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Keywords:

  • cadherin;
  • osteoblast;
  • bone density;
  • null mutant;
  • calcification

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

The migration and adhesion of osteoblasts requires several classical cadherins. Cadherin-11, one of the classical cadherins, was expressed in mouse osteoblasts in skull bone and femur, revealed by immunohistochemistry. To elucidate the function of cadherin-11 in osteoblastogenesis, cadherin-11 null mutant mice were investigated. Although apparently normal at birth, Alizarin red staining of null mutant mice showed a reduced calcified area at the frontal suture that caused a round-shaped calvaria with increasing animal age to 3 months. Consequently, there was a reduction in bone density at the femoral metaphyses and the diploë of calvaria in null mutant mice. In the in vitro culture of newborn calvarial cells, the calcified area of mutant cells was smaller than those derived from wild-type littermates. These results show that absence of cadherin-11 leads to reduced bone density in some parts of skeletons including calvaria and long bone metaphyses, and thus suggest that cadherin-11 plays roles in the regulation of osteoblast differentiation and in the mineralization of the osteoid matrix.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

OSTEOBLASTS ARE the skeletal cells responsible for the synthesis, deposition, and mineralization of the bone matrix.(1) Based on morphological and histological studies, osteoblastic cells develop in a presumed linear sequence progressing from osteoprogenitors to preosteoblasts, osteoblasts, and lining cells or osteocytes. The process also has been subdivided into three developmental time stages: proliferation, extracellular matrix development, and mineralization. Investigation of the extracellular matrix during development and, specifically, how cell adhesion molecules are involved in the destiny of cells is important.

Cell adhesion molecules include members of the cadherin, integrin, immunoglobulin, selectin, and proteoglycan superfamilies.(2) Cadherins are Ca2+-dependent homophilic adhesion receptors that play important roles in cell recognition and cell sorting during development.(3, 4) One of the most important and ubiquitous types of adhesive interactions required for the maintenance of solid tissues is that mediated by the classical cadherins, subgrouped into type I and type II.(5, 6) A type II molecule, cadherin-11,(7) is expressed in stromal cells and mesenchymal cells. The mouse cadherin-11 gene, termed osteoblast (OB)-cadherin, has been isolated from the mouse osteoblastic cell line MC3T3-E1,(8) and two types of the human cadherin-11 genes also have been isolated from the human osteosarcoma complementary DNA (cDNA) library.(9) Human cadherin-11 was reported to be expressed in osteoblast cell lines(10) and in human bone tissues,(11) suggesting that cadherin-11 expressed in osteoblasts of calvaria and reactive bone but not in mature osteocytes may participate in osteoblast differentiation. Recently, we and others reported that cadherin-11 expression was associated with the osteoblast lineage, suggesting the involvement of cadherin-11 in osteoblast differentiation.(12, 13)

In this report, to investigate the function of cadherin-11 in bone formation in vivo, we examined the defects in cadherin-11 knock-out mice. The results show a reduction in bone density following cranial defects in mutant mice, and show that cadherin-11 regulates osteoblast differentiation on function in cell-cell interactions for osteoblasts, which is important for mineralization of the osteoid matrix.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

Animals

To generate cadherin-11 knockout mice, a targeting vector was constructed as follows: the portion of the cadherin-11 gene encoding the last 56 amino acids of the extracellular domain through most of the transmembrane was replaced with a PGKNeopA gene cassette.(14) The targeting construct was used to transfect embryonic stem cells, and then heterozygous animals were established by mating germline-transmitting chimeras with C57BL/6 mice. Western blotting showed that E10.5 cadherin-11 mutant embryos expressed a 97-kDa protein. DNA sequencing of this splicing variant revealed that it lacked the transmembrane domain but contained the cytoplasmic domain including the β-catenin binding site; however, transfection of the cDNA of this truncated molecule into L-cells established that it could not be displayed on the cell surface and it lacked adhesion properties.(14)

Genomic PCR and Western blotting

Mice tail genomic DNAs were subjected to the polymerase chain reaction (PCR) analysis to detect genotype using the following primers: KTP91, 5′-ttcagtcggcagaagcaggac-3′; KTP92, 3′-gtgtattggttgcaccatg-5′; and KTP93, 5′-tctatcgccttcttgacgagttc-3′. Wild-type and targeted alleles were amplified with combinations of KTP91 and -92 and KTP92 and -93 primers, respectively. Thirty-eight cycles of amplification were done (30 s at 94°C, 30 s at 60°C, and 30 s at 72°C). For detection of cadherin-11 protein, 10 μg of cell lysates from calvarial cells was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). After transfer to nylon membrane and incubation with a cadherin-11 antibody, signals were detected by an enhanced chemiluminescence (ECL) system (Amersham Pharmacia Biotech, Buckinghamshire, UK).

Immunohistochemistry

Paraformaldehyde fixed and decalcified bone tissues were cut into 4-μm-thick sections. Immunohistochemical studies were performed by the peroxidase streptavidin-biotin method (Nichirei, Tokyo, Japan). The mouse monoclonal antibody against cadherin-11 (Ola-9) was obtained by immunization of the recombinant N-terminal EC-1 domain of mouse cadherin-11 into a cadherin-11 null mutant mouse. A streptavidin-conjugated antibody of Ola-9 was used for detection. The biotin-conjugated mouse immunoglobulin G (IgG; Cedarlane Labs., Ontario, Canada) was used as a control antibody.

Skeletal preparation

Bone and cartilage tissues of mice were stained as described by Komori et al.(15) with minor modifications. In brief, eviscerated newborn mice were fixed in ethanol and acetone for 1 day each and stained by Alizarin red and Alcian blue 8GX for 5 days. After incubation in phosphate-saturated buffer with 1% trypsin, specimens were kept in 40% glycerol/1% KOH at 37°C until skeletons become clearly visible. They were transferred to 60, 80, 90, and finally 100% glycerol, for storage.

Radiographic and microcomputed tomography scanning analyses

Radiographs of the wild-type and mutant mice were taken by mFX-1000 (Fuji Photo Film Co., Ltd. Tokyo, Japan). The cross-sectional tomogram produced by the microfocus X-ray computed tomography (CT) equipment (model NX-CP-C80H-IL) was developed by Nittetsu ELEX Co., Ltd. (Osaka, Japan). To evaluate the bone structure of femoral metaphysis, serial 100 slices from the growth plate of femur were examined by using the three-dimensional reconstruction of 100 tomograms obtained with amplification of a 20-μm-thick slice and reconstructed at 512 × 512 pixels performed by use of an acceleration voltage of 30 kV and a current of 0.1 mA in the volume-rendering method (VIP-station with SUN SPARK-5). The threshold of each image was kept at the same level, when trabecular bone and cortex in femur were observed.

Histology

Mice were injected with calcein at 20, 19, 18, 3, 2, and 1 days before death. Then, decalcified 4-μm sections of 3-month-old mice were stained with hematoxylin and eosin and tartrate-resistant acid phosphatase (TRAP) following basic procedures. In brief, sections were embedded in either methylmethacrylate or glycol methacrylate. For TRAP activity, sections were incubated with a mixture of 0.1 mg/ml naphthol AS-MX phosphate (Sigma, St. Louis, MO, USA), 0.5% N,N-dimethylformamide, and 0.6 mg/ml fast red LB salt (Sigma) in 0.1 M acetate buffer solution (pH 5.0) at 37°C.

Analysis of calvarial cells

Calvariae isolated from newborn mice were incubated for 40 minutes in collagenase solution (phosphate-buffered saline [PBS] containing 0.1% collagenase and 0.2% dispase) at 37°C with agitation. Calvarial cells in the suspension were collected by centrifugation and then cultured in α modified essential medium (α-MEM) containing 10% fetal calf serum (FCS) for 2 days in 60-mm dishes. After removal of nonadherent cells, cells were dissociated and inoculated into 6-well plates (2 × 105 cells/well) or 24-well plates (2 × 104 cells/well) to detect calcification and alkaline phosphatase (ALP) activity in α-MEM containing 10% FCS, 50 μg/ml ascorbic acid, and 10 mM β-glycerophosphate. Cells were fixed in 10% formalin and mineralization was visualized by incubation with a 2% Alizarin red solution.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

To analyze the specific expression of cadherin-11 in mouse osteoblasts, immunohistochemical staining of bone tissue of 3-month-old mice was performed. Strong expression was observed at the outer periosteal layer of calvaria (Fig. 1A), in the sagittal suture (Fig. 1C), and in the periosteal surfaces of the femoral diaphysis (Fig. 1E) and at the primary spongiosum in femoral trabecular bone (Fig. 1I), whereas fainter expression of cadherin-11 was observed in the endosteal surfaces of the femoral diaphysis (Fig. 1G, shown by an arrow). Cadherin-11 was not clearly detectable in osteocytes. These results suggest that cadherin-11 plays a role as a homophilic cell adhesive molecule on osteoblasts. However, the function of cadherin-11 in osteoblast development and subsequent osteogenesis has not yet been elucidated.

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Figure FIG. 1.. Distribution of cadherin-11-positive cells in bone tissues. Immunohistochemistry of cadherin-11 at skull bone and at femur. The upper row of panels shows outer layer of (A) calvaria, (C) sagittal suture, (E) periosteum, and (G) endosteum of femoral diaphysis and (I) primary spongiosa of femur in a 3-month-old C57BL/6 mouse were immunostained with anti-cadherin-11 antibody. The lower panels (B, D, F, H, and J) show the corresponding negative control sections stained with biotin-conjugated IgG. Bar indicates 10 μm and an arrow shows cadherin-11-positive cells.

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The targeting strategy for the null mutation of cadherin-11 reported earlier was designed to delete the entire transmembrane and cytoplasmic domains; thus, the mutant cadherin-11 (97 kDa) was detected in E10.5 embryos.(14) In our experiments, a truncated form of cadherin-11 was detected in the primary culture of calvarial osteoblasts from the null mutant mice (−/−) and the heterozygous mice (+/−; Fig. 2), which is the mutant cadherin-11. On the other hand, the intact form was found as a 116-kDa band in calvarial osteoblasts from wild-type mice (+/+) and heterozygous (+/−) cadherin-11mice (Fig. 2). Fluorescent staining of calvarial osteoblasts showed that cadherin-11 was localized to the adherens junctions in wild-type mice but showed a diffuse staining pattern in the null mutant mice although β-catenin was localized to the adherens junctions in both wild-type mice and mutant mice (data not shown). These data suggest that the adhesive function of cadherin-11 is disrupted in osteoblasts from the mutant mice. This conclusion is in accord with the previous finding that the mutant cadherin-11 had no effect on the cadherin-mediated cell aggregation.(14)

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Figure FIG. 2.. Identification of cadherin-11 protein. Cell lysates were prepared from calvarial cells isolated form newborn mice of wild (+/+), heterozygote (+/−), and mutant (−/−) and were subjected to Western blotting by using anti-mouse cadherin-11 antibody.

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Because previous observations showed a low survival rate of inbred crosses (data not shown), heterozygous mice backcrossed five times to a C57BL/6 strain were used in this study and were intercrossed to generate null mutant mice. Cadherin-11 (−/−) mice have a normal external appearance indistinguishable from that of wild-type littermates at birth (Fig. 3A). Alizarin red and Alcian blue staining of the newborn mutant mice showed that skeletal components including the shape of the calvaria, tibia, and femur also were apparently normal (Fig. 3B), whereas a clear reduction in the calcified area at the sagittal suture of calvaria was observed in the mutant mice (Figs. 3C and 3D). These results indicate that the interparietal bone may be slightly hypoplastic in the mutant mice, resulting in widened foramen and open sutures.

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Figure FIG. 3.. Skeletal structure of wild-type (+/+) and mutant (−/−) newborn mice. (A) Gross appearance; (B) skeletal staining by Alizarin red and Alcian blue; (C) total view of calvaria from top. (D) magnification of frontal suture. Note that calcification was delayed at the frontal suture in the null mutant mice.

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The skeletal phenotype of the null mutant mice at 3 months old was further investigated using soft X-ray and CT scanning analyses. Soft X-ray examination revealed that the mutant mice had a round-shaped skull in comparison with normal littermates (NLMs) as shown in Fig. 4A. Other bones including the femurs were generally normal in the mutant mice (data not shown). Concerning calvaria, the diploë of mutant mice was reduced drastically on CT scanning analysis (Figs. 4B and 4C). The bone volume/total tissue volume (BV/TV) ratio, a histomorphometric index of trabecular bone mass, in the femoral diaphysis was almost the same between wild-type mice and mutant mice (Figs. 4D and 4F); however, that of the metaphysis in mutant mice was reduced to 75% of the normal value (Figs. 4E and 4F). The histological analyses using tibial metaphyses from the mutant and wild-type mice confirmed the reduced bone density in the long bone metaphyses of the mutant mice (Fig. 5). Therefore, these data suggest that the mutant mice show a reduction in bone density following a phenotype of cranial defects.

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Figure FIG. 4.. Radiological and CT-scanning analysis of skeletons of wild-type (+/+) and mutant (−/−) mice at 3 months old. (A) Total view from wild-type (+/+) or mutant (−/−) mice at 3 month old was examined by X-ray. (B) Lateral section of calvaria, (C) high magnification of panel B, (D) intersection of femur diaphysis, and (E) metaphysis from wild-type (+/+) or mutant (−/−) mice at 3 months old were examined by CT scanning. (F) The bone volume (%) at the site of diaphysis or metaphysis shown in panel D or E is represented as ratio of BV/TV, and bars represent mean ± SD for wild-type (+/+) and mutant (−/−) mice at 3 months old. *p < 0.05; n = 4. The mutant mice have a round-shaped calvaria, and bone mass is reduced at vertebrate.

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Figure FIG. 5.. The histological analysis of long bone in mutant mice. The histological analyses of tibial metaphyses from 3-month-old wild-type (+/+) or mutant (−/−) mice were performed by using the Goldner's trichrome stain.

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Established histomorphometric parameters estimating bone mass and bone cell numbers were obtained by analyzing undecalcified plastic sections of tibia from the mutant and wild-type mice. Mice were labeled with calcein in vivo to measure the amount of bone deposited in the time interval defined by the administration of the fluorescent marker. The BV/TV ratio was reduced to 72% of the normal value at 3 months of age (Fig. 6A), which agrees with the reduced bone volume of metaphysis shown in Figs. 4 and 5. The mineralizing surface (MS; which estimates the proportion of bone surface undergoing mineralization; Fig. 6B) was decreased to 50% in the mutant mice compared with NLMs. On the other hand, the level of erosion surface (ES/BS; %; Fig. 6D) or the number of TRAP+ osteoclasts (OcS/BS%; Fig. 6F) in the mutant mice was equivalent to that in NLMs, as were other parameters such as the mineral apposition rate and the osteoblast surface (Figs. 6C and 6E). This result indicates that the reduced bone density is not attributed to increases in bone resorption by OcS but probably to defects for mineralization of the osteoid matrix, because the mineralizing surface was reduced although the mineral apposition rate was not changed.

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Figure FIG. 6.. Bone histomorphometry. Calcein was injected into 3-month-old wild-type (+/+) and mutant (−/−) mice at 20, 19, 18, 3, 2, and 1 day before death and then decalcified 4-μm sections of tibia from mice were stained with hematoxylin and eosin and TRAP. (A) BV/TV (%) represents the ratio of bone volume to tissue volume. (B) MS/BS (%), (D) ES/BS (%), and (E) Ob.S/BS (%) indicate the proportion of bone surface covered with mineralizing area, erosion area, and osteoblasts, respectively. (C) Mineral apposition rate measures the amount of bone that is mineralized per time unit and is based on the measurement of the distance between the two calcein labels. (F) OcS/BS (%) estimates bone resorption as osteoclast surface and number, respectively, over bone surface. Bars represent mean ± SD for wild-type (diagonal bars) and the mutant (open bars) mice of 3 months. *p < 0.05; n = 4.

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To investigate further the alteration in osteoblast function in the mutant mice, primary calvarial cells were prepared from the neonatal mutant mice and NLMs, and calcification was compared after in vitro differentiation (Fig. 7A). The calcified area as estimated by Alizarin red staining of the cultures was reduced in the mutant mice (Fig. 7B). The number of nodules was not significantly different between them, but the number of large nodules from the mutant mice was lower, probably because osteoblast formation was disrupted by the inactivation of homophilic cell adhesion caused by the lack of cadherin-11. However, proliferation of calvarial cells was not significantly different between wild-type cells and mutant cells (data not shown). These results indicate that cadherin-11 functions directly in the mechanism of osteoblast differentiation via cell adhesion, which is reflected by the reduction of bone density in vivo in the mutant mice.

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Figure FIG. 7.. Calcification of long-term-cultured calvarial cells isolated from wild-type (+/+) and mutant (−/−) newborn mice. (A) Calvarial cells isolated from wild-type (+/+) and mutant (−/−) newborn mice were cultured for 10, 17, or 30 days, and then Alizarin red staining was performed. The representative result of three independent experiments was shown. Similar results were obtained from seven different wild-type and the mutant littermates (data not shown). (B) The diagrammatic representation of the calcified area (%) of each cultured calvarial cells indicated in panel A compared with the total area was shown by using data from seven different wild-type and the mutant littermates.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

Classical cadherins have been considered as homophilic cell adhesion receptors. Interestingly, restoration of E-cadherin to the tumor cell line caused mesenchymal to epithelial transition.(16) Also, constitutive expression of E-, N-, or R-cadherin restricted the differentiation pathway of embryonic stem cells into epithelial, neuroepithelial/chondroblasts, or muscle cells.(17, 18) These observations suggest that classical cadherins are important for tissue formation or cell differentiation through their adhesive activities. Recently, it was reported that several cadherins including N-cadherin and cadherin-11 were crucial for the adhesive properties of osteoblasts.(9–11) However, function of the adherens junctions formed by osteoblasts has not yet been explained clearly. Because N-cadherin null mutant mice are embryonic lethal at E10 before calcification, it is impossible to investigate the function of N-cadherin in osteoblastogenesis.(19) However, it is noted that double null mutations of cadherin-11 and N-cadherin showed a more severe abnormality of the somites than N-cadherin single null mutant mice. These results indicated that both cadherin-11 and N-cadherin compensated for each other during the process of somatogenesis,(14) and they may compensate each function in differentiation from mesenchymal cells to osteoblasts.

In this article, we revealed that cadherin-11 contributes to bone formation in calvaria and long bone metaphyses. Cadherin-11 may function in the late stage of osteoblast differentiation through its adhesive properties: cell-cell and cell-matrix interactions, facilitated by cadherins, which is important for mineralization of the osteoid matrix. In the cadherin-11 mutant mice, the function of cadherin-11 in bone formation may be compensated by N-cadherin because immunohistochemistry revealed that the mouse N-cadherin was expressed in the osteoblasts (data not shown).

Here, we investigated that absence of a cell adhesion molecule leads to skeletal defects; thus, the results show that a cell adhesion molecule acts as a positive regulator of increasing bone mass. Interestingly, the abnormal phenotype in cadherin-11 mutant mice was preferentially observed in the skull bone, suggesting that the mutant mouse is a good tool for investigating the molecular mechanism of cranium construction, especially suture formation. Lincecum et al. reported that Msh genes (Msx-1 and -2) regulate cadherin-mediated adhesion,(20) and Msx-1- and Msx-2-deficient mice showed abnormal skull structure.(21, 22) Thus, cadherin molecules themselves possibly control osteoblast differentiation.

A similar result was observed in chondrogenesis in which the perturbation of N-cadherin activity by an antibody resulted in malformation of primordial cartilage(23); therefore, specific classical cadherins generally may affect the late stage of maturation, formation, and function of related cells including osteoblasts. The precise mechanism of cadherin-11 regulation together with N-cadherin in bone formation remains to be resolved.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

We thank Dr. A. Grigoriadis for critical reading of the manuscript. This work was supported by grants from the Ministry of Education, Science, Sports and Culture of Japan.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES
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