
unmineralized bone matrix, termed osteoid. Certain OBs become entrapped in osteoid (osteoid osteocytes) and these differentiate further into
mature OCy, a process concomitant with bone mineralization. The effects of 25(OH)D3 to 1,25(OH)2D3 conversion, both those reported in the text,
and those inferred from the known effects of exogenous 1,25(OH)2D3, at each stage are listed below each target cell type.

DIRECT ACTIONS OF VITAMIN D osteocalcin, possess vitamin-D-responsive elements

IN BONE
Osteoblast
The osteoblast regulates bone matrix synthesis and
contributes to the coordination of bone resorption
during remodeling in response to a large number of
regulatory signals of which 1,25(OH)2D3 appears to
be an important and pleiotropic member. A primary
function of the osteoblast is to secrete a specialized
organic extracellular matrix, which consists mainly of
type I collagen but also a number of noncollagenous
proteins, such as osteocalcin, osteopontin, osteonectin,
bone sialoprotein-1, proteoglycans such as versican,
and the small chondroitin sulfate proteoglycans,
decorin and biglycan. This organic matrix is ultimately
mineralized at discrete sites by the incorporation of
calcium and phosphate, to form a mature bone matrix
(for an excellent review of this topic, see [48]). In vitro
studies have shown that 1,25(OH)2D3 is capable
of regulating osteoblast gene transcription, prolifera-
tion, differentiation, and mineralization [49e51]. The
genes of matrix proteins, such as osteopontin and
(VDRE) within their promoter regions, suggesting
a direct action for 1,25(OH)2D3 on their expression.
Other important bone-matrix-associated genes, such
as type I collagen and osteonectin, may have nonclas-
sical VDREs in their promoters or be indirectly regu-
lated by 1,25(OH)2D3 [52].
As mentioned above, in vitro evidence suggests that
1,25(OH)2D3 exerts effects during both the resorptive
and synthetic phases of bone remodeling. In association
with other factors including PTH, 1,25(OH)2D3 can also
indirectly induce osteoclastogenesis by stimulating the
differentiation of bone-marrow-derived promyelocytes
and monocytes to active osteoclasts [53,54]. The tumor
necrosis factor (TNF) ligand member, RANKL, itself
a 1,25(OH)2D3-inducible protein, has been shown to be
a critical mediator of 1,25(OH)2D3, PTH or inflammatory
cytokine-induced osteoclastogenesis [55,56].
Thus, a paradox exists in that 1,25(OH)2D3 can poten-
tially induce a proresorptive expression pattern, by
increasing expression of RANKL, or a pro-osteogenic
pattern in osteoblast lineage cells. One possibility is
that the support of osteoclastogenesis and osteogen-
esis are performed by different types of specialized

III. MINERAL AND BONE HOMEOSTASIS

 DIRECT ACTIONS OF VITAMIN D IN BONE
osteoblasts. A second possibility is that these two
diverse osteoblast functions are performed at different
stages of osteoblast differentiation, as depicted in
Figure 23.3. Our previous study [51] showed that in
primary human osteoblasts, 1,25(OH)2D3 induced the
expression of RANKL in phenotypically immature osteo-
blast precursors, identified by their expression of the
marker STRO-1 [57]. However, in phenotypically mature
osteoblasts, negative for STRO-1 expression, an osteocal-
cin response predominated [51]. This differential
response was not related to levels of VDR expression,
nor to the overall ability of the cells to respond to 1,25
(OH)2D3, evidenced by the expression of other
“synthetic phase” 1,25(OH)2D3-responsive genes such
as type I collagen and bone sialoprotein-1, which were
found to be expressed independently of differentiation
stage. Similar results were obtained in mineralizing
cultures of primary mouse osteoblasts, where the 1,25
(OH)2D3 induction of RANKL expression decreased
with increasing maturation of the osteoblast [58]. A third
and combinatorial model is possible, with the sum of
signals received by the osteoblast within a permissive
window of its differentiation program determining
either an osteoclastogenic or osteogenic response.
The above studies imply that the particular cohort of
genes expressed in response to 1,25(OH)2D3 in the osteo-
blast is regulated according to their stage of differentia-
tion. Differential responses of osteoblasts to 1,25(OH)2D3
may result from different VDR signaling complexes. As
detailed elsewhere in this volume, upon ligation of 1,25
(OH)2D3 with VDR and translocation to the nucleus, the
complex forms a heterodimer with the retinoid X
receptor (RXR). This induces a VDR conformation that
is essential for effective binding to the VDRE. This asso-
ciation serves to recruit nuclear proteins as coactivators
or corepressors, necessary for VDR-mediated transcrip-
tional regulation. In short, the interaction of the 1,25
(OH)2D3-bound VDReRXR complex with nuclear
proteins forms a so-called “pre-initiation complex,”
which regulates the rate of transcription of the target
gene [59]. The constitution, and therefore the promoter
specificity, of this complex may change with differentia-
tion stage of the cell. It remains to be seen whether other
mechanisms of modifying the 1,25(OH)2D3 response,
such as CpG methylation and inactivation of the
VDRE, as has been shown for the RANKL promoter
[60], occur for other bone protein genes.
Osteoblast Proliferation and Matrix Synthesis
In general, 1,25(OH)2D3 inhibits the proliferation of
osteoblasts. This antiproliferative activity is associated
with the ability of 1,25(OH)2D3 to induce osteoblast
differentiation [61,62]. Numerous studies have shown
the inhibition by 1,25(OH)2D3 of osteoblast proliferation
in the human [51,63,64], rat [65,66], and mouse [67e69].
III. MINERAL AND BONE HOMEOSTASIS
415
The effects of 1,25(OH)2D3 on osteoblast proliferation
are, however, dependent on species and maturity of
the cell. For example, van den Bemd and coworkers
[64] found that in human MG-63 cells, 1,25(OH)2D3
could suppress proliferation, whereas in rat osteosar-
coma (ROS 17/2.8) cells, 1,25(OH)2D3 stimulated
growth. Murray and coworkers [66] found that 1,25
(OH)2D3 inhibited proliferation of the rat osteoblast
cell line, G2, and stimulated proliferation in rat osteo-
blast cell line, C12. The differences in the effects of 1,25
(OH)2D3 on osteoblast proliferation are unclear but
may be due to the differentiation state of the osteo-
blast-like cell line tested. A further complication of the
above studies is that immortalized cell lines, in general,
proliferate in an uncontrolled fashion, making interpre-
tation of the data difficult. Using carboxy-fluorescein
succinimidyl ester (CFSE), a fluorescent dye that enables
the number of cell divisions to be tracked with respect to
other fluorescently tagged proteins [70], Atkins et al. [51]
showed that while growth was inhibited overall in
a heterogeneous population of human primary osteo-
blast-like cells in the presence of 1,25(OH)2D3, immature
cells proliferated more than phenotypically mature cells.
This implies that the degree of growth inhibition by 1,25
(OH)2D3 relates to the inherent growth potential of
a particular cell type. Moreover, the effects of 1,25
(OH)2D3 on osteoblast proliferation appear to be dose-
dependent. For example, treatment of human osteo-
blasts with a low dose of 1,25(OH)2D3 (5
10e12 M)
increased proliferation, whereas a pharmacological
dose of 1,25(OH)2D3 (5
10e6 M) showed decreased
proliferation [63].
Type I collagen is expressed in the proliferative stage
of osteoblast development and is essential for the tensile
strength of bone. Stein and coworkers [71] suggested
that the inhibition of type I collagen gene expression
prevents subsequent extracellular matrix development.
The effect of 1,25(OH)2D3 on type I collagen expression
during osteoblast proliferation, however, is dependent
on the cell model of osteoblast studied. In human
MG-63 osteosarcoma cells, 1,25(OH)2D3 stimulated the
synthesis of type I collagen [64,72]. In rats and chickens,
treatment of osteoblasts with 1,25(OH)2D3 reduced type
I collagen mRNA transcription and protein synthesis
[65,73e75]. The effects of 1,25(OH)2D3 on type I collagen
synthesis in osteoblasts also appears to be conditional on
the differentiation and proliferation state of the cells. In
proliferating rat osteoblasts, acute 1,25(OH)2D3 treat-
ment inhibited the high levels of type I collagen expres-
sion found at this stage of osteoblast development.
During mineralization, however, low basal levels
of type I collagen mRNA were stimulated by acute
1,25(OH)2D3 treatment and were unaltered by chronic
1,25(OH)2D3 treatment [65]. In the mouse, while 1,25
(OH)2D3 treatment was shown to promote type I
416 23. TARGET GENES: BONE PROTEINS
collagen breakdown in calvarial osteoblasts [74], it has
also been shown to stimulate type I collagen synthesis
in early-phase MC3T3-E1 cells and have no effect in
late-phase MC3T3-E1 cells [49].
Alkaline Phosphatase
Alkaline phosphatase activity is important for the
mineralization of bone and represents a useful
biochemical marker of bone formation [76,77]. Osteo-
blasts express the bone- or tissue-non-specific isoform
of alkaline phosphatase (TNAP), which is a glycosyl-
phosphatidylinositol (GPI) anchored cell surface
protein [78]. Treatment of rat osteoblast-like cells with
1,25(OH)2D3 promoted mineralization, which was asso-
ciated with high alkaline phosphatase activity [79e81].
While the alkaline phosphatase gene promoter has no
classical VDRE, 1,25(OH)2D3 was also shown to have
a stimulatory effect on alkaline phosphatase mRNA
levels, protein synthesis, and activity in human osteo-
blasts [82,83]. The stage of differentiation of osteoblasts
has been shown to determine the response of alkaline
phosphatase expression to 1,25(OH)2D3. During the
proliferative period of osteoblast development, 1,25
(OH)2D3 inhibited the expression of alkaline phospha-
tase, whereas during mineralization, 1,25(OH)2D3 stim-
ulated alkaline phosphatase mRNA expression [65]. In
the mouse, however, 1,25(OH)2D3 stimulated alkaline
phosphatase activity only in the early phase of osteo-
blast differentiation and not in the mineralization
phase [49].
Matrix Gla Protein
Matrix Gla protein (MGP), like osteocalcin, requires
vitamin-K-dependent gamma-carboxylation for its func-
tion. MGP has been identified as a calcification inhibitor
in cartilage and vasculature since MGP-null mice die
soon after birth due to aberrant cartilage and arterial
calcification [84]. In both rat UMR 106-01 and ROS17/
2.8 cells, 1,25(OH)2D3 treatment markedly increased
MGP mRNA and protein levels [85,86]. The stimulation
of MGP mRNA by 1,25(OH)2D3 was shown to be less in
the late stages of rat osteoblast differentiation [87] than
in earlier stages of osteoblast growth [87].
Osteoid/Pre-osteocytes and Bone Mineralization
Following cessation of proliferation and production
of the collagenous matrix, certain osteoblasts become
embedded in the matrix, signaling their differentiation
into osteoid- or pre-osteocytes, sometimes referred to
as mineralizing osteocytes. Matrix mineralization is an
active process and evidence suggests that it is tightly
regulated by protein products of the osteoid-osteocyte
and mature osteocyte. It has been demonstrated that
vitamin D augments matrix mineralization in vivo
III. MINERAL AND BONE HOMEOSTASIS
and in vitro [11]. This may be due to effects of 1,25
(OH)2D3 on the expression of proteins known to
be nucleators and regulators of mineralization, and
possibly others, as will be discussed in the ensuing
sections.
Osteopontin
Osteopontin (OPN), an extracellular glycosylated
bone phosphoprotein, is one such gene that, in bone,
is secreted by late-stage osteoblasts at the mineraliza-
tion front [65,88]. In human bone marrow cultures,
and in MG-63 cells, 1,25(OH)2D3 administration was
associated with increased levels of OPN mRNA [89].
Cultured rat bone cells and ROS17/2.8 cells were both
shown to increase OPN mRNA expression and protein
secretion in response to 1,25(OH)2D3 administration
[90]. Low basal levels of OPN mRNA were seen in rat
calvarial cultures of intermediate maturity, which
were markedly up-regulated by 1,25(OH)2D3 [91].
However, 1,25(OH)2D3-mediated stimulation of OPN
mRNA in rat calvarial osteoblasts was shown to be far
greater in premineralization cells than in mature miner-
alizing cells, where levels of OPN mRNA were already
high [65]. OPN is a member of the small integrin-
binding N-linked glycoprotein (SIBLING) family since
it contains an ASARM (acidic serine- and aspartate-
rich motif) [92]. The OPN ASARM peptide with three
phosphoserines can inhibit in vitro mineralization
and is a substrate for PHEX which can rescue
mineralization.
It has been found that the helix-loop-helix-type tran-
scription factor (HES-1) is expressed in osteoblastic cells
and is suppressed by 1,25(OH)2D3. Overexpression of
HES-1 in ROS17/2.8 cells suppressed the vitamin-D-
dependent up-regulation of osteopontin gene expres-
sion in these cells [90]. TGF-b and PTH were also shown
to abrogate 1,25(OH)2D3-mediated induction of OPN in
ROS17/2.8 cells [93,94], suggesting that multiple tran-
scription factors and hormones may be involved in regu-
lating OPN activity.
Bone Sialoprotein
Bone sialoprotein (BSP) is largely specific for mineral-
ized tissues and is highly expressed during the initial
formation of bone and cementum [95]. The expression
of BSP is suppressed by 1,25(OH)2D3 treatment in rat
calvaria and ROS 17/2.8 cells [96]. A VDRE that is inte-
grated with an inverted TATA box in the rat BSP
promoter mediates the suppression of BSP transcription
[97e99]. In human bone marrow stromal cells, 1,25
(OH)2D3 treatment alone did not significantly affect
the expression of BSP mRNA [89]. However, data from
our laboratory demonstrate a positive induction of
BSP-1 mRNA by 1,25(OH)2D3 in normal human
osteoblast-like cells (G.J. Atkins et al., unpublished
 DIRECT ACTIONS OF VITAMIN D IN BONE
data), suggesting further differences between human
and rodent responses to 1,25(OH)2D3.
Osteocalcin
In the mature osteoblast, 1,25(OH)2D3 down-
regulates the expression of BSP and type I collagen
[96,100] and increases the expression of OPN and osteo-
calcin (OCN) [101,102]. OCN has a high affinity for
calcium ions of hydroxyapatite and is the most abun-
dant noncollagenous protein in bone [103]. OCN is
expressed in postproliferative osteoblasts as well as in
osteocytes. Ablation of the OCN gene in mice increases
both bone formation and bone mass, although the mech-
anism for this remains unclear [104]. While the precise
function of this protein is not well defined, it has been
shown to be a chemotactic factor for osteoclasts and their
precursors [105]. OCN has been widely used as a marker
of bone formation [77]. It has recently been reported to
be released from the matrix in the intact form rather
than as fragments during remodeling as a result of
osteoclast activity [106].
A number of studies have reported the induction of
both OCN mRNA and protein synthesis by 1,25
(OH)2D3 in human and rat bone cells [63,65,82,83,
91,107e111], although the pattern of 1,25(OH)2D3-
induced expression of the OCN gene seems to depend
on the culture system and the stage of maturity of the
cells. For example, in human MG-63 cells, 1,25(OH)2D3
induction of OCN was highest in subconfluent cultures
and decreased in confluent cultures [112]. Similarly,
OCN gene expression was found to have a decreased
responsiveness to 1,25(OH)2D3 in mineralizing human
osteoblasts, which was suggested to be due to an accu-
mulation of OCN in the extracellular matrix [113]. There
are, however, reports of 1,25(OH)2D3 down-regulating
OCN expression both in chicken embryonic osteoblasts
[75] and in mouse osteoblast cultures [114,115]. Recent
studies in our laboratories (H.A. Morris & G.J. Atkins
et al., unpublished data), using mouse primary osteo-
blasts derived from adult mouse cortical bone, indicate
that 1,25(OH)2D3 may stimulate OCN expression in
this species, in contrast to previously published findings,
and the direction of OCN expression in response to 1,25
(OH)2D3 may depend on the differentiation stage of the
cells in question.
The characterization of the OCN gene has identified
a number of factors that are potentially involved in the
development of the osteoblast phenotype. Besides
the identification of a VDRE in the distal promoter of
the OCN gene, several other promoter sites have been
shown to be critical in the expression of the OCN and
in osteoblastic differentiation. For example, the require-
ment for the osteoblast transcription factor, core binding
factor alpha (CBFA)-1/AML-3, is best demonstrated in
the CBFA-1/AML-3-null mutant mouse. These mice
III. MINERAL AND BONE HOMEOSTASIS
417
died at birth and had major skeletal deformations char-
acterized by disrupted mineralization of osteoblasts
[116,117]. Mutations of the three CBFA-1 motifs identi-
fied on the osteocalcin promoter were found to lead to
abrogation of responsiveness to 1,25(OH)2D3 [118].
Another important site identified in the promoter of
the OCN gene is the AP-1 site juxtaposed to the VDRE
of the OCN gene. While this AP-1 site is essential for
1,25(OH)2D3 induction of the OCN gene [119], the
suppression of OCN gene expression at the onset of
mineralization appears to be related to the interaction
of specific transcription factors at this AP-1 site. In
proliferating osteoblasts, the repression of OCN gene
expression is partly due to the expression of c-fos/
c-jun, which binds as a heterodimer to the AP-1 site
and blocks the binding of the VDR/RXR complex
[120,121]. In contrast, in postproliferative osteoblasts
approaching mineralization, the expression of fra-2
results in binding to the AP-1 site, which facilitates
VDR/RXR binding to the VDRE and 1,25(OH)2D3-
mediated expression of the OCN gene [121,122].
Osteocytes
Osteocytes are the most long-lived and numerous cell
type in bone tissue and have emerged over recent years
as key controllers of osteoblast behavior, bone minerali-
zation, and potentially of osteoclast activity [123,124].
While it has not been studied in detail, it is likely that
osteocytes are responsible for the majority of osteocalcin
synthesis, which as discussed above is under the control
of 1,25(OH)2D3. Other key osteocyte derived proteins
appear also to respond to 1,25(OH)2D3.
Fibroblast Growth Factor (FGF) 23
FGF23 is a bone-derived hormone with known endo-
crine activities in regulating the renal expression of
CYP27B1, having a negative effect on this enzyme and
thus inhibiting the renal synthesis of 1,25(OH)2D3. The
osteocyte is a major source of FGF23 although its expres-
sion has also been linked to osteoblasts. Deletion of the
DMP1 gene in mice has revealed a complex relationship
with FGF23, with DMP1-null cells resembling immature
osteocytes and expressing excessive levels of FGF23
[125]. It is not yet known whether immature osteocytes
predominantly express FGF23 in wild-type mice or in
human bone. Importantly, FGF23 has been demon-
strated to be 1,25(OH)2D3-responsive [126,127]. Addi-
tionally, in a recent study, Tang and coworkers
demonstrated that 25(OH)D3 metabolism in cultured
neonatal rat calvarial osteoblasts also resulted in the
up-regulation of FGF23 expression [127]. Interestingly,
the action of FGF23 on kidney tubule cells is to decrease
their expression of Cyp27b1 and inhibit their synthesis of
1,25(OH)2D3 [128]. However, FGF23 has been shown to
418 23. TARGET GENES: BONE PROTEINS
increase Cyp27b1 expression in bovine parathyroid cells
[129]. It remains to be seen whether FGF23 also regulates
osteoblast/osteocyte synthesis of 1,25(OH)2D3. Overex-
pression of FGF23 has been linked to rickets in X-linked
hypophosphatemia (XLH) and in mouse models of this
disease, such as the Hyp mouse and DMP1 mutant
mice [130e133]. The pathology in these cases appears
due to the increase in renal phosphate wasting and the
decrease in 1,25(OH)2D3 levels. Because of the reciprocal
relationship between FGF23 and circulating 1,25
(OH)2D3 levels, it might seem attractive to treat patients
with XLH with 1,25(OH)2D3. A recent study, however,
showed that treatment of these patients with calcitriol
and phosphate resulted in increased FGF23 levels indi-
cating that such a treatment may be counter-productive
[134].
Dentin Matrix Protein 1 (DMP1)
DMP1 is an acidic phosphorylated extracellular
matrix protein, and like OPN, is a member of the
SIBLING family [135]. First described as a product of
odontoblasts [136], it is now recognized to be highly
expressed in osteocytes and important for the differenti-
ation of these cells [125]. Roles for DMP1 and its proteo-
lytic cleavage products, the N-terminal 37 kDa and
C-terminal 57 kDa fragments, are not fully understood
but include nucleation of mineralization, cell attach-
ment, and possibly as a transcriptional regulator [137].
As discussed above, DMP1, FGF23, and the CYP27B1
have a complex interplay made more complex per-
haps by the findings that DMP1 is itself 1,25(OH)2D3-
responsive [138]. The in vivo significance of this
observation has yet to be determined.
Osteoclasts
Osteoclasts are multinucleated cells of the monocyte/
macrophage lineage whose primary function is to resorb
bone during bone remodeling. They accomplish this by
attaching to the mineral surface forming a tight sealing
zone, and creating a basolateral membrane termed the
ruffled border, from which bone-matrix-degrading
enzymes, such as cathepsin K, and protons are secreted
via proton pumps such as the vacuolar ATP-ase complex
(V-ATPase) and carbonic anhydrase II, to break down
the collagenous matrix and release calcium and phos-
phate ions, respectively. Osteoclasts may also be
involved in the active coupling of bone resorption to
bone formation by inducing osteoblast proliferation.
Osteoclasts are generated by the proliferation and fusion
of mononuclear precursors. These complex processes
include several key proteins whose genes are known
to be 1,25(OH)2D3-responsive. More recent data indicate
that osteoclasts, like macrophages, are a site of extrare-
nal 1,25(OH)2D3 synthesis by virtue of their expression
III. MINERAL AND BONE HOMEOSTASIS
of CYP27B1, and that the prevailing level of blood 25
(OH)D3 may govern aspects of both osteoclast formation
and their resulting activity.
Effects of Exogenous Vitamin D
Evidence suggests that 1,25(OH)2D3 has direct effects
on osteoclast precursors, increasing the expression of the
key adhesion molecule, aVb3 integrin, in both avian osteo-
clast precursor cells [139e141] and in the human myelo-
monocytic cell line, HL-60 [142], thus potentially
promoting osteoclast adhesion and the formation of the
sealing zone. 1,25(OH)2D3 has been shown to facilitate
adhesion of osteoclast precursors to stromal osteoblasts
by increasing the expression of the intercellular adhesion
molecule, ICAM-1 [143]. 1,25(OH)2D3 has also been
shown to increase the expression of the receptor for
RANKL, RANK, in HL-60 cells [144]. We have recently
demonstrated a direct effect of 1,25(OH)2D3 on
RANKL-induced osteoclast formation from the mouse
preosteoclast cell line, RAW 264.7, where 1,25(OH)2D3
in the copresence of RANKL, increased the resulting
numbers of multinucleated TRAP-positive osteoclasts
and significantly increased osteoclast multinucleation
[145].
Effects of Endogenous Vitamin D
It was recently described that, similar to other macro-
phage cell lines and primary cells, the RAW 264.7 cell
line expresses CYP27B1 and also that CYP27B1 mRNA
levels increased during their differentiation into
osteoclast-like cells [9]. We recently confirmed that
human PBMC-derived osteoclasts possess the molecular
machinery to both respond to and metabolize 25(OH)D3,
as they express cytoplasmic CYP27B1 and nuclear VDR
proteins [26]. Furthermore, CYP27B1 mRNA expression
increased in response to M-CSF/RANKL-induced
differentiation of PBMC, suggesting that 25(OH)D3
metabolism plays a role in osteoclast differentiation. In
a subsequent study, we confirmed that the capacity of
osteoclasts to synthesize 1,25(OH)2D3 increases substan-
tially with their differentiation [27]. Metabolism of 25
(OH)D3 into 1,25(OH)2D3 in human PBMC-derived osteo-
clasts resulted in the increased expression of a number
of genes, principal among these being the osteoclast
transcription factor NFATc1 [27]. This may or may
not be responsible for the observed concomitant
increase in expression of a number of osteoclastic
genes, including calcitonin receptor, tartrate-resistant
acid phosphatase (TRAcP), cathepsin K, carbonic
anhydrase, and V-ATPase. Notably, 1,25(OH)2D3 treat-
ment also up-regulated the expression of these genes
but generally did so to a lesser extent. It remains to
be determined if some or all of these genes are
vitamin-D-responsive in the classical sense or whether
their expression was as a consequence of cell
 REFERENCES
differentiation and the expression of transcription
factors, such as NFATc1, that created a permissive
environment for their expression. In the case of V-
ATPase, which is a complex multisubunit enzyme
[146], Lee et al. [147] have demonstrated that its
activity is up-regulated by exogenous 1,25(OH)2D3 by
an unidentified post-translational mechanism, suggest-
ing that 1,25(OH)2D3 effects on osteoclast behavior
may not be simply transcriptional. In terms of the overall
effect of 1,25(OH)2D3 on the osteoclast, studies pub-
lished to date [26,27] suggest that intracellular accumu-
lation of 1,25(OH)2D3 promotes osteoclast formation
but inhibits the resorptive capacity of these cells.
CONCLUDING REMARKS
Overwhelming clinical evidence suggests that
vitamin D is important for calcium/phosphate and skel-
etal homeostasis. Numerous direct and indirect effects
of 1,25(OH)2D3 have been demonstrated on a range of
critical bone proteins and 1,25(OH)2D3 appears to be
involved in their regulation at all stages of osteoblast
differentiation and, indeed, bone remodeling. Future
studies will need to unravel the complexities
surrounding the involvement of 1,25(OH)2D3 in the
coordination of these processes. However, the evidence
for a fundamental and nonredundant role for 1,25
(OH)2D3 in skeletal biology in vivo, namely, gene-
ablated mouse models, is currently lacking. As sug-
gested in earlier, this is in part due to an incomplete
analysis of these potentially informative models, rather
than a convincing lack of a biological effect. Addition-
ally, some of the apparently contradictory data
surrounding this question arise from the plethora of
model systems (immortalized cell lines versus primary
cells, the use of different mammalian and other verte-
brate species) which have been utilized to investigate
this issue, many of which may have significant and con-
founding limitations. Recent studies using 1,25(OH)2D3
analogs that show potent anabolic effects, for
example, the compound 2-methylene-19-nor-(20S)-1-a,25-
dihydroxyvitamin D3 (2-MD) when administered to
sham-operated or ovariectomized mice under otherwise
normal dietary conditions [148], and other examples
discussed elsewhere in this volume, must provide addi-
tional evidence for the bone-specific effects and impor-
tance of the natural hormone. The combination of
clinical [149,150] and preclinical [47] studies provides
strong evidence that adequate circulating 25(OH)D3
levels and not 1,25(OH)2D3 are required to optimize
skeletal health. Such findings support the concepts
described above that the local metabolism of vitamin D
and the subsequent biological activity of autocrine/
paracrine-derived 1,25(OH)2D3 enhance the maturation
III. MINERAL AND BONE HOMEOSTASIS
419
and activities of each of the major bone cell types to opti-
mize skeletal structure and strength.
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