C H A P T E R
22
Vitamin D Regulation of Type I Collagen
Expression in Bone
Barbara E. Kream 1, Alexander C. Lichtler 2
1
Departments of Medicine, the University of Connecticut Health Center, Farmington, Connecticut 06030, USA,
2
Reconstructive Sciences, the University of Connecticut Health Center, Farmington, Connecticut 06030, USA
INTRODUCTION
Vitamin D has multiple functions in humans and
animals [1e3]. It is probably best known as a nutrient
required for adequate growth and mineralization of
bone. Vitamin D, like parathyroid hormone (PTH), is
an important calcium-regulating hormone. PTH is
primarily responsible for the acute physiologic mainte-
nance of serum calcium levels. In the presence of pro-
longed hypocalcemia, PTH increases the renal
production of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3),
the active metabolite of vitamin D. 1,25(OH)2D3 together
with PTH, normalizes serum calcium levels by
increasing intestinal calcium transport, bone resorption,
and renal calcium reabsorption. To carry out many of its
biological actions in its target cells, 1,25(OH)2D3 binds
with high affinity to a nuclear receptor, the vitamin D
receptor (VDR), which in turn binds to DNA promoter
elements and recruits coactivators to regulate gene tran-
scription [4e6].
The mechanisms by which 1,25(OH)2D3 affects bone
mineralization have been the subject of intense study.
Some of the most well-recognized features of vitamin D
deficiency are undermineralized bone in children
(rickets) and adults (osteomalacia), a reduction in bone
matrix formation [7] and mineralization [8] and an alter-
ation in the pattern of collagen crosslinking [9]. Calcium
deficiency also decreases bone formation and minerali-
zation in rats [10]. Defective bone formation and miner-
alization in vitamin-D-deficient rats can be largely
corrected by the administration of calcium and phos-
phate, suggesting that the trophic effect of vitamin D
on the skeleton may be due to its ability to stimulate
intestinal calcium absorption [11e13]. Accordingly,
Vitamin D, Third Edition DOI: 10.1016/B978-0-12-381978-9.10022-8
preservation of mineral homeostasis in VDR-null mice
reverses the abnormal skeletal phenotype (including
excessive osteoid production) seen in these animals,
suggesting that a key effect of vitamin D on bone miner-
alization may be due to its stimulation of intestinal
calcium absorption [14].
The discovery of high-affinity 1,25(OH)2D3 receptors
(VDRs) in cytosolic extracts of embryonic chick and fetal
rat calvariae over three decades ago suggested that 1,25
(OH)2D3 also has direct effects on osteoblast function
[15,16]. One notable effect of 1,25(OH)2D3 action in
bone is its ability to stimulate osteoclastic bone resorp-
tion [17]. 1,25(OH)2D3 increases osteoclast formation
and bone resorption by signaling in cells of the osteoblast
lineage [18]. 1,25(OH)2D3 increases the expression of
macrophage-colony-stimulating factor, which enhances
proliferation and differentiation of osteoclast precursors,
and receptor activator of nuclear factor-kappaB ligand
(RANKL), which increases osteoclast differentiation
and survival [18,19].
In addition to its effect on osteoclast formation and
resorption, 1,25(OH)2D3 regulates the expression of
matrix proteins in osteoblasts, including type I collagen
[20], osteocalcin [21], and osteopontin [22]. Type I
collagen is the most abundant protein in the body and
comprises at least 90% of the organic component of the
bone matrix. The biochemistry, molecular biology, and
hormonal regulation of collagen genes have been exten-
sively reviewed [23e28]. Type I collagen is produced at
high levels by differentiated osteoblasts and is required
for the formation of the mineralized bone matrix. Many
other cell types synthesize and secrete type I collagen,
although in lesser amounts. Each type I collagen mole-
cule consists of three polypeptides; two a1(I) chains
403 Copyright Ó 2011 Elsevier Inc. All rights reserved.
404 22. VITAMIN D REGULATION OF TYPE I COLLAGEN EXPRESSION IN BONE
and one a2(I) chain. These polypeptides are encoded by
separate genes (Col1a1 and Col1a2, respectively)
expressed in a 2:1 ratio [29].
Collagen synthesis in bone is modulated by a variety
of hormones, growth factors, and cytokines, some of
which are produced locally by osteoblasts [28,30e32].
Insulin [33], insulin-like growth factor [34], and trans-
forming growth factor-b [35] increase type I collagen
synthesis whereas PTH [36], interleukin-1 [37], and
tumor necrosis factor [37] are inhibitory. Glucocorticoids
[38] and prostaglandins [39] can be either stimulatory or
inhibitory in vitro depending on the model and culture
conditions. Because of the critical role of type I collagen
in maintaining the structure and function of the skel-
eton, it is important to understand the mechanisms by
which 1,25(OH)2D3 regulates type I collagen expression.
An overview of the effects of vitamin D on bone biology
has been recently reviewed [3].
REGULATION OF BONE COLLAGEN
SYNTHESIS
Collagen synthesis in organ cultures of rodent calvar-
iae and cell cultures has been assessed using several
different assays [40,41]. In the most widely used assay,
calvariae and cells are incubated with radiolabeled
proline for several hours prior to the end of culture.
The incorporation of radiolabeled proline into collage-
nase-digestible protein (CDP labeling) and noncollagen
protein (NCP labeling) is measured in extracts of the
cultures using highly purified bacterial collagenase
[42]. The percent collagen synthesis is calculated from
the CDP and NCP values after correcting for the greater
abundance of proline in collagen relative to noncollagen
proteins [43]. Collagen production can also be deter-
mined by measuring the hydroxyproline content of cell
or organ cultures, since hydroxyproline is virtually
unique to collagens. These methods do not distinguish
between different types of fibrillar collagen. However,
the collagen synthesized by bone organ cultures and
most osteoblastic cell cultures is largely type I (>95%)
so that the CDP labeling value usually reflects type I
collagen synthesis. If desired, the production of different
collagen types can be distinguished by ion-exchange
chromatography and polyacrylamide gel electrophoresis
of radiolabeled extracts of cell or organ cultures [44,45].
Type I collagen expression in human cell cultures has
also been assessed by measuring secretion of the procol-
lagen I C-terminal propeptide [46,47]. Finally, specific
cDNA probes in Northern blotting and allele-specific
primers in reverse transcriptase-polymerase chain reac-
tion assays have been used to assess collagen mRNA
expression in bone models. The measurement of the
effect of 1,25(OH)2D3 on collagen synthesis and mRNA
III. MINERAL AND BONE HOMEOSTASIS
levels has given comparable results using these different
assays.
1,25(OH)2D3 inhibits collagen synthesis in organ
cultures of 21-day fetal rat calvariae [20] and neonatal
mouse calvariae [48] with little or no effect on noncolla-
gen protein synthesis. Maximal inhibition of collagen
synthesis by 1,25(OH)2D3 in rat calvariae (about 50%)
occurs at 10 nM [20]. 1,24R,25-(OH)3D3 also inhibits
collagen synthesis but is less potent than 1,25(OH)2D3
[20]. 25-(OH)D3 and 24R,25(OH)2D3 do not alter
collagen synthesis below 100 nM [20,48]. Vitamin D
metabolites inhibit collagen synthesis and stimulate
resorption of fetal rat long bones with similar relative
potencies that correlate with the affinity of the metabo-
lites for the skeletal VDRs [49]. To determine the cell
selectivity of the 1,25(OH)2D3 inhibition of collagen
synthesis, organ cultures of fetal rat calvariae were
treated with 1,25(OH)2D3 for 22 h and then incubated
with tritiated proline for the final 2 h of culture. The
central bone (mature osteoblasts) was dissected free of
the periosteum (less mature osteoprogenitors and fibro-
blasts) and both compartments were analyzed sepa-
rately for the incorporation of tritiated proline. 1,25
(OH)2D3 decreases collagen synthesis in the central
bone but not the periosteum, indicating selectivity of
the 1,25(OH)2D3 effect for mature osteoblasts [50,51].
Using an in vivo protocol in which neonatal rats were
given multiple injections of tritiated proline to radio-
label newly synthesized bone matrix, 25 ng of 1,25
(OH)2D3 given on days 1, 3, and 5 inhibited bone matrix
synthesis as assessed by histomorphometry of autora-
diographs of tibia and calvariae [52].
1,25(OH)2D3 also inhibits collagen production in rat
osteoblastic osteosarcoma ROS 17/2.8 cells [53], primary
rat [54,55] and mouse osteoblastic cells [56], and an
immortalized murine osteoblast cell line (MMB-1) [57].
1,25(OH)2D3 has a greater inhibitory effect on type I
collagen synthesis during log phase growth of primary
murine osteoblastic cells than at confluence, perhaps
because proliferating cells contained more VDRs [58].
Likewise, 1,25(OH)2D3 inhibition of collagen synthesis
is greater in sparse cultures of MMB-1 cells that have
higher VDR levels than confluent MMB-1 cells [59].
1,25(OH)2D3 inhibition of collagen synthesis is equiva-
lent in sparse and confluent rat primary osteoblastic
cells [54], but VDR number did not change during
growth of the cells [60]. Taken together, these data
show that the extent of inhibition of collagen synthesis
by 1,25(OH)2D3 is largely determined by the cellular
quantity of VDRs. 1,25(OH)2D3 inhibits collagen
mRNA levels during the proliferative phase of long-
term cultures of rat primary osteoblastic cells [61] and
prevents the formation of mineralized bone nodules by
these cultures [61,62]. These studies show that 1,25
(OH)2D3 inhibits the differentiation of osteoprogenitors
 MOLECULAR MECHANISMS OF REGULATION
that form mineralized nodules in primary rat osteo-
blastic cell cultures [62]. However, the inhibition of
nodule formation by 1,25(OH)2D3 may be secondary to
the suppression of type I collagen synthesis in the
cultures.
In contrast to the inhibitory effects described above,
1,25(OH)2D3 transiently stimulates collagen and noncol-
lagen protein synthesis (about twofold), which peaks
between 12 and 24 h, in the immortalized murine osteo-
blastic cell line MC3T3-E1 [63]. In this study, the percent
collagen synthesized by the cultures (collagen relative to
total protein synthesis) was not reported; as a result, it
was not possible to determine the selectivity of the
1,25(OH)2D3 effect for collagen synthesis. 1,25(OH)2D3
also increases collagen expression in the human osteo-
blastic osteosarcoma cell line MG-63 [46,64,65] and
primary cultures of human osteoblastic cells [66]. Inter-
estingly, the increase in collagen synthesis by 1,25
(OH)2D3 in MG63 cells is blocked by insulin-like growth
factor binding protein 5, which interacts directly to the
VDR and prevents heterodimerization with the retinoid
X receptor RXR [65]. However, in other studies, 1,25
(OH)2D3 has been shown to decrease the percent
collagen synthesis in MC3T3-E1 cells [67,68]. MC3T3-E1
and MG-63 represent preosteoblastic cells that undergo
in vitro osteogenic differentiation with ascorbic acid;
1,25(OH)2D3 inhibits cell growth and increases osteocal-
cin expression and alkaline phosphatase activity in
both cell lines. MC3T3E1 cells, like most immortalized
osteoblastic cell lines, display significant phenotypic
variation [69]. Therefore some of these discrepant results
may be due to variations in the cells used for the exper-
iments. Collectively, these data suggest that 1,25(OH)2D3
may act as a differentiating hormone in early cells of the
osteoblast lineage, which results in increased type I
collagen expression. In contrast, 1,25(OH)2D3 inhibits
type I collagen expression in mature osteoblasts.
MOLECULAR MECHANISMS
OF REGULATION
Some early studies showed that 1,25(OH)2D3
represses collagen synthesis in mature osteoblasts at
a pretranslational level [50]. Measurements of procolla-
gen mRNA activity by translation of total RNA in a retic-
ulocyte lysate first showed that 1,25(OH)2D3 inhibited
collagen mRNA in the osteoblast-rich central bone but
not the periosteum of 21-day fetal rat calvariae [50].
1,25(OH)2D3 at 10 nM inhibited procollagen mRNA
activity at 6 h; maximal inhibition of about 50% occurred
at 24 h [50]. A single subcutaneous injection of 1,25
(OH)2D3 (1.6 ng/g body weight) also decreased procol-
lagen mRNA activity in calvariae [50]. Subsequently,
specific cDNA probes were used to show that 1,25
III. MINERAL AND BONE HOMEOSTASIS
405
(OH)2D3 inhibited Col1a1 mRNA levels in ROS 17/2.8
cells [53], primary rat [55], and chick calvarial osteo-
blastic cells [70,71]. Nuclear run-on assays in ROS
17/2.8 cells showed that 1,25(OH)2D3 represses Col1a1
and Col1a2 mRNA levels by a transcriptional mecha-
nism [72]. 1,25(OH)2D3 at 1 and 10 nM decreased the
rate of Col1a1 and Col1a2 transcription by about 50%,
similar to its effect on collagen synthesis and type I
collagen mRNA levels, while actin and tubulin tran-
scription were unaffected. 1,25(OH)2D3 repressed
Col1a1 and Col1a2 transcription as early as 4 h with
maximal inhibition at 24 h [72].
DNA motifs that mediate stimulatory effects of 1,25
(OH)2D3 on gene expression have been well character-
ized for several genes [6,73,74]. Vitamin-D-responsive
elements (VDREs) that mediate 1,25(OH)2D3 induction
of target genes such as human [75] and rat [76] osteocal-
cin, mouse osteopontin [77], rat 24-hydroxylase [78], and
rat calbindin D-9K [79] contain two perfect or imperfect
direct hexameric repeats of the consensus AGGTCA
motif separated by three spacer nucleotides [6,73,74].
The consensus VDRE binds a heterodimer of the VDR
and the retinoic acid X receptor (RXR) [80]. Negative
promoter elements have also been identified. The nega-
tive VDRE in the avian PTH promoter is analogous to
the consensus VDRE, since it contains two imperfect
direct repeats separated by three spacer nucleotides
and binds VDR and RXR [81]. In contrast, the negative
VDRE in the human PTH gene contains a single
AGGTTC motif, and binding of the VDR to this site
does not require RXR [82,83]. The negative VDRE of
the parathyroid-hormone-related protein (PTHrP) gene
contains two potential VDREs, one similar to the nega-
tive VDRE in the human PTH gene and another identical
to the stimulatory VDRE; both motifs bind the VDR [84].
To characterize the regions of the Col1a1 gene that are
involved in its repression by 1,25(OH)2D3, we produced
a chimeric gene containing a fragment of the rat Col1a1
gene extending from e3518 to þ116 bp fused to the
chloramphenicol acetyl transferase (CAT) reporter
gene termed ColCAT3.6 [85]. 1,25(OH)2D3 inhibited Col-
CAT3.6 activity in transiently transfected ROS 17/2.8
cells by 50%, similar to its effect on the endogenous
Col1a1 gene [85]. We then generated a series of ColCAT
constructs containing progressive 50 promoter deletions
of the Col1a1 promoter to map 1,25(OH)2D3 response
elements [86,87]. In stably transfected cells, 1,25
(OH)2D3 inhibited a Col1a1 promoter fragment deleted
to e2295 bp (ColCAT2.3) but did not affect a promoter
fragment deleted to e1670 bp [87]. These experiments
localized an inhibitory 1,25(OH)2D3 element to a region
of the Col1a1 promoter from e2295 to e1670 bp.
Sequence analysis of the Col1a1 promoter revealed
a site between e2240 and e2234 bp that had high
homology to both the human and rat osteocalcin VDREs.
406 22. VITAMIN D REGULATION OF TYPE I COLLAGEN EXPRESSION IN BONE
We hypothesized that the VDR binding to this motif
would inhibit Col1a1 transcription. Electrophoretic
mobility shift assays using VDR expressed in COS cells
or by an adenovirus vector demonstrated that the VDR
bound to this sequence in vitro [87]. However, deletion
of the sequence between e2256 and e2216 bp from the
ColCAT3.6 or ColCAT2.3 constructs did not affect the
inhibitory effect of 1,25(OH)2D3 on promoter activity
[87]. Therefore, 1,25(OH)2D3 does not inhibit Col1a1
transcription in ROS 17/2.8 cells solely via the e2240/
e2234 bp site.
To determine the effect of 1,25(OH)2D3 on Col1a1
promoter activity in vivo, we previously produced a series
of transgenic mouse lines carrying ColCAT constructs
[88,89]. 1,25(OH)2D3 inhibited ColCAT3.6 activity in
organ cultures of 6e8-day-old transgenic mouse calvariae
[90]. 1,25(OH)2D3 inhibited CAT mRNA as early as 3 h,
and maximal inhibition of CAT mRNA (50%) was seen
at 24 h. The inhibition of CAT mRNA by 1,25(OH)2D3
was not affected by cycloheximide, suggesting that new
protein synthesis is not required for the effect. A series
of Col1a1 promoter fragments deleted to e1719 bp were
fully inhibited by 1,25(OH)2D3. However, a Col1a1
promoter construct deleted to e1670 could not be
analyzed because it did not have detectable basal activity
in transgenic calvariae [91].
Subsequently, we showed that the rat Col1a1
promoter contains a homeodomain protein motif imme-
diately downstream from e1683 bp that is required for
high levels of promoter expression in osteoblasts in
vivo [89]. A similar element is also present in the rat
Col1a1 promoter [92]. In organ cultures of transgenic
mouse calvariae carrying ColCAT constructs, we showed
that 1,25(OH)2D3 inhibited CAT activity when the
promoter was further deleted to e1683 bp. Moveover,
in a transgene having the e1719 bp promoter with a large
internal deletion extending from e1284 to e318 bp, the
inhibitory action of 1,25(OH)2D3 promoter activity was
maintained (A. Ivkovic, A. C. Lichtler and B. E. Kream,
unpublished). Taken together, studies in ROS 17/2.8 cells
and transgenic calvariae suggest that down-regulation of
the Col1a1 promoter by 1,25(OH)2D3 involves sites
located between e1683/e1284 bp or in the proximal
promoter downstream from e318 bp. There are no
good matches to consensus VDREs within these regions,
suggesting several possible mechanisms. For one, 1,25
(OH)2D3 repression of Col1a1 could involve binding of
the VDR to a novel negative VDRE. Another possibility
is that 1,25(OH)2D3 inhibition of Col1a1 expression
involves displacement of a stimulatory transcription
factor(s) from its cognate DNA-binding site, similar to
the mechanism by which 1,25(OH)2D3 inhibits the inter-
leukin-2 gene [93]. It is also possible that 1,25(OH)2D3
inhibition of Col1a1 involves interaction of the VDR
with other transcription factors rather than binding of
III. MINERAL AND BONE HOMEOSTASIS
the VDR to DNA. Such a mechanism has been described
for the inhibition of collagenase expression by glucocor-
ticoids [94,95]. Finally, 1,25(OH)2D3 repression of Col1a1
expression could be mediated by alternative signal
transduction pathways. It has been suggested that
some biological effects of 1,25(OH)2D3 may be mediated
by the protein kinase C (PKC) signaling pathway [74].
We have shown that stimulation of PKC with phorbol
myristate acetate inhibits collagen synthesis in fetal rat
calvariae [96] and ColCAT3.6 expression in transgenic
mouse calvariae [97]. Therefore, 1,25(OH)2D3 activation
of the PKC pathway might inhibit Col1a1 expression.
This could be mediated by a putative 1,25(OH)2D3
membrane receptor, which activates intracellular signal
transduction pathways leading to alteration of gene tran-
scription. Future experiments to identify 1,25(OH)2D3
response elements in the Col1a1 gene will involve the
analysis of additional constructs having selected site-
directed mutations and internal promoter deletions in
cultured osteoblastic cells and transgenic mice.
The previous data provide evidence of a direct action
of vitamin D on type I collagen expression in osteoblasts.
In other systems, the effects of vitamin D may be indi-
rect. For example, vitamin D blocks the fibrotic effects
of TGFb in lung fibroblasts and epithelial cells, and
although the precise mechanism is not clear, it may
involve vitamin D inhibition of TGFb transcriptional
activation [98]. Vitamin D has also been shown to inhibit
5-azacytodine induction of TGFb and type I collagen
expression in C3H10T1/2 multipotent mesenchymal
cells [99].
CONCLUSIONS AND PERSPECTIVES
The effect of 1,25(OH)2D3 on collagen expression,
either inhibitory or stimulatory, may depend in part on
in vitro culture conditions such as cell density, the
timing and concentration of 1,25(OH)2D3 addition, the
presence of ascorbic acid, and the state of maturation
of the model. A model has been proposed based on
the premise that cells of the osteoblast lineage differ in
their response to 1,25(OH)2D3 depending on their state
of maturation [100]. 1,25(OH)2D3 stimulates osteoblast
markers in immature osteoprogenitor cells (MC3T3-E1
and MG-63 cells) but inhibits these markers in mature
osteoblasts such as rodent calvarial organ cultures,
primary rodent osteoblastic cell cultures, and ROS
17/2.8 cells [100]. Such a model is consistent with the
effects of 1,25(OH)2D3 on bone remodeling during
periods of calcium and phosphate deficiency. When
serum calcium and phosphate are low, PTH increases
the synthesis of 1,25(OH)2D3. Both hormones increase
bone resorption to increase the supply of calcium and
phosphate for soft tissues. During periods of mineral
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at sites of new osteoid formation. At the same time,
1,25(OH)2D3 may stimulate the differentiation of early
osteoprogenitors to differentiation into a new cohort of
osteoblasts that would initiate the phase of coupled
formation [100].
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