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. 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


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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 ({alpha}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-{alpha}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
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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 {alpha}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 ({alpha}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.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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. 1Go)
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. 1Go). 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: A–F, x400.

 
rHox/MHox mRNA Expression in Newborn Mouse Tissues (Fig. 2Go)
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. 2Go), 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. 2Go) 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).

 
rHox/MHox mRNA Expression in Adult Mouse Tissue (Fig. 3Go)
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. 3Go) 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).

 
Northern Blot Analyses
Expression of rHox/MHox mRNA in Tissues of Newborn and Adult Mouse (Fig. 4Go)
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

 
Regulation of rHox/MHox mRNA Expression by BMP2 (Fig. 5Go)
UMR 201 or UMR 106–06 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 106–06, 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 106–06 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.

 
Regulation of rHox/Mhox mRNA Expression by PTHrP (Figs. 6Go and 7Go)
rHox/MHox mRNA expression in response to PTHrP was determined in UMR 106–06 cells but not in UMR 201 cells because UMR 201 cells do not express receptors for PTH/PTHrP. UMR 106–06 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. 6Go). 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. 7Go). 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. 7AGo). PTHrP mRNA was not detected in osteoblasts obtained from PTHrP -/- mice (Fig. 7BGo).



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Figure 6. Northern Blot Analysis for rHox/MHox mRNA Expression in Osteoblastic UMR 106–06 Cells

Cells were treated with 100 ng/ml PTHrP for 2–24 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.

 
Regulation of pro-{alpha}1(I) Collagen mRNA Expression in UMR 106–06 by PTHrP (Fig. 8Go)
Treatment of UMR 106–06 cells resulted in a reduction in expression of pro-{alpha}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-{alpha}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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Figure 8. Regulation of pro-{alpha}1(I) Collagen mRNA by PTHrP

Osteoblastic UMR 106–06 cells were treated with 100 ng/ml PTHrP for 2–24 h. The relative quantities of pro-{alpha}1(I) collagen mRNA are expressed as the collagen/18S ratios. PTHrP, which had increased rHox mRNA expression, suppressed pro-{alpha}1(I) collagen mRNA expression.

 
Regulation of rHox/MHox mRNA in Primary Calvarial Osteoblasts by BMP2 and PTHrP (Fig. 9Go)
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.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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 {alpha}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.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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. Vincent’s Institute of Medical Research, Melbourne, Australia).

Cell Culture
UMR 201 and UMR 106–06 cells were grown in {alpha}-modified Eagle’s medium ({alpha}-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."


    FOOTNOTES
 
Address requests for reprints to: Kong Wah Ng, Department of Medicine, The University of Melbourne, St. Vincent’s 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
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 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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