Expression of Rat Homeobox Gene, rHOX, in Developing and Adult Tissues in Mice and Regulation of Its mRNA Expression in Osteoblasts by Bone Morphogenetic Protein 2 and Parathyroid Hormone-Related Protein
Yun Shan Hu,
Hong Zhou,
Vicky Kartsogiannis,
John A. Eisman,
T. John Martin and
Kong Wah Ng
Department of Medicine (Y.S.H., H.Z., V.K., T.J.M., K.W.N.) The
University of Melbourne St. Vincents Hospital Fitzroy,
Victoria, 3065 Australia
The Garvan Institute of Medical
Research (J.A.E.), St. Vincents Hospital Darlinghurst, New
South Wales 2010, Australia
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ABSTRACT
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The rat homeobox gene, rHox, was cloned from a rat
osteosarcoma cDNA library. Southwestern and gel mobility shift analyses
showed that rHox binds to the promoter regions of collagen (
1)I and
osteocalcin genes while transient transfection with rHox resulted in
repression of their respective promoter activities. In situ
hybridization studies showed that rHox mRNA was widely expressed in
osteoblasts, chondrocytes, skeletal muscle, skin epidermis, and
bronchial and intestinal epithelial cells, as well as cardiac muscle in
embryonic and newborn mice. However in 3-month-old mice, rHox mRNA
expression was restricted to osteoblasts, megakaryocytes, and
myocardium. Bone morphogenetic protein 2, a growth factor that
commits mesenchymal progenitor cells to differentiate into osteoblasts,
down-regulated rHox mRNA expression by 4050% in UMR 201, a rat
preosteoblast cell line, in a time- and dose-dependent manner. In
contrast, PTH-related protein (PTHrP), recently shown to be a
negative regulator of chondrocyte differentiation, significantly
enhanced rHox mRNA expression in UMR 10606 osteoblastic cells by
3-fold at 24 h while at the same time down-regulating expression
of pro-
1(I) collagen mRNA by 60%. Expression of rHox mRNA in
calvarial osteoblasts derived from PTHrP -/- mice was approximately
15% of that observed in similar cells obtained from normal mice. In
conclusion, current evidence suggests that rHox acts as a negative
regulator of osteoblast differentiation. Furthermore, down-regulation
of rHox mRNA by bone morphogenetic protein 2 and its up-regulation by
PTHrP support a role of the homeodomain protein, rHox, in osteoblast
differentiation.
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INTRODUCTION
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A network of protein products encoded by developmental genes
controls the coordinated expression of structural genes in normal
tissue development. Homeobox genes in Drosophila were among
the first developmental genes to be identified. Homeodomain proteins,
characterized by a conserved DNA-binding domain, function as regulatory
factors in tissue differentiation and proliferation by committing cells
to specific developmental pathways as well as playing important roles
in pattern formation (reviewed in Refs. 1, 2).
The role of homeobox genes in limb development has been
extensively studied (3, 4, 5, 6). A number of homeobox genes, regionally
expressed in the developing vertebrate limb, have been shown to be
involved in the regulation of axial skeletal structure as well as
formation of the limb bud and its organization into limb structure. For
example, in the Hox d cluster, Hox d4 d5, d6, d7,
and d8 are expressed in the posterior-most regions of the
developing embryo, including the hind limb bud (7, 8). A group of
homeobox genes related to the Drosophila gene
msh, may be involved in mediating the interaction between
the apical ectodermal ridge (AER) and the underlying mesenchyme in the
progress zone of the developing limb bud to set up the proximal-distal
axis of limb development. At least two msh-related genes are
expressed in the limb bud: Hox7 (Msx1) in both the AER
region and the progress zone mesoderm (9, 10) and Hox 8
(Msx2) in the AER distal to Hox 7 (11). The involvement of
Msx2 in limb development is suggested not only by its
expression pattern, which is slightly different from that of
Msx1 (12, 13, 14), but also by a dominantly inherited
Msx2 mutation in a kindred with Boston-type craniosynostosis
in which some affected individuals also exhibit limb abnormalities
(15). Recently, Msx1 and Msx-2 were shown to be
expressed in adult osteoblast cells (16, 17). A group of homeobox genes
homologous to Drosophila paired and gooseberry as
well as mouse Pax-3, but lacking a paired-box sequence, is
also associated with limb development. This group includes chick
Prx-1 and mouse S8 and MHox (also
known as Pmx) (18, 19, 20, 21). The expression patterns of
Prx-1, S8, and MHox overlap with those of
Msx1 and Msx2.
Bone morphogenetic proteins (BMPs) are members of the transforming
growth factor-ß superfamily that are important in the
determination of pattern formation as well as cell differentiation.
In vivo, the implantation of BMPs into muscular tissues
induces ectopic bone formation via the endochondral route at the site
of implantation. In vitro, BMP2 can stimulate osteoblastic
differentiation in mesenchymal progenitor cells (22), converting
myoblastic and fibroblastic differentiation pathways into that of
osteoblasts. BMP2 also induces osteoblastic phenotype-related protein
expression, such as collagen
1(I), alkaline phosphatase, and
osteocalcin, in osteoblastic as well as nonosteoblastic cells (23, 24, 25, 26).
These results suggest that BMP2 not only induces the commitment of
undifferentiated mesenchymal cells into osteoblasts, but also
stimulates the maturation of committed osteoblast progenitors.
PTH-related protein (PTHrP) is expressed by chondrocytes in the resting
and prehypertrophic zones of the epiphyseal growth plate and in the
perichondrium of the developing cartilage elements of rodent limb,
predominantly in the periarticular regions early in bone development,
as well as in cells of the osteoblast lineage in the diaphyses (27, 28). Several lines of evidence have shown that PTHrP plays a major role
in modulating the rate of cartilage differentiation. For example,
targeted disruption of the PTHrP gene in mice results in premature
ossification in chondrocytes, suggesting that PTHrP has a pivotal role
in retarding the differentiation of chondrocytes (29, 30). Further
evidence was provided by Vortkamp et al. (31) to show that
PTHrP acts downstream to Indian Hedgehog in a regulatory pathway that
results in slowing down the rate of hypertrophic cartilage
differentiation.
PTHrP is expressed by osteoblasts in vivo (28) as well
as in cultured newborn rat calvarial cells, UMR 201, and UMR
10606 cells (32). However, the role of PTHrP in osteoblast
differentiation is less clearly defined. It influences osteoblasts
directly by acting through the PTH/PTHrP receptor expressed by these
cells. There is the intriguing possibility that PTHrP may also
indirectly influence osteoblast differentiation through its effects on
chondrocyte differentiation. Several lines of evidence suggest that
hypertrophic chondrocytes can transdifferentiate to osteoblasts.
In vitro cultures of explants have shown that hypertrophic
chondrocytes have osteogenic potential (33, 34, 35, 36, 37, 38). Other in
vitro studies have demonstrated that immortalized mouse
chondroblastic cells, TMC23, could spontaneously differentiate into
cells that form mineralized bone nodules and express several osteoblast
markers in culture (39) while Galotto and co-workers (40) have reported
that hypertrophic chondrocytes can differentiate toward an
osteoblast-like phenotype in vivo. It has also been shown,
in a transgenic mouse model, that the cis-acting regulatory
elements present in the flanking regions of the human osteocalcin gene
directed expression of a reporter gene specifically to cells of the
osteoblastic lineage and to hypertrophic chondrocytes (41). Therefore,
it is possible that factors such as PTHrP, which modulate chondrocyte
differentiation, could directly as well as indirectly influence
osteoblast differentiation.
We have previously identified and characterized a homeobox gene, termed
rHox, from a cDNA library derived from rat osteoblast-like cells. rHox
cDNA is 1.3 kb in length and identical at the amino acid level to the
mouse MHox gene. rHox will be referred to as rHox/MHox from
here on. rHox/MHox mRNA is expressed in Northern blot analyses of
mature as well as premature osteoblasts. Southwestern and gel mobility
shift analyses showed that rHox/MHox binds to response elements in the
promoter regions of collagen (
1)I and osteocalcin genes that are
strongly expressed in mature osteoblasts. Transient transfection assays
revealed that promoter activity in both genes were repressed by
rHox/MHox (42).
In this study, the temporal and spatial expression patterns of
rHox/MHox mRNA in the embryonic, newborn, and adult mouse were examined
by in situ hybridization and Northern blot analysis. We also
studied the manner in which rHox/MHox mRNA expression is influenced by
BMP2 and PTHrP in primary calvarial cells as well as in two
osteoblast-like cell lines, UMR 201 and UMR 10606, representing early
and late stages in osteoblast differentiation. The results suggest a
role of rHox/MHox in the control of osteoblast and chondrocyte
differentiation.
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RESULTS
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Localization of rHox/MHox mRNA Expression in Mouse Tissue
by in Situ Hybridization
The expression pattern of rHox/MHox mRNA in the
embryonic, newborn, and adult mouse was investigated by in
situ hybridization to show its tissue distribution in these three
stages of development.
rHox/MHox mRNA Expression in Mouse Embryo (Fig. 1
)
In skeletal tissues from a day 15 (D15) embryonic mouse in which only
cartilage is present, rHox/MHox mRNA was expressed in chondrocytes.
rHox/MHox mRNA was present in bronchi of lung and strongly expressed in
all layers of epidermis (Fig. 1
). This
mRNA was also expressed in skeletal muscle and intestine, just
detectable in myocardium and renal tissue, and not expressed in brain
or liver (data not shown).

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Figure 1. Expression of rHox/MHox mRNA Expression in Day 15
Embryonic Mouse Tissues
The tissues in panels A, C, and E were hybridized with antisense (AS)
rHox/MHox probe with rHox/MHox mRNA shown in dark
blue-purple. The tissues in panels B, D, and F were probed with
the sense (S) rHox/MHox probe as negative controls with a fast-green
counterstain. rHox/MHox mRNA was expressed in chondrocytes (ch) of long
bone in panel A, epithelium of bronchi (b) in panel C, and all layers
of epidermis (ep) in panel E. Original magnification: AF, x400.
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rHox/MHox mRNA Expression in Newborn Mouse Tissues (Fig. 2
)
In the newborn mouse, expression of rHox/MHox mRNA was similar to
that observed in D15 embryo with some notable differences. rHox/MHox
mRNA was still expressed in proliferating, prehypertrophic, and
hypertrophic chondrocytes. By this stage, bone formation was evident
and rHox/MHox mRNA was strongly expressed in the cuboidal multilayered
osteoblasts lining bone trabeculae but not in osteocytes (data not
shown). As in embryonic mouse, rHox/MHox mRNA was also detected in
skeletal muscle and all cellular layers of the epidermis of skin. The
expression of rHox/MHox mRNA in skeletal muscle and epidermis was much
stronger in newborn mouse compared with day 15 embryo. The level of
expression of rHox/MHox mRNA in bronchial epithelium, renal glomeruli,
and tubules (Fig. 2
), as well as
intestinal epithelium (data not shown), was similar to that observed in
the embryo. The expression of rHox/MHox mRNA was detectable in
myocardium but not in liver (data not shown). The major difference
between newborn and embryonic mouse tissues was the strong expression
of rHox/MHox mRNA in the cerebral cortex (Fig. 2
) and in other parts of
brain (data not shown).

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Figure 2. Expression of rHox/MHox mRNA in Newborn Mouse
rHox/MHox mRNA was expressed in the following tissues: A, chondrocytes
(ch); B, osteoblasts (ob); C, skin (sk); D, skeletal muscle (skm); E,
kidney (kid) where it was expressed in glomeruli (g) and proximal
tubules (p); F, Unlike embryonic mouse, rHox/MHox mRNA was strongly
expressed in newborn mouse brain (br). G, Expression of rHox/MHox mRNA
in terminal bronchioles (b) and alveoli (a) in lung tissue (lu).
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rHox/MHox mRNA Expression in Adult Mouse Tissue (Fig. 3
)
The expression of rHox/MHox mRNA in adult tissues was examined in
the 3-month-old mouse, and major differences were observed when
compared with its pattern of expression in embryonic or newborn mouse
tissues. In adult mouse bone, rHox/MHox mRNA was expressed in
osteoblasts but not in osteocytes. In bone marrow, rHox/MHox mRNA was
expressed by megakaryocytes. In the adult mouse, expression of
rHox/MHox mRNA was no longer detected in kidney, brain, or lung tissue
(Fig. 3
) and neither was it expressed in
intestine or liver (data not shown).

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Figure 3. Expression of rHox/MHox mRNA in a 3-Month-Old Adult
Mouse
rHox/MHox mRNA was strongly expressed in osteoblasts (ob) (panel A) and
megakaryocytes (m) (panel B) but not in kidney (kid) (panel C), lung
(lu) (panel D), or brain (br) (panel E).
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Northern Blot Analyses
Expression of rHox/MHox mRNA in Tissues of Newborn and Adult Mouse
(Fig. 4
)
rHox/MHox mRNA expression was further examined in tissues of newborn
and adult mouse by Northern blot analysis. These results correlated
with those demonstrated by in situ hybridization. In newborn
mouse, rHox/MHox mRNA was strongly expressed in skeletal muscle and
skin. Its expression was detected in kidney, brain, myocardium, and
lung but not in liver. In the adult mouse, rHox/MHox mRNA was expressed
in myocardium but not in skin, lung, liver, kidney, skeletal muscle,
gut, or brain.

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Figure 4. Northern Blot Analysis of rHox/MHox mRNA Expression
in Newborn (A) and Adult (B) Mouse Tissues
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Regulation of rHox/MHox mRNA Expression by BMP2 (Fig. 5
)
UMR 201 or UMR 10606 cells were treated with increasing
concentrations of human recombinant BMP2 ranging from 50 ng/ml to 600
ng/ml for 24 h. BMP2 down-regulated the steady state level of
rHox/MHox mRNA expression in a dose-dependent manner by approximately
50% in UMR 201 and by 90% in UMR 10606, respectively.

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Figure 5. BMP2 Regulation of rHox/MHox mRNA Expression in
Cells of Osteoblastic Lineage
Both preosteoblastic UMR 201 and UMR 10606 cells were treated for
24 h with increasing concentrations of BMP2. The relative
quantities of rHox/MHox mRNA expression are represented by the
rHox/MHox to 18S mRNA ratios. rHox/MHox mRNA expression was decreased
in both cell lines by BMP2.
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Regulation of rHox/Mhox mRNA Expression by PTHrP (Figs. 6
and 7
)
rHox/MHox mRNA expression in response to PTHrP was determined in
UMR 10606 cells but not in UMR 201 cells because UMR 201 cells do not
express receptors for PTH/PTHrP. UMR 10606 cells were treated with
100 ng/ml PTHrP (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34) for varying times or for 24 h with
increasing concentrations of PTHrP (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34) ranging from 1 ng/ml to 100
ng/ml. PTHrP increased rHox/MHox mRNA expression by 2- to 3-fold
compared with control in a time and dose-dependent manner (Fig. 6
). The increase of rHox/MHox mRNA
expression in response to PTHrP was first observed at 2 h and
increased up to 24 h. rHox/MHox mRNA expression was also examined
in osteoblasts from control and PTHrP -/- mouse calvaria (Fig. 7
). Expression of rHox/MHox mRNA in
calvarial osteoblasts derived from PTHrP -/- mice was approximately
15% of that observed in similar cells obtained from normal mice (Fig. 7A
). PTHrP mRNA was not detected in osteoblasts obtained from PTHrP
-/- mice (Fig. 7B
).

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Figure 6. Northern Blot Analysis for rHox/MHox mRNA
Expression in Osteoblastic UMR 10606 Cells
Cells were treated with 100 ng/ml PTHrP for 224 h (A) and for 24
h (B) with increasing concentrations of PTHrP. There was a time- and
dose-dependent stimulation of rHox/MHox mRNA expression by PTHrP.
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Figure 7. Expression of rHox/MHox mRNA in Calvaria Derived
from Control and PTHrP Knockout Mice
A, rHox/MHox mRNA expression in calvarial osteoblasts of control (+/+)
and PTHrP knockout mice (-/-). B, PTHrP was present by RT-PCR in
control (+/+) mouse calvarial osteoblasts but not in PTHrP knockout
mouse calvarial osteoblasts (-/-). The two flanking lanes are the
negative controls. The RT-PCR product in the gel was stained by
ethidium bromide and viewed under UV light.
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Regulation of pro-
1(I) Collagen mRNA Expression in UMR 10606
by PTHrP (Fig. 8
)
Treatment of UMR 10606 cells resulted in a reduction in expression of
pro-
1(I) collagen mRNA by approximately 60%. This effect was time
dependent and was evident by 4 h. In UMR 201 and UMR 10606
cells, no significant change in pro-
1(I) collagen mRNA expression
level was observed when treated with concentrations of BMP2 up to 400
ng/ml (data not shown).
Regulation of rHox/MHox mRNA in Primary Calvarial Osteoblasts by
BMP2 and PTHrP (Fig. 9
)
Primary calvarial osteoblasts from newborn mice were treated for
24 h with increasing concentrations of BMP2 (100 ng/ml to 600
ng/ml) or PTHrP (1 ng/ml to 100 ng/ml). BMP2 decreased expression of
rHox/MHox mRNA in primary mouse calvarial osteoblasts in a
dose-dependent manner by approximately 40%, while the effect of PTHrP
was minimal.

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Figure 9. Regulation of rHox/MHox mRNA in Primary Calvarial
Osteoblasts by BMP2 (A) and PTHrP (B)
Cells were treated with increasing concentrations of either factor for
24 h. The relative quantities of rHox/MHox mRNA expression are
represented by the rHox/MHox to 18S mRNA ratios. BMP2 decreased
rHox/MHox mRNA expression in a dose-dependent manner while PTHrP had
minimal effect.
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DISCUSSION
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In vertebrates, homeobox genes participate in morphogenesis, and
establishment of the body plan and influence segmentation processes
(2). For example, the mouse homeobox genes engrailed-1 and
-2 are expressed in the ventral half of the limb ectoderm
and play a role in determining dorsal-ventral polarity during limb
development (43, 44).
In this study, the high level of expression of rHox/MHox mRNA in
hypertrophic chondrocytes in embryonic mouse is consistent with the
hypothesis that homeobox genes play a major role in embryonic limb
formation (5, 6, 45). As development proceeded to the newborn stage
(day 1), rHox/MHox mRNA was expressed in osteoblasts as well as in
prehypertrophic and hypertrophic chondrocytes. With elongation of long
bone in the adult, rHox/MHox mRNA continued to be expressed in
osteoblasts. The persistent expression of rHox/MHox mRNA in
osteoblasts, but not in osteocytes, may suggest that rHox/MHox has a
role in modulating the differentiation of osteoblasts during
active stages of bone formation. The expression of rHox/MHox mRNA in
megakaryocytes within the marrow environment of adult long bone
suggests a possible role as a local growth factor not only in bone but
also in marrow cell development.
Although the physiological role of rHox/MHox in bone remains to be
further elucidated, the functional significance of rHox/MHox may be
inferred from a number of functional studies of related genes. A
transgenic mouse in which Hoxb-8 is expressed across the
entire proximal region of the forelimb bud forms a mirror-image
duplication of the resultant limb (46). Mice that are homozygous for
the gene knock-out of MHox, the mouse homolog of rat rHox,
die at the neonatal stage displaying multiple craniofacial defects. In
addition, the ossified shafts of the radius and ulna of the forelimb,
as well as the tibia and fibula of the hindlimb, in MHox
mutant neonates are bowed, shortened, and broader than wild-type bones.
Analysis of the cartilaginous precursors showed that in the long bones
of the mutants, formation of the diaphyseal ossification center was
delayed. Thus, the abnormalities evident in the limbs of the mutant
neonates can be traced to a defect in the formation and subsequent
ossification of specific cartilaginous precursors (21). In a study by
Liu et al. (47) of a transgenic mouse that overexpressed a
point mutation of Msx-2 gene, it was noted that the animal
had premature suture closure and ectopic bone formation in the
developing skull, suggesting that the Msx-2 point mutation
resulted in a constitutively active gene. These abnormalities resemble
those found in the clinical syndrome of autosomal dominant
craniosynostosis, which is also the result of a point mutation in the
human MSX-2 gene (15).
In the study by Cserjesi et al. (20), mRNA expression of
Mhox was examined in tissues derived from 9.5 days
postcoitum to 15.5 days postcoitum embyronic mouse using in
situ hybridization with a 35S-labeled RNA probe. They
concluded that MHox expression was restricted to
mesodermally derived cells in the embryo, predominantly in developing
limb bud, visceral arches, frontonasal processes, somites, and skeletal
and perichondrial tissue. In the present study, in situ
hybridization was performed in three distinct developmental stages in
the embryonic, newborn, and adult mouse, using a digoxigenin
(DIG)-labeled rHox/MHox riboprobe that enabled the study of tissues in
much greater detail at the cellular level. The results in this study
showed that rHox/MHox mRNA was expressed in a wide range of tissues and
was not confined to mesodermally derived tissues. RHox/MHox mRNA was
expressed in skin, skeletal muscle, cartilage, bone, and visceral
organs in the embryonic and newborn mouse. This may imply an action of
rHox/MHox as a general regulator essential for tissue development at
the early stages of development. RHox/MHox mRNA was also
developmentally regulated in the brain in that it was only expressed in
newborn mouse brain and not in embryonic or adult brain. Certainly the
wide distribution of rHox/MHox implies a role in tissue development
that is not confined to the skeleton. RHox/MHox expression and function
became progressively restricted during ontogeny and in the adult mouse,
rHox/MHox mRNA expression was largely restricted to osteoblasts and
marrow cells correlating with the cessation of differentiation of most
tissues by this stage.
Northern blot analysis of rHox/Mhox mRNA expression in tissues derived
from newborn and adult mice correlated with the in situ
hybridization findings. In newborn mice, rHox/MHox mRNA was strongly
expressed in skeletal muscle and skin. It was also detected in brain,
myocardium, and lung and just discernible in kidney. In adult mice
tissue, rHox/MHox mRNA was expressed in myocardium but was no longer
observed in skeletal muscle, brain, kidney, skin, or lung. In the study
conducted by Cserjesi et al. (20), MHox mRNA was strongly
expressed by adult mice myocardium, but the major difference between
the two studies lies in the absence of rHox/MHox mRNA expression in
adult skeletal muscle in this study. There is no obvious explanation
for this discrepancy.
The reduction in osteoblast rHox/MHox mRNA expression by BMP2 is
consistent with the actions of BMP2 in committing mesenchymal cells
along the osteoblast differentiation pathway. In this model, the
maturation of osteoblast progenitor cells would be facilitated by a
reduction in the expression of inhibitory factors such as rHox/MHox.
Yokouchi et al. (48) have shown that a related homeobox
gene, msx-1, is down-regulated by retinoic acid in the
course of limb bud differentiation and development. Although it was
considered that BMP2 might increase the baseline type I collagen mRNA,
which is already highly expressed in UMR 201 cells, in the course of
promoting the differentiation of these preosteoblasts, this was not
observed. However PTHrP did increase the rHox/MHox mRNA expression. In
previous studies in preosteoblastic UMR 201 cells, PTHrP mRNA
expression was down-regulated by retinoic acid in conjunction with
their differentiation (32). This suggests that PTHrP may act as an
inhibitor of osteoblast as well as chondrocyte differentiation, and
this would be consistent with the reduction of collagen
1 (I) mRNA
expression in UMR 10606 cells by PTHrP. This action of PTHrP as an
inhibitor of osteoblast differentiation may be mediated through a rise
in rHox/MHox mRNA expression. RHox/MHox mRNA was also expressed in
primary mice calvarial osteoblasts. Their response to BMP2 and PTHrP
more closely resembled those of the preosteoblastic UMR 201 than the
more differentiated 10606 in that BMP2 down-regulated rHox/MHox mRNA
expression while PTHrP had only a minimal effect.
In conclusion, rHox/MHox is the only homeobox gene to date, whose
expression has been detected from the embryonic to adult stages in
chondrocytes and osteoblasts in vivo by in situ
hybridization. The continued expression of rHox/MHox in cartilage and
osteoblasts from the embryonic to the adult stage may suggest an
important role of rHox/MHox in the modulation of bone formation.
Furthermore, the expression of rHox/MHox in actively synthesising
osteoblasts implies a regulatory function of rHox/MHox in osteoblast
differentiation. In the functional studies, BMP2, which acts to commit
undifferentiated mesenchymal progenitor cells into osteoblasts,
decreased rHox/MHox mRNA expression, while PTHrP, an inhibitor of
chondrocyte differentiation and possibly also of osteoblast
differentiation, increased its expression. These results thus provide
support for a role of rHox/MHox in regulating bone cell differentiation
that includes mediating the opposing actions of BMP2 and PTHrP in
osteoblast differentiation. However, the regulation of endochondral
bone formation is rather more complex, and this is illustrated by the
diametrically opposite phenotypic features of the MHox and PTHrP
knockout mice. The PTHrP knockouts show premature diaphyseal
ossification (29), while the MHox knockouts show a delay in diaphyseal
ossification (21). Yet if rHox/MHox acts downstream to the effects of
PTHrP on chondrocyte or osteoblast differentiation, then it might be
expected that the MHox knockout mouse should have features similar to
those of the PTHrP knockout mouse. Obviously, the regulation of
endochondral bone formation involves the interaction of many different
factors. The phenotypic differences between the two knockout strains
strongly suggest that rHox/Mhox acts on targets and is in turn,
regulated by factors that have yet to be identified. By the same
reasoning, rHox/MHox may be one of several effectors of PTHrP action,
and the final result depends on how they interact with each other. A
more thorough understanding of the regulation and functions of
rHox/MHox is therefore required before its role in chondrocyte and
osteoblast differentiation can be properly appreciated.
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MATERIALS AND METHODS
|
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BMP2 was kindly provided by the Immunology Department, Genetics
Institute, Inc. (Cambridge, MA). HPTHrP (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34) peptide was
synthesized and kindly supplied by Professor B. E. Kemp (St.
Vincents Institute of Medical Research, Melbourne, Australia).
Cell Culture
UMR 201 and UMR 10606 cells were grown in
-modified
Eagles medium (
-MEM) with 10% FCS. All cell lines were cultured
as monolayers in T-150 flasks and maintained in a humidified incubator
at 37 C with 5% CO2. Culture medium was replaced every
23 days. FCS was purchased from Commonwealth Serum Laboratories
(Melbourne, Australia).
Primary Calvarial Osteoblasts
Osteoblastic cells were prepared from the calvaria of newborn
mice by digestion with 0.1% collagenase (Worthington Biochemical Co.,
Freefold, Australia) and 0.2% dispase (Godo Shusei, Tokyo, Japan) as
previously described (49).
Synthesis of Riboprobes
Riboprobes were generated by cloning a 270-bp open reading
fragment of rHox, without the homeodomain region, into a pCMV plasmid
(Invitrogen Corp., Carlsbad, CA). The plasmid was either linearized
with HindIII and transcribed with Sp6 RNA polymerase to
generate an antisense riboprobe or linearized with ApaI and
transcribed with T7 to generate a sense riboprobe. Probes were labeled
with DIG using a RNA-labeling kit (Boehringer Mannheim GmbH, Mannheim,
Germany).
Northern Blot Analyses
Total RNA was isolated with the method described by
Chomczynskzi and Sacchi (50), dissolved in Tris-EDTA buffer,
and stored at -70 C. Total RNA was separated in a 1.5%
agarose-formaldehyde gel and then transferred to nylon membranes
(Amersham International, Buckinghamshire, UK). The membranes were
hybridized with DIG-labeled rHox riboprobe overnight at 65 C in
hybridization buffer containing 50% formamide, 5x saline-sodium
citrate (SSC), 2% blocking reagent, 0.1%
N-lauroyl-sarcosine, and 0.02% SDS. The membranes were
washed sequentially twice for 5 min in 2xSSC with 0.1% SDS at room
temperature followed by two 15-min washes with 0.1xSSC with 0.1% SDS
at 65 C. Alkaline phosphatase-coupled anti-DIG antibody was used
to detect hybridized probe according to the manufacturers
instructions (Boehringer Mannheim Gmbh). The membranes were exposed to
x-ray film for 216 h, and the density of signals was analyzed with a
densitometer (model 300 A, Molecular Dynamics, Sunnyvale, CA).
In the regulatory studies, cells were treated with BMP2 or PTHrP for 2,
4, 8, and 24 h or with increasing concentrations of these factors
for 24 h when the cells grew to reach 80% confluency. These cells
were then harvested and RNA was extracted. All experiments were
performed at least two to three times with representative results
shown.
In Situ Hybridization
Mouse tissues were obtained at three different stages of growth.
Day 15 embryonic mouse tissue was obtained and prepared for in
situ hybridization as described previously (28). Tissues from
newborn and adult mice were immediately fixed in a sterile solution of
4% paraformaldehyde in PBS and kept on ice for 24 h. Tissues were
dehydrated with ethanol and then embedded in paraffin (51). Bone
tissues were transferred to a sterile decalcifying EDTA solution [15%
EDTA, 0.5% paraformaldehyde in PBS (pH 8.0)]. The decalcifying
solution was changed every day until the tissues were completely
decalcified. Tissues were washed three times with PBS before processing
and embedding in paraffin.
In situ hybridization was performed as described (52) with
minor modifications. Paraffin sections, collected on slides pretreated
with 3-aminoprophyltriethoxysilane, were dewaxed with xylene and then
rehydrated in ethanol with sequential concentrations from 100% to 30%
before rinsing in sterile water treated with pyrocarbonic acid diethyl
ester. Tissues were then deproteinized with 0.2 M HCl for
20 min, followed by digestion with 2 µg/ml of proteinase K in 0.1
M Tris buffer (pH 8.0) with 50 mM EDTA for 30
min at 37 C. Digestion was stopped with 2 mg/ml of glycine in PBS for 5
min. The tissues were then fixed in 4% paraformaldehyde in PBS for 15
min before acetylation with 0.25% acetic anhydride in 0.1
M triethanolamine (pH 8.0) for 10 min. Slides were rinsed
in PBS between each treatment and all procedures were carried out at
room temperature unless indicated otherwise. After prehybridization in
hybridization solution containing 50% formamide in 5xSSC
[1xSSC = 0.15 M NaCl and 0.15 M sodium
citrate (pH 7.0), 2% block reagent, 0.1%
N-lauroyl-sarcosine, 0.02% SDS], the sections were
hybridized overnight at 42 C in a humidifying box with hybridization
solution (20 to 40 µl/slide) and the DIG-labeled probe at a
concentration of 8 ng/µl. To compare the expression intensity between
tissue sections under the same experimental conditions, three or four
pairs of newborn and adult tissues were placed on the same glass slide
for in situ hybridization. Slides were protected with
coverslips during the hybridization period. After hybridization, the
sections were washed with 2xSSC at 37 C for 30 min, then 1x SSC, and
finally 0.1x SSC for 30 min each at 37 C. The alkaline
phosphatase-coupled anti-DIG antibody was used to detect hybridized
probe (Boehringer Mannheim GmbH). To ensure reproducibility, the
experiments were performed at least two times for each tissue. In each
experiment, tissues probed with the sense orientation riboprobe were
used as the negative controls.
Experimental Animals
All animal studies were conducted in accord with the principles
and procedures outlined in "Guidelines for the Care and Use of
Experimental Animals."
 |
FOOTNOTES
|
---|
Address requests for reprints to: Kong Wah Ng, Department of Medicine, The University of Melbourne, St. Vincents Hospital, Fitzroy, Victoria, 3065, Australia. E-mail:
k.ng{at}medicine.unimelb.edu.au
This project was supported by a program grant from the National Health
and Medical Research Council (Australia).
Received for publication December 22, 1997.
Revision received June 29, 1998.
Accepted for publication August 7, 1998.
 |
REFERENCES
|
---|
-
Levine M, Hoey T 1988 Homeobox proteins as
sequence-specific transcription factors. Cell 55:537540[Medline]
-
Shashikant CS, Utset MF, Violette SM, Wise TL, Einat P, Einat
M, Pendleton JW, Schughart K, Ruddle FH 1991 Homeobox genes in mouse
development. Crit Rev Eukaryot Gene Express 1:207245[Medline]
-
McGinnis W, Krumlauf R 1992 Homeobox genes and axial
patterning. Cell 68:283302[Medline]
-
Gendron-Maguire M, Mallo M, Zhang M, Gridley T 1993 Hoxa-2
mutant mice exhibit homeotic transformation of skeletal element derived
from cranial neural crest. Cell 75:13171331[Medline]
-
Rijli FM, Mark M, Lakkaraju S, Dierich A, Dolle P,
Chambon P 1993 A homeotic transformation is generated in the
rostral branchial region of the head by disruption of Hoxa-2, which
acts as a selector gene. Cell 75:13331349[Medline]
-
Dolle P, Dierich A, LeMeur M, Schimmang T, Schuhbaur B,
Chambon P, Duboule D 1993 Disruption of the Hoxd-13 gene induces
localized heterochrony leading to mice with neotenic limbs. Cell 75:431441[Medline]
-
Dolle P, Izpisua-Belmonte JC, Falkenstein H, Renucci A,
Duboule D 1989 Coordinate expression of the murine Hox-5 complex
homeobox-containing genes during limb pattern formation. Nature 342:767772[CrossRef][Medline]
-
Izpisua-Belmonte JC, Tickle C, Dolle P, Wolpert L, Duboule D 1991 Expression of the homeobox Hox-4 genes and the specification of
position in chick wing development. Nature 350:585589[CrossRef][Medline]
-
Hill RE, Jones PF, Rees AR, Sime CM, Justice MJ, Copeland
NG, Jenkins NA, Graham E, Davidson DR 1989 A new family of mouse homeo
box-containing genes: molecular structure, chromosomal location, and
developmental expression of Hox 7.1. Genes Dev 3:2637[Abstract]
-
Robert B, Sasson D, Jacq B, Gehring W, Buckingham M 1989 Hox-7, a mouse homeobox gene with a novel pattern of expression during
embyrogenesis. EMBO J 8:91100[Abstract]
-
Tabin CJ 1991 Retinoids, homeoboxes, and growth factors:
toward molecular models for limb development. Cell 66:199217[Medline]
-
Robert B, Lyons G, Simandl BK, Kuroiwa A, Buckingham M 1991 The apical ectodermal ridge regulates Hox-7and Hox-8 gene expression in
developing chick limb buds. Genes Dev 5:23632374[Abstract]
-
Coelho CN, Sumay L, Rodgers BJ, Daridson DR, Hill RE, Upholt
WB, Kosher RA 1991 Expression of the chicken homeobox-containing gene
GHox-8 during embryonic chick limb development. Mech Dev 34:143154[CrossRef][Medline]
-
Coelho CND, Sumoy L, Kosher RA, Upholt WB 1992 GHox-7: a
chicken homeobox-containing gene expressed in a fashion consistent with
a role in patterning events during embryonic chick limb development.
Differentiation 49:8592[Medline]
-
Jabs EW, Muller U, Li X, Ma L, Luo W, Haworth IS, Klisak I,
Sparkes R, Warman ML, Mulliken JB, Snead ML, Maxson R 1993 A
mutation in the homeodomain of the human MSX2 gene in a family affected
with autosomal dominant craniosynostosis. Cell 75:443450[Medline]
-
Towler DA, Bennett CD, Rodan GA 1994 Activity of the rat
osteocalcin basal promoter in osteoblastic cells is dependent upon
homeodomain and CP1 binding motifs. Mol Endocrinol 8:614624[Abstract]
-
Hoffman HM, Catron KM, van Wijnen AJ, McCabe LR, Lian JB Stein
GS, Stein JL 1994 Transcriptional control of the tissue-specific,
developmentally regulated osteocalcin gene requires a binding motif for
the Msx family of homeodomain proteins. Proc Natl Acad Sci USA 91:1288712891[Abstract/Free Full Text]
-
Nohno T, Koyama E, Myokai F, Taniguchi S, Ohuchi H, Saito T,
Noji S 1993 A chicken homeobox gene related to Drosophila
paired is predominantly expressed in the development limb. Dev Biol 158:254264[CrossRef][Medline]
-
Opstelten DJ, Vogels R, Robert B, Kalkhoven E, Zwartkruis F,
de Laaf L, Destree OH, Deschamps J, Lawson DA, Meijlink F 1991 The
mouse homeobox gene, S8, is expressed during embryogenesis
predominantly in mesenchyme. Mech Dev 34:2941[CrossRef][Medline]
-
Cserjesi P, Lilly B, Bryson L, Wang Y, Sassoon DA, Olson EN 1992 MHox: a mesodermally restricted homeodomain protein that binds an
essential site in the muscle creatine kinase enhancer. Development 115:10871101[Abstract/Free Full Text]
-
Martin JF, Bradley A, Olson EN 1995 The paired-like homeo box
gene MHox is required for early events of skeletogenesis in multiple
lineages. Genes Dev 9:12371249[Abstract]
-
Katagiri T, Lee T, Takeshima H, Suda T, Tanaka H, Omura S
1990a Transforming growth factor-ß modulates proliferation and
differentiation of mouse clonal osteoblastic MC3T3E1 cells depending
on their maturation stages. J Bone Miner Res 11:285293
-
Katagiri T, Yamaguchi A, Komaki M, Abe E, Takahashi N, Ikeda
T, Rosen V, Wozney JM, Fujisawa-Sehara A, Suda T 1994 Bone
morphogenetic protein-2 converts the differentiation pathway of C2C12
myoblasts into the osteoblast lineage. J Cell Biol 127:17551766[Abstract]
-
Katagiri T, Yamaguchi A, Ikeda T, Yoshiki S, Wozney JM, Rosen
V, Wang EA, Tanaka H, Omura S, Suda T 1990b The non-osteogenic mouse
pluripotent cell line, C3H10T1/2, is induced to differentiate into
osteoblastic cells by recombinant human bone morphogenetic protein-2.
Biochem Biophys Res Commun 172:295299
-
Ahrens M, Ankenbauer T, Schroder D, Hollnagel A, Mayer H,
Gross G 1993 Expression of human bone morphogenetic proteins-2 or -4 in
murine messenchymal progenitor C3H10T1/2 cells induces differentiation
into distinct mesenchymal cell lineages. DNA Cell Biol 12:871880[Medline]
-
Yamaguchi A, Katagiri T, Ikeda T, Wozney JM, Rosen V, Wang EA,
Kahn AJ, Suda T, Yoshiki S 1991 Recombinant human bone morphogenetic
protein-2 stimulates osteoblast maturation and inhibits myogenic
differentiation in vitro. J Cell Biol 113:681687[Abstract]
-
Lee K, Deeds JD, Segre GV 1995 Expression of parathyroid
hormone-related peptide and its receptor messenger ribonucleic acids
during fetal development of rats. Endocrinology 136:453463[Abstract]
-
Kartsogiannis V, Moseley J, McKelvie B, Chou ST, Hards DK, Ng
KW, Martin TJ, Zhou H 1997 Temporal expression of PTHrP mRNA and
protein during both endochondral and intramembranous bone formation.
Bone 21:18[CrossRef][Medline]
-
Karaplis AC, Luz A, Glowacki J, Bronson RT, Tybulewicz VL,
Kronenberg HM, Mulligan RC 1994 Lethal skeletal dysplasia from targeted
disruption of the parathyroid hormone-related peptide gene. Genes Dev 8:227289
-
Amizuka N, Warshawsky H, Henderson JE, Goltzman D, Karaplis AC 1994 Parathyroid hormone-related peptide-depleted mice show abnormal
epiphyseal cartilage development and altered endochondral bone
formation. J Cell Biol 126:16111623[Abstract]
-
Vortkamp A, Lee K, Lanske B, Segre GV, Kronenberg HM, Tabin CJ 1996 Regulation of rate of cartilage differentiation by indian hedgehog
and PTH-related protein. Science 273:613621[Abstract]
-
Suda N, Gillespie MT, Traianedes K, Zhou H, Ho PWM, Hards DK,
Allan EH, Martin TJ, Moseley JM 1996 Expression of parathyroid
hormone-related protein in cells of osteoblast lineage. J Cell
Physiol 166:94104[CrossRef][Medline]
-
Roach HI, Shearer JR 1989 Cartilage resorption and
endochondral bone formation during the development of long bones in
chick embryo. Bone Miner 6:289309[Medline]
-
Kahn AJ, Simmonds DJ 1977 Chondrocyte-to-osteocyte
transformation in grafts of perichondrium-free epiphyseal
cartilage. Clin Orthop 129:299404[Medline]
-
Richman JM, Diewert VM 1988 The fate of Mechels cartilage
chondrocytes in ocular culture. Dev Biol 129:4860[Medline]
-
Yoshioka C, Yagi T 1988 Electron microscopic observations on
the fate of hypertrophic chondrocytes in condylar cartilage of rat
mandible. J Craniofac Genet Dev Biol 8:253264[Medline]
-
Strauss PG, Closs EI, Schmidt J, Erfle V 1990 Gene expression
during osteogenic differentiation in mandibular condyles in
vitro. J Cell Biol 110:13691377[Abstract]
-
Thesingh CW, Croot CG, Wassenaar AM 1991 Transdifferentiation
of hypertrophic chondrocytes in murine metatarsal bones, induced by
cocultured cerebrum. Bone Miner 12:2540[Medline]
-
Xu C, Ji X, Harris MA, Mundy GR, Harris SE 1997 Transcriptional regulation of the BMP-2 gene in murine chondroblasts.
J Bone Miner Res 12:S293
-
Galotto M, Campanile G, Robino G, Cancedda FD Bianco P,
Cancedda R 1994 Hypertrophic chondrocytes undergo further
differentiation to osteoblast-like cells and participate in the initial
bone formation in developing chick embryo. J Bone Miner Res 9:12391249[Medline]
-
Sims NA, White CP, Sunn KL, Thomas GP, Drummond ML, Morrison
NA, Eisman JA, Gardiner, EM 1997 Human and murine osteocalcin gene
expression: conserved tissue restricted expression and divergent
responses to 1,25-dihydroxyvitamin D3 in vivo. Mol
Endocrinol 11:16951708[Abstract/Free Full Text]
-
Hu Y, Flanagan J, Brennan DP, Zhou H, Ng KW, Eisman JA,
Morrison NA 1995 rHox: a homeobox gene expressed in osteoblastic cells.
J Cell Biochem 59:486497[Medline]
-
Davis CA, Joyner AL 1988 Expression patterns of the homeo
box-containing genes En-1 and En-2 and the proto-oncogene int-1 diverge
during mouse development. Genes Dev 2:17361744[Abstract]
-
Davis CA, Holmyard DP, Millen KJ, Joyner AL 1991 Examining
pattern formation in mouse, chicken and frog embryos with an
en-specific antiserum. Development 111:287298[Abstract]
-
Le Mouellic H, Lallemand Y, Brulet P 1992 Homeosis in the
mouse induced by a null mutation in the homeogene Hox-3.1. Cell 69:251264[Medline]
-
Charite J, de Graaff W, Shen S, Deschamps J 1994 Ectopic
expression of Hoxb-8 causes duplication of the ZPA in the forelimb and
homeotic transformation of axial structures. Cell 78:589601[Medline]
-
Liu F, Malaval L, Gupta AK, Aubin JE 1994 Simultaneous
detection of multiple bone-related mRNAs and protein expression during
osteoblast differentiation: polymerase chain reaction and
immunocytochemical studies at the single cell level. Dev Biol 166:220234[CrossRef][Medline]
-
Yokouchi Y, Ohsugi K, Sasaki H, Kuroiwa A 1991 Chicken
homeobox gene Msx-1: structure, expression in limb buds and effect of
retinoic acid. Development 173:431444
-
Udagawa N, Horwood NJ, Elliot J, Mackay A, Owens J, Okamura H,
Kurimoto M, Chambers TJ, Martin TJ, Gillespie, MT 1997 Interleukin-18
(Interferon-Gamma-Inducing Factor) is produced by osteoblasts and acts
via granulocyte macrophage-colony stimulating factor and not via
interferon-gamma to inhibit osteoclast formation. J Exp Med 185:10051012[Abstract/Free Full Text]
-
Chomczynski P, Sacchi N 1987 Single-step method of RNA
isolation by acid guanidium thiocyanate-phenol-chloroform extraction.
Anal Biochem 162:156159[CrossRef][Medline]
-
Noji S, Yamaai T, Koyama E, Nohno T, Taniguchi S 1989 Spatial
and temporal expression pattern of retinoic acid receptor genes during
mouse bone development. FEBS Lett 257:8396
-
Zhou H, Choong P, McCarthy R, Chou ST, Martin TJ, Ng KW 1994 In situ hybridization to show sequential expression of
osteoblast gene markers during bone formation in vivo.
J Bone Miner Res 9:14891499[Medline]