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Department of Medicine (Y.S.H., H.Z., V.K., T.J.M., K.W.N.) The
University of Melbourne St. Vincent’s Hospital Fitzroy,
Victoria, 3065 Australia
The Garvan Institute of Medical
Research (J.A.E.), St. Vincent’s Hospital Darlinghurst, New
South Wales 2010, Australia
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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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 40–50% 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 106–06 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. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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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 106–06 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 106–06, 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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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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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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)), 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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)) and neither was it expressed in
intestine or liver (data not shown).
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)
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)
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and 7)). 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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1(I) Collagen mRNA Expression in UMR 106–06
by PTHrP (Fig. 8)1(I) collagen mRNA by approximately 60%. This effect was time
dependent and was evident by 4 h. In UMR 201 and UMR 106–06
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).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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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 106–06 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 106–06 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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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Cell Culture
UMR 201 and UMR 106–06 cells were grown in -modified
Eagle’s 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
2–3 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 manufacturer’s
instructions (Boehringer Mannheim Gmbh). The membranes were exposed to
x-ray film for 2–16 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."
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FOOTNOTES |
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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.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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